Proteases of Infectious Agents

PREFACE

As the decade of the 1990s draws to a close, it is appropriate to assess the changes that have taken place in the drug discovery process. Previously, an infectious agent was identified and cultured for two purposes: (1) so that tests could be established to screen compounds for inhibition of growth of the infectious organism, and (2) to permit isolation of proteins that could serve as drug targets in favorable cases where sufficient quantities could be obtained. In this decade a new paradigm has emerged. Isolation of the genetic material of the infectious organism, followed by sequence analysis at the DNA level by rapid, automated methods, can reveal the entire genomic structure in a short time. Straightforward analysis using sequence-searching algorithms can lead to the identification of possible critical functional activities. Cloning of the specific region of the genome can yield the target for new drug development. With the exploding database of protein structure, and with homology modeling programs, it is now possible to predict structure and initiate the drug discovery process while waiting for solution of the X-ray structure. During the period following the identification of sequences within the HIV genome that fit the template for the structure of an aspartic protease, drugs were designed and synthesized, and the effect on viral growth was demonstrated. Now that FDA-approved drugs have been shown to suppress viral growth and improve the health of thousands of patients, we can conclude that the new paradigm for drug discovery has been successful. Of course, this account should not fail to point out the continuing problems of patient noncompliance, low bioavailability, and rapid development of resistant viral strains. Nonetheless, the value of protease-directed drugs, as well as the best pathway to finding them, is clear. Fittingly then, this book begins with an account of the development of antiviral agents targeted for AIDS. Important lessons derived from this work in-

xv

XV1

Preface

clude the demonstration, through catalytic mutation, that an active protease was essential for viral replication. Also, the value of the early determination of the three-dimensional structure of the enzyme and enzyme-inhibitor complexes has been established. Next, the iterative design process, based on structure determination, marks the HIV-1 protease case as a defining example for future work. Finally, the problems associated with drug metabolism and the resistance question complete the catalog of lessons learned in this case. Other chapters in this book summarize current knowledge regarding new drug targets from other infectious organisms. A significant new effort is directed toward a serine protease encoded by the hepatitis C virus. Due to the relatively recent identification of this virus and the lack of a cell-culture system, progress was very slow. In the past three years, however, expression of the protease and the solution of its structure have dramatically stimulated the search for drugs in this area. In addition, a shift occurred when many pharmaceutical companies cut back their HIV efforts because of the success of the Roche, Abbott, and Merck compounds. Hepatitis C was the next disease likely to have a significant public health impact in the United States and elsewhere due to the spread of the infection through transfusion before its discovery. As detailed in the chapter by Urbani, De Francesco, and Steink~hler on this subject, a strategy similar to that in the HIV case was followed. A huge body of work, conducted largely in pharmaceutical companies, is summarized by Qiu and Abdel-Meguid in their chapter on the human herpesviruses. Here again, the paradigm of discovery of a viral sequence, cloning, expression, and structure determination has proven to be the route to drug design. In this case especially, the structural insights reveal novel mechanisms of action that could provide clues for potent and selective inhibitor design. The Candida genus provides an example of an infectious agent that is not a problem for a healthy human. However, in the case of a patient whose immune system has been impacted by infectious agents such as HIV or through treatment with immune-suppressing agents to avoid transplant rejection, severe systemic infections can be the ultimate cause of death. In the chapter by Stewart, Goldman, and Abad-Zapatero, several related crystal structures derived from protease variants cloned from C. albicans are described. Unique insights that point the way toward selective inhibitor design are derived. Work in the picornarvirus arena has been under way for some time; however, the new information described by Bergmann and James seems likely to stimulate this area significantly. Progress in this field will have widespread impact, as the picornavirus family includes the rhinoviridae, which bring us the common cold, as well as many others. The chapters by Berry, on malaria, and by Cazzulo, on Chagas disease, represent the field of protozoan infectious species. Berry provides a thorough summary of current knowledge on hemoglobin degradation during the blood-borne

Preface

xvii

stage of malaria. The parasite is somewhat unique in presenting two targets for drug discovery: a cysteine protease (falcipain) and an aspartic protease (plasmepsin). The interplay of these two enzymes in the complicated process of breakdown of the globin chain provides several strategies for attack. In the case of T. cruze, the organism that causes Chagas disease, the major antigen is a cysteine protease, cruzipain (or cruzain). Cazzulo describes the involved life cycle, the properties of cruzipain, and other putative serine proteases of the organism. Successful infection by foreign agents frequently requires some function of the host. In the chapter by Kido, Chen, Murakami, Beppu, and Towatari, the role of cellular proteases in infection by influenza A and Sendai viruses in the respiratory tract and tryptase TL2 in T lymphocytes is described. The involvement of a cellular enzyme in the entry of HIV-1 into cells is also discussed. The implications for future therapeutic intervention are clear, but the complications of attempting to alter the function of a normal cellular enzyme are also significant. While it has been clearly established that polyprotein processing in the case of viruses is an essential feature of their life cycle, the world of bacteria is not so straightforward. One point of attack is the necessity for processing newly synthesized proteins targeted by the bacteria for export. Lively discusses the bacterial signal peptidases in a chapter that precedes structure determination. Nonetheless, the ground is well-prepared for the anticipated structural information. In this case as in the others, the differences between the bacterial enzyme and the related human enzyme will be critical to development of selective inhibitors. The world of plants is represented by the chapter of Garcia, FernandezFern~indez, and L6pez-Moya describing plant viruses. This area is unique in that a wide range of protease mechanisms are found, lacking only the metalloproteases. Given the huge impact of plant viruses on crop production, this is a field with a large potential for future growth. While this compilation is not encyclopedic, the chapters presented cover a wide range of infectious agents and mechanisms. There also are obvious differences in the state of knowledge of the different proteases described. This is appropriate, as efforts in the area of proteases will continue to expand into research in new diseases and infectious agents as they are discovered. The task before us is clear: Find the critical protease and develop a potent and selective inhibitor. Ben M. Dunn

CONTRIBUTORS

Numbers in parentheses indicate the pages on which the authors' contributions begin.

CELE ABAD-ZAPATERO (117) Protein Crystallography D-46Y, Abbott Laboratories, Abbott Park, IL 60064 SHERIN S. ABDEL-MEGUID (93) Department of Macromolecular Science, SmithKline Beecham, King of Prussia, PA 19406 YOSHIHITO BEPPU (205) Department of Enzyme Chemistry, Tokushima University of Medical School, Tokushima 770, Japan ERNST M. BERGMANN (139) Dept. of Biochemistry, University of Alberta/ Edmonton, Edmonton, Alberta T6G 2H7, Canada COLIN BERRY (165) Cardiff School of Biosciences, Cardiff University, Cardiff CF1 3US, Wales, UK JUAN JOSl~ CAZZULO (189) Instituto de Investigaciones Biotechnol6gicas, Universidad Nacional de Beneral San Marin, San Martin 1650, Buenos Aires, Argentina YE CHEN (205) Department of Enzyme Chemistry, Tokushima University of Medical School, Tokushima 770, Japan RAFFAELE DE FRANCESCO (61) Instituto di Ricerche di Biologia Moleculare, 00040 Pomezia, Rome, Italy MICHAEL A. EISSENSTAT (1) Structural Biochemistry Program, NCIFCRDC, Frederick, MD 21702 JOHN W. ERICKSON (1) Structural Biochemistry Program, NCI-FCRDC, Frederick, MD 21702 xiii

xiv

Contributors

MARIA ROSARIO FERNANDEZ-FERNANDEZ (233) Centro Nacional de Biotecnologia, Campus de la Universidad Autonoma, 28049-MADRID, Spain JUAN ANTONIO GARCIA (233) Centro Nacional de Biotecnologia, Campus de la Universidad AutOnoma, 28049-MADRID, Spain ROBERT C. GOLDMAN (117) Anti-infective Group, D-47, AP-9A, Abbott Laboratories, Abbott Park, IL 60064 MICHAEL N. G. JAMES (139) Department of Biochemistry, University of Alberta/Edmonton, Edmonton, Alberta T6G 2H7, Canada HIROSHI KIDO (205) Department of Enzyme Chemistry, Tokushima University of Medical School, Tokushima 770, Japan MARK O. LIVELY (219) Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, NC 27157 JUAN JOSI~ LOPEZ-MOYA (233) Centro Nacional de Biotecnologia, Campus de la Universidad Autonoma, 28049-MADRID, Spain MEIKO MURAKAMI (205) Department of Enzyme Chemistry, Tokushima University of Medical School, Tokushima 770, Japan XIAYANG QIU (93) Department of Macromolecular Science, SmithKline Beecham, King of Prussia, PA 19406 CHRISTIAN STEINKOHLER (61) Instituto di Ricerche di Biologia Moleculare, 00040 Pomezia, Rome, Italy KENT STEWART (117) Molecular Modeling Group, D-46Y, AP-10, Abbott Laboratories, Abbott Park, IL 60064-3500 TAKAE TOWATARI (205) Department of Enzyme Chemistry, Tokushima University of Medical School, Tokushima 770, Japan ANDREA URBANI (61) Instituto di Ricerche di Biologia Moleculare, 00040 Pomezia, Rome, Italy

HIV Protease as a Target for the Design of Antiviral Agents for AIDS JOHN W. ERICKSON AND MICHAEL A. EISSENSTAT Structural Biochemistry Program, National Cancer InstitutemFrederick Cancer Research and Development Center, Frederick, Maryland 21702

I. II. III. IV. V. VI.

Introduction Retroviruses, HIV, and AIDS HIV Protease: Biology, Biochemistry, and Structure Design of HIV Protease Inhibitors Drug Resistance Future Challenges in HIV Protease Inhibitor Design VII. Summary and Conclusions References

I. I N T R O D U C T I O N The design of clinically effective inhibitors of HIV protease has been a major success story for m o d e r n antiviral therapy and has raised the awareness and attractiveness of viral proteases in general as drug design targets. 2 C u r r e n t interest in viral proteases as drug design targets is highlighted by the chapters in this b o o k devoted to the proteases of herpesviruses, adenoviruses, plant viruses, picornaviruses, hepatitis C viruses, and HIV. A c o m m o n strategy in the life cycle of viruses is the utilization of polycistronic messenger RNAs that can be translated into precursor polyproteins which subsequently are processed The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services nor does mention of trade names, commercial products, or organization imply endorsement by the U.S. Government. 2There are two distinct types of human immunodeficiency virus, HIV-1 and HIV-2. In addition, multiple subtypes have been identified for HIV-1. For clarity, HIV will be used throughout this chapter to denote HIV-1, subtype B unless otherwise indicated. Proteasesof InfectiousAgents

Copyright9 1999byAcademicPress.Allrightsof reproduction in anyform reserved.

2

John W. Erickson and Michael A. Eissenstat

enzymatically into mature, functional proteins during virus assembly (Kr~iusslich et al., 1988; Kay and Dunn, 1990). Processing enzymes may be either cellular or viral encoded and offer novel targets for intervention. Virus-encoded proteases afford particularly attractive therapeutic targets for the design of antiviral agents that are highly specific and nontoxic to their host cells. Viral proteases can be classified according to their mechanism of action as serine, cysteine, aspartic, or metalloproteinases. This chapter discusses the key biological and structural features of HIV protease (HIV PR) that define its usefulness as an antiviral target. A brief review as well as an update on efforts to discover and design inhibitors of HIV PR inhibitors is presented. Finally, the problem of drug resistance to HIV PR inhibitors is discussed with an emphasis on viral strategies for resistance and on implications for future drug discovery efforts in this area.

II. R E T R O V I R U S E S , HIV, A N D A I D S The human immunodeficiency virus type 1 (HIV) causes acquired immunodeficiency syndrome (AIDS) in humans. This disease was recognized only recently in the U.S., around 1981, as a unique clinical syndrome manifested by opportunistic infections or malignancies associated with an underlying defect of the immune system characterized by the progressive loss of CD4 helper T cells. Nearly always fatal unless treated, AIDS cases currently number upward of 2.0 million reported cases worldwide and may be closer to 10 million based on recent estimates from the 1998 AIDS meeting held in Geneva, Switzerland. The number of HIV-infected individuals is much higher, around 30 million, and current projections of global prevalence predict 4 0 - 1 0 0 million HIVinfected individuals by the year 2000. Human immunodeficiency virus is a lentivirus and belongs to the family Retroviridae, which are enveloped, positive-sense, single-stranded RNA viruses. Retroviruses induce a variety of neoplastic diseases and are widely distributed among vertebrate species. A defining characteristic of these viruses is their use of an RNA-dependent DNA polymerase, or reverse transcriptase (RT), for replication of the viral RNA. Many of the molecular events specific to HIV-1 infection have been characterized including functions common to the retrovirus life cycle (Fig. 1). Infection by a retrovirus results in the synthesis of one or more double-stranded DNA intermediates by the action of RT and at least one DNA copy of the viral genome is integrated into the host DNA. This proviral DNA serves to direct its own transcription, translation, and assembly of new virions. This ability of retroviruses to incorporate their genomes into that of their host cells endows them with the capability to be stably maintained during the life of the host and even to be transmitted through the germ line. While most germline "endogenous" retroviruses found in animals and humans are believed to be

HIV

Protease

3

nonpathogenic, tumor-causing retroviruses are fairly common in animals, and their discovery dates back to 1901. However, the first definitive reports that associated a retrovirus with disease in humans would not appear until 1980 when it was discovered that adult T-cell leukemia (ATL) was caused by human T-cell leukemia virus (HTLV-I) (Poiesz et al., 1980). First described in 1977, ATL is highly malignant; median survival is measured in months (Kawano et al., 1985). Current prevalence estimates of ATL are around 1 0 - 2 0 million cases worldwide. There is currently no effective therapy for ATL. Human T-cell leukemia virus has also been attributed to be the cause of a myelopathy disorder as well as an aggressive form of non-Hodgkins T-cell lymphoma. Shortly after the discovery of HTLV-I, another C-type retrovirus closely related to HTLV-I, named HTLV-II, was isolated from a patient with hairy cell leukemia (Kalyanaraman et al., 1982). Two years later a third human retrovirus, initially called HTLV-III, was linked to AIDS (Gonda et al., 1985) and was later found to be nearly identical to lymphadenopathy-associated virus isolated a year earlier (Wain-Hobson et al., 1991). When the capsid morphology and genetic structure clearly identified HTLV-III as a lentivirus (Gonda et al., 1985), its name was changed to HIV-1. The lentiviruses comprise a subfamily of retroviruses that characteristically cause chronic infections and disease in animals and include bovine, feline, and simian immunodeficiency viruses, equine infectious anemia virus, and visna virus of sheep. A second human AIDS virus, HIV-2, was discovered in the mid-1980s (Clavel et al., 1986). Human immunodeficiency virus 2 has a distinct geographic distribution from HIV-1 and is found mainly in West Africa. Human immunodeficiency virus type 2 appears to have a lower morbidity and longer clinical latency than HIV-1 and is closely related in sequence to SIV, a lentivirus that causes an AIDS-like disease in macaques (Gao et al., 1994). This brief history underscores the fact that the identification of disease-associated retroviruses in humans is fairly recent. Thus, much of our current understanding of HIV infection and, in particular, the identification and characterization of targets for antiviral drug design have drawn heavily on basic virology studies obtained with animal retroviruses prior to the discovery of HIV (for a comprehensive review, see Coffin et al., 1997). The discovery of HIV-1 in 1984 led to a parallel explosion of research on the molecular virology of this infectious agent and to an intensive, global search for a cure for this fatal disease that continues to the present. Efforts to inhibit HIV continue to represent one of the most active areas of antiviral research today, and nearly every aspect of the viral life cycle has become a target for antiviral drug discovery (Mitsuya and Broder, 1987; De Clercq, 1995). Modern antiviral strategies are turning more and more to the use of structure- and mechanismbased approaches for the design of safer, more specific, and effective drugs. The successful introduction of HIV PR inhibitors for AIDS treatment is a testimonial to the importance of structure- and mechanism-based approaches in the

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HIV Protease

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discovery and design of more effective, less toxic antiviral therapies. To date, there are four FDA-approved PR inhibitorsmSaquinavir, Ritonavir, Indinavir, and Nelfinavir. These compounds are highly potent and play a central role in the development of the highly active antiretroviral therapies that comprise the current standard of care and that, for the first time in the brief history of HIV infection, provide dramatic and durable suppression of HIV replication. It is probably safe to assume that much of the attention currently being paid by the pharmaceutical industry to other viral proteases as drug design targets is a "coattail" effect based on the HIV success story. It can be argued that much of the current focus on viral proteases as targets for antiviral therapy really stems from the collision of two disparate disciplines--protease biochemistry and virology. Thus, it is worth reflecting on the scientific developments that led up to the identification of HIV PR as an attractive target for drug design.

III. H I V P R O T E A S E : BIOLOGY, B I O C H E M I S T R Y , AND STRUCTURE A. ROLE OF H I V PROTEASE IN THE VIRAL LIFE CYCLE The HIV genome, like all other retroviral genomes, is a single-stranded, positivesense RNA molecule that is organized into three major coding elements: the gag, pol, and env genes. The gag and pol gene products are translated from a single unspliced polycistronic mRNA that encodes both genes (Fig. 2). A stop codon in the unspliced RNA leads to the translation of a 55-kDa Gag polyprotein, Pr55 gag,that contains sequences of the structural proteins of the virionm matrix (MA), capsid (CA), and nucleocapsid (NC)malong with the peptides p2, p 1, and p6 that are involved in the assembly and morphogenesis of mature capsids. The pol gene encodes the viral enzymes necessary for replicationm protease (PR), reverse transcriptase (RT), and integrase (IN). These proteins

FIGURE 1 The HIV life cycle. Human immunodeficiency virus infects a T cell via recognition of the CD4 receptor on the cell surface. Fusion of the viral envelope and cell membranes leads to cytoplasmic invasion by the nucleoprotein core of the virus. Proviral DNA is synthesized using the virion-associated reverse transcriptase and tRNA as a primer. Integration of proviral DNA is mediated by a viral integrase, also present in the infecting virion, and host factors. Transcription of proviral DNA into spliced and unspliced RNAs provides mRNAs for translation of the gag, pol, and env gene products as well as viral RNA for packaging. Assembly of the Gag and Gag-Pol precursor proteins and packaging of the viral RNA occurs at the cell membrane. Extracellular budding of virions results in the acquisition of an envelope which contains the viral env proteins required for subsequent rounds of receptor recognition and fusion. Processing of the Gag and Gag-Pol polyproteins occurs during budding and release and is mediated by a viral protease. Reprinted from Coffin (1996) with permission.

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John W. Erickson and Michael A. Eissenstat

FIGURE 2 Genome organization and translational strategy for HIV. Structural (gag, pol, and env) genes are shaded; regulatory (tat and rev) and accesory (vif, nef, vpr, and vpu) genes are clear. Common to all retroviruses, the gag and pol gene products are translated on free ribosomes in the cytoplasm from newly synthesized unspliced viral RNA. Translation usually occurs through to a stop codon at the 3' end of the gag gene resulting in the structural polyprotein Pr55 gag.About 5% of the time ribosome frameshifting during translation of gag results in the synthesis of a Gag-Pol fusion protein, Pr160gag-p~ The frameshift site (fs) is located upstream of the Gag p6 protein such that a transframe polypeptide, TF, is incorporated into Gag-Pol in place of p6. The functions of the p6 and TF proteins are unclear. The total number of amino acids contained by each polyprotein is indicated at the end of each molecule. See text for individual protein abbreviations. Reprinted from Swanstrom and Wills (1997) with permission.

are also translated as part of a larger polyprotein precursor, Pr160g "g-p~ which results from ribosomal frameshift and readthrough during translation of the gag gene. The frameshift site has been mapped to lie between the N C and p6 coding sequences. The gag and gag-pol gene products in mature virions are found in a ratio of 20:1, which represents the frequency of ribosomal frameshifting, about 5%. Thus, frameshifting is used as a regulatory mechanism to ensure that large numbers of the structural proteins of the virion are synthesized relative to the viral enzymes, which are needed in only catalytic amounts. The N-termini of Pr55 and Pr160 both contain a covalently attached myristic acid moiety that is added cotranslationally and targets the polyproteins to the cellular membrane where virus assembly and budding takes place (Fig. 3). The Pr55 precursor protein is believed to play a central role in directing virion assembly and RNA packaging based on studies with other retroviruses that show that enveloped nucleoprotein core particles can form from Gag precursor proteins in the absence of pol and env gene products. Proteolytic processing of Pr55 g~gand Pr160 g~g-p~during virus assembly and maturation is performed by the viral PR, which is itself encoded by the pol gene (Swanstrom and Wills, 1977, and references therein). The env gene product, gp160, is processed into gp 120 and gp41 by a cellular protease. The processing products of HIV PR include the gag-encoded structural proteins and peptides-MA, CA, NC, pl, p2, and p 6 w a n d the pol enzymes--RT, IN, and PR. All of

7

HIV Protease

FIGURE 3 Budding and maturation of HIV-1 from an infected cell. Both immature (third panel from left) and mature (fourth panel from left) forms are shown. The mature virion exhibits the elongated capsid morphology typical of most lentiviruses. Inactivating mutations in the viral protease gene, or the presence of protease inhibitors, blocks the morphogenesis of immature to mature virion. Reprinted from Swanstrom and Wills (1997) with permission.

these products are found in mature infectious viral particles and result from cleavages at unique amino acid sequences that span the N- and C-termini of the mature proteins (Debouck, 1992) (Fig. 4). The sequences recognized by HIV PR are diverse, but certain general features emerge. Hydrophobic amino acids are preferred at the P1-PI' residues that flank the scissile peptide bond, aliphatic and Glu/Gln residues are often found at P2', aromatic residues are almost never found at P3', and small residues are preferred at P2. Several sequences contain an aromatic residue at P 1 followed by a Pro at P 1'. Although

PROCESSING SITES FOR HIV-1 PROTEASE Site

P4 P3

P2

P1

PI" P2"P3" P4"

MA/CA

-Ser-Gln-Asn-Tyr/Pro-Ile-Val-Gln-

CA/p2

-Ala-Arg-Val-Leu/Ala-Glu-Ala-Met-

p2/NC

-Ala-Thr-Ile-Met/Met-Gln-Arg-Gly-

NC/pl

-Arg-Gln-Ala-Asn/Phe-Leu-Gly-Lys-

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-Pro-Gly-Asn-Phe/Leu-Gln-Ser-Arg-

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-Ser-Phe-Asn-Phe/Pro-Gln-Ile-Thr-

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-Thr-Leu-Asn-Phe/Pr~176

RT/IN

-Arg-Lys-Val-Leu/Phe-Leu-Asp-Gly-

RT (internal)

-Alu-Glu-Thr-Phe/Tyr-Val-Asp-Gly-

FIGURE 4 Cleavage site sequences in Gag and Gag-Pol polyproteins recognized by HIV PR. Cleavage occurs between residues in the P1/PI' positions and they are indicated in bold and separated by a slash. The RT (internal) represents a PR-mediated cleavage at the junction of the p51/ RNase H domains which yields the active p66/p51 heterodimer found in isolated virus particles (Tomasselli et al., 1993). The nomenclature of Schechter and Berger (1967) is used to designate residue positions in the substrate sequence.

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John W. Erickson and Michael A. Eissenstat

all retroviral proteases appear to be structurally and functionally related, their cleavage site preferences vary widely. Efforts to predict cleavage sites for HIV PR have met with limited success (Poorman et al., 1991; Chou and Zhang, 1993) and our understanding of the basis of PR specificity is incomplete (Dunn et al., 1994; Katz and Skalka, 1994). However, identification of cleavage site sequences quickly led to the successful generation of a variety of synthetic substrates that facilitated the design of rapid and quantitative assays of PR activity (Hellen, 1994; Krafft and Wang, 1994). The HIV PR has long been known to be toxic to cells and this has prompted a search to identify cellular proteins that may be cleaved by HIV PR. Several investigators have shown that key proteins, such as NF-KB and certain cytoskeletal proteins, are cleaved in HIV-infected cells (Shoeman et al., 1993). The possible involvement of PR in the early stages of retroviral replication was suggested initially on the basis of observations with equine infectious anemia virus (Roberts and Oroszlan, 1989) and later with HIV (Baboonian et al., 1991; Nagy et al., 1994). However, recent data from several groups have demonstrated that PR inhibitors fail to block the synthesis of proviral DNA, its integration into cellular DNA, and transcription (Jacobsen et al., 1992; Uchida et al., 1997). Similar conclusions were reached using conditional lethal HIV-1 PR mutants as a probe (Kaplan et al., 1996). In 1988, the late I. Segal and co-workers observed that deletion mutagenesis of the HIV PR gene resulted in the production of virus particles that had an immature morphology and were noninfectious (Kohl et al., 1988). These results were confirmed by mutation of the active site aspartic acids and subsequently by chemical inhibition with PR inhibitors (Seelmeier et al., 1988; McQuade et al., 1990). This seminal experiment provided conclusive proof that HIV PR is essential for the life cycle of HIV and defined this enzyme as an important target for the design of specific antiviral agents for AIDS. A similar conclusion had been reached for the PR of murine leukemia virus in 1985 (Crawford and Goff, 1985; Katoh et al., 1985). However, the HIV PR studies provided the boost needed by many groups to launch drug discovery programs for this target.

B. STRUCTURE AND MECHANISM OF H I V PROTEASE Identification of the mechanistic family that a viral protease belongs to is the key to predicting its structure and function and may unlock strategies for inhibitor design that were previously developed for homologous members of the family. This concept was extremely valuable for the design of HIV PR inhibitors and led directly to the design of the first approved drug, Saquinavir, long before

HIV Protease

9

the structure of the enzyme was known. Homology modeling and biochemical inhibition studies on retroviral proteases had led to the hypothesis that these enzymes were related mechanistically to the aspartic proteinase family, typified by pepsin (Toh et al., 1985; Katoh et al., 1987). The active site of these bilobed enzymes contains two aspartic acids, one from each lobe, that participate in the catalysis of peptide bond breakage. Crystal structures of aspartic proteinases from cellular organisms revealed that the N- and C-domains associate to form an active site with approximate twofold symmetry at the protein backbone level (Davies, 1990). These observations led to the suggestion that the cellular enzymes had evolved via a duplication event of a primordial aspartic protease gene (Tang et al., 1978). Since the sequence length of retroviral proteases is about one-third that of typical aspartic proteases, it was proposed that the former enzymes are composed of two identical subunits, each of which contributes a single aspartic acid to the active site (Pearl and Taylor, 1987). This hypothesis was verified by the crystal structure determination of Rous sarcoma virus (RSV) protease by Wlodawer and colleagues (Miller et al., 1989) and subsequently of HIV PR by several laboratories (Lapatto et al., 1989; Navia et al., 1989; Wlodawer et al., 1989; Spinelli et al., 1991). So far, retroviruses are the only family of viruses whose proteases have adopted an aspartic proteinase mechanism. The HIV PR dimer consists of two identical, noncovalently associated subunits of 99 amino acid residues associated in a twofold (C2) symmetric fashion (Wlodawer and Erickson, 1993) (Fig. 5a). The dimer is stabilized by a fourstranded antiparallel/3-sheet formed by the interlocking N- and C-termini of each subunit. The active site of the enzyme is actually formed at the dimer interface and contains two conserved catalytic aspartic acid residues, one from each monomer. The substrate binding cleft is composed of equivalent residues from each subunit and is bound on one side by the active site aspartic acids, Asp25 and Asp125, and on the other by a pair of twofold related, antiparallel /3-hairpin structures, or "flaps." Comparison of the structure of HIV PR with that of a complex with a peptide-based inhibitor (Miller et al., 1989) shows that the flap undergoes significant structural changes upon binding and that it makes several direct interactions with inhibitor (Fig. 5b). Molecular dynamics studies indicate that the flaps are highly flexible and must undergo large localized conformational changes during the binding and release of inhibitors and substrates (Collins et al., 1995). Crystal packing forces apparently maintain the flap in a conformation that is unsuitable for substrate binding in the structure of the uncomplexed form of the enzyme (York et al., 1993). The crystal structures of RSV and HIV PR revealed that, despite the apparent lack of sequence homology, aspartic proteinases and retroviral proteases display considerable structural homology at the backbone level (Rao et al., 1991). Fully one-third of the main chain atoms of RSV PR can be superposed onto the backbone of

10

John W. Erickson and Michael A. Eissenstat

FIGURE 5 Chain tracings of HIV PR from crystal structures of (A) unbound and (B) inhibited forms of the enzyme. Inhibitor is drawn as stick figure.

porcine pepsin to within 1.5 A root-mean-square deviation. As expected, most of the structural c o r r e s p o n d e n c e is in the active site region. However, the overall chain topologies of the two families of enzymes are more similar than a simple superposition analysis reveals and are indicative of a distant but definite relationship to a c o m m o n , ancestral aspartic proteinase gene.

HIV Protease

11

C. STRUCTURE OF H I V PROTEASE INHIBITOR COMPLEXES To date, several hundred crystal structures have been solved for various HIV protease/inhibitor complexes~a testimony to the importance placed on structural information in the process of inhibitor design (Fitzgerald and Springer, 1991; Huff, 1991; Tomasselli et al., 1991; Meek, 1992; Abdel-Meguid, 1993; Appelt, 1993; Erickson, 1993; Wlodawer and Erickson, 1993). Structural comparison of the inhibitor complexes reveals certain common features (Fig. 6). The inhibitor and enzyme make a pattern of complementary hydrogen bonds between their backbone atoms. In some instances, these hydrogen bonds are mediated by bridging water molecules. A unique feature found in the structure of HIV PR/inhibitor complexes is the presence of a water molecule that forms bridging hydrogen bonds between the NH atoms of Ile50 and Ile150 in the two flaps and the P2 and P 1' backbone carbonyl groups of the inhibitor. This water is close to the twofold axis of the enzyme and is distinct from the water molecule that has been identified in the active site of uncomplexed structures of aspartic proteinases, including HIV PR. The latter has been implicated in substrate catalysis. The enzyme also contains a number of well-defined pockets, or subsites, in its active site region into which inhibitor side-chains protrude, resulting in tight binding interactions between enzyme and inhibitor. Since a similar pattern of hydrogen bonds is believed to be made for both substrates and peptidomimetic inhibitors, specificity is believed to reside in the pattern of largely nonpolar subsite interactions between inhibitor and enzyme side-chain atoms. Overall, knowledge of the structure and function of HIV PR and its relationship to other aspartic proteinases has led to the successful development of a wide variety of potent and chemically diverse inhibitors as is discussed in the next section.

IV. D E S I G N O F H I V P R O T E A S E I N H I B I T O R S The design of clinically effective HIV PR inhibitors has been a major success story for structure-based design (for reviews on clinically effective protease inhibitors see Vacca and Condra, 1997; Flexner, 1998). Several HIV PR inhibitors are currently in widespread use for the treatment of patients with AIDS (Mous et al., 1994; Ho et al., 1995; Wei et al., 1995; Markowitz et al., 1998). These compounds represent a new class of therapeutic agents that complement already-licensed antivirals--AZT, ddI, ddC, and d4T--all of which inhibit HIV RT. Although the initial lead compounds have been generated in various ways, the availability of protein crystal structures of these leads with

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L///

,,,,"

k" "- ~

L//,

--

o.'- :

L///

..,,,,,,llmlllllll e

0----o

',.~~IIIIIIII

\... o

9 ,,,,,IIillllll

.

~ 1 7..... 6 =~ z:z:

C,):

1 \

=

0

....

.....

.....

"/..

:=Z

0 : 0

---

0 ~ 0

---'rZ

~

\z= /

\

:C..Z

.... 0 ~ 0

. . . . . . . .

Z=

---~ 0

~"

e~ 0 : 0

. . . .

~, z:z: . . . .

S / ~ . , ~

//'~/I

,~, ~ I

\z=

~

~~0

~

_>=

I.T-I ~

HIV Protease

13

HIV PR facilitated structure-based approaches to the optimization of interactions to increase potency. Once potent inhibitors of the enzyme were generated, considerations such as improving antiviral potency, improving bioavailability (Kempf, 1994), and reducing cost could be addressed within the known structural limitations provided by these crystal structures. We make no attempt to describe in detail the enormous amount of medicinal chemistry that has been done over the past decade that has led to the development of these successful drugs. Rather, we describe the various approaches that have been used to generate potent HIV PR inhibitors with an emphasis on recent developments and in particular how they might address the two major challenges remaining for drugs of this class: pharmacokinetics and effectiveness against resistant mutants. The inhibitors can be classified on the basis of the rationale used for their design as substrate-based, structure-based, nonpeptidic, and irreversible inhibitors. These distinctions are somewhat arbitrary since many inhibitors can fall into more than one category. We apologize in advance for placing compounds into a class that others may not agree with. One indication of the enormous activity in this field is the large number of cores or templates that have been used for elaboration of HIV protease inhibitors. Table I lists the various cores along with representative examples of potent inhibitors. Cores are arranged approximately by size according to the portion delimited by a heteroatom (usually nitrogen) or carbonyl connected by atoms to another heteroatom or carbonyl, allowing for a shorthand nomenclature for grouping purposes. When looking at the compounds in this way one sees that there are close similarities between the structures of the series pursued by the various groups. As will become apparent (see below), the clinically successful compounds often resulted from incorporating pieces discovered by various groups onto a proprietary core or introducing a proprietary group onto an established core. In the discussions below structural references to a specific core in Table I are made whenever possible.

A. SUBSTRATE-BASEDINHIBITORS The close structural and functional relationships between the retroviral and cellular aspartic proteases, together with knowledge of the HIV PR cleavage site sequences on the Gag and Gag-Pol polyproteins, immediately opened the avenue of peptidomimetic substrate-based approaches that had been developed for designing inhibitors of human renin, an aspartic protease that was a popular target for the design of antihypertensive agents in the 1980s. Screening compounds generated in these programs rapidly identified reasonably potent inhibitors of HIV PR. Many of the cores shown in Table I had been previously generated as substrate mimetics for renin programs. Substrate peptidomimetic

14

John W. Erickson and Michael A. Eissenstat

TABLE I HIV Inhibitor Cores Core

Structure

Source

NCCN NH(CH2)2NH

Glaxo (Humber et al., 1993)

PhCH2CONHH PhCH2NHCO~HN ~~Or S~ N~

H NHCOCH2Ph

HN.~ NH O NCCC--O NHCH(R)CH(OH)CO

O",11.N

O - H MeS KNI-272

NHCH(R)COCO

H2N,tO

N~S O~

~./

Japan Energy (Mimoto et al., 1992; Kageyama et al., 1993; E1-Farrash et al., 1994) Syntex (Tam et al., 1992) Takeda (Kitazaki et al., 1994)

~ N H Takeda (Kitazaki et al., 1994) Scripps (Slee et al., 1995)

O NHtBu NCCCN NHCH(R) CH(OH) CH(R')NH

oWH OHH~O

Abbott (Erickson et al., 1990; Kempf et al., 1990)

A-74704 (continues)

15

HIV Protease TABLE I

(continued)

Core

Structure

Source

NHCH(R) CH(OH) CH2N(R)CO

Monsanto-Searle (Getman et al., 1993)

~ H oN.~ 0 ~I ONH2

~ ON41

SC-52151 NHCH(R) CH(OH) CHzN(R)SO2

~O

Monsanto-Searle (Getman et al., 1993) Vertex (Kim et al., 1995)

O

N"SO2-p-NH2Ph

OH~ VX-478 Amprenavir NHCH(R) CH(OH) CH2NR~

,~.N~H

'~~~-~~'~ ==_ . N O

o-

N'~ H [~.~

,[

O' NH20HH

Roche (Roberts et al., 1990; Thomas et al., 1994) Boehringer,Biomega

..,..199~.

Beaulieu et al., 1997) Agouron, Lilly (Kalish et al., 1995; Kaldor et al., 1997)

Ro 31-8959 Saquinavir

sO O~.-4NH

0 ~N:

AG 1343 Nelfinavir (continues)

16 TABLE I

John W. Erickson and Michael A. Eissenstat (continued)

Core

Structure

Source

"4-

0.,.. NH

OH

'0

%0

BILA 2011 Palinavir NH CH (R) CH (OH) CH2NR' NH

Mer

..N"~rN..,,'~ N" \ 0 NHCH(R) CH(OH)CH2NR'R' 'NH

Abbott (Sham et al., 1993, 1995) Ciba-Geigy (Fassler et al., 1993) Narhex (Grobelny et al., 1997)

UCSF (Rutenber et al., 1996)

O OH IX

Me CH2Ph AQ-148

NCPCN NH CH (R) PO (OH) CH (R') NH

HO

OHNN~H l H .VO OJ'l' ~ "Nv- E~~"X N11'O~ H

SKB (Abdel-Meguid et al., 1993a)

SB-204,144 (continues)

17

HIV Protease TABLE I

(continued)

Core

Structure

Source

NCCCC~---O NHCH(R)CH(OH)CH2CO

Glaxo (Holmes et al., 1993)

PhCH23NH PhCH2NH S N H ~ NHCH2Ph O

OH U

NHCH(R) CH(OH) CH(NHR') CO

Sandoz (Billich et al., 1994; Scholz et al., 1994)

CbzNHLH - ~ ~ N~ NHCbz = HN "

n

0

NHCH(R)COCF2CO

MMD

(Taylor et al., 1997)

o- b

O~F. F H .~

H ~ ~

O

MDL-74695 NCCCS NHCH(R)CH(OH)CH2S

Lilly (Cho et al., 1994)

O@NH O NH2 (continues)

18

John W. Erickson and Michael A. Eissenstat

TABLE I

(continued)

Core

Structure

Source

OCCCO OCH(R)CH(OH)CH(R')O

0

~

0

IL.

x~

Lederle (Babine et al., 1992)

OH

v

0

NCCCCN

NHCH(R)CH(OH)CH(OH)CH(R')NH

Me H O ~

H.0

H ~ --

0

N o ,&"

6HI,

o

"

0

N

N

Abbott (Kempf et al., 1990, 1993, 1995a) SKB (Dreyer et al., 1993) Hoechst, Bayer (Budt et al., 1995; Lange-Savage et al., 1997) NCI (Hosur et al., 1994; Randad et al., 1996)

A-77003 NH CH (R) CH (O H) CH2CH (R') NH

Abbott (Kempf et al., 1993)

H

~,~O O

A-80987

NwN 0

NwO 0

A-84538 (ABT-538) Ritonavir (continues)

19

HIV Protease

TABLE I

(continued)

Core

Structure

Source Abbott (Kempf et al., 1991)

NHCH(R) CH(OH) CF2CH(R')NH

H O~ F F , , V O 0 fN H ( ~ H b O

H Abbott (Kempf et al., 1991)

NHCH(R)COCF2CH(R')NH

~ M e H O~

FF H V . O N 1.fN_~ _N_1 , ~ N~,~ N J.kN

oz,.o

N

A-79295 Lilly (Munroe and Hornback, 1993) LG Chemical (Park et al., 1995)

NHCH(R)CH(O)CHCH2NH

O,rfNH H 0

%'0

H HN,rfO 0

O ..(~ 0

H HN,I1,0 0 (continues)

20 TABLE I

John W. Erickson and Michael A. Eissenstat (continued)

Core

Structure

NCPCCN NHCH(R)P(O) (OH) CHOHCH(R')NH

~

Source

Hoechst (Stowasser et al., 1992)

O0 H 0 0 HN.~N~ IpIJ,~H ~ N

6. )o -

NCCCCC--O NHCH(R) CH(OH) CH2CH(R')CO

HO ~ OH BocHN. ~ ~ N,,

Merck (Vacca et al., 1991) SKB (Dreyer et al., 1992) Upjohn (Mulichak et al., 1993)

o

Merck (Dorsey et al., 1994a,b)

NR2CH2CH(OH)CH2CH(R')CO

~N'~

OH~H OH NHtBu

MK-639 (L-735,524) Indinavir LG Chemical (Lee et al., 1996; Yoon et al., 1997)

NHCH(R)CH(O)CHCH2CO

,,,/.o~

H 0 H 0 I"v N,.,,.~ N J,~ ~,~ N~,,~ _ H

o~,

b"

S02Me

o /.,, ~

LB-71350 (continues)

21

HIV Protease TABLE I

(continued) Core

Structure

Source

NCCNCC---O NHCH(R)CH2NHCH(R')CO

BocHN

N H

U

Czech Academy of Science (Urban et al., 1992)

qO O ~

H

O

NCCCCCN NHCH(R) CH(OH) CH2CH2CH(R')NH

U. Montreal (Hanessian and Devasthale, 1996)

O o.~~ o..~or .o

~~-o~

Q

O--CCCCCCC~-----O COCH(R) CH2CH(OH) CH2CH(R') CO

OH H'~-N

Merck (Bone eta/., 1991) kederle (Babine eta[., 1992)

OH

O

H

OH

O

L-700417 NCCCNCCCN NHCH(R) CH(OH)CH2NHCH2CH(OH) CH(R)NH

OH BocNH ~ -

U

BMS (Patick et al., 1995)

OH N

-

NHB oc

/~O

"~oy ~-~ BMS-186318

(continues)

22 TABLE I

John W. Erickson and Michael A. Eissenstat (continued)

Core

Structure

Source

Nonpeptide Agouron (Kalish et al., 1995) Roche (Thomas et al., 1994)

NHCH(R)CH(OH)CH2R'

S O

~ OOH NH

AG-1254 SKB (Thompson et al., 1994)

NHCH(R) CH(OH) CH2CH(R')Ar

H

o

O

N...*.N ..%.....~. ,j

,

SB-206343

LG Chemical

NH CH (R) CH (O) CH2CH2SO2NR'

(Choy et al., 1997)

ON~N ~,~ H ~" o

~N' ..<.

SO2Me (continues)

23

HIV Protease TABLE I

(continued)

Core

Structure

Source Agouron, Lilly (Kaldor et al., 1994; Reich and Pino, 1994; Kalish et al., 1995; Reich et al., 1995; Melnick et al., 1996)

ArCHzCH(OH)CH2CH2Ar

N.O

if-OH

HOrN

O

% AG-1284 Imidazolone

Dupont-Merck (De Lucca, 1997)

HO G.

O

N

~t

OH N

Pyrimidin-2-one

Dupont-Merck (De Lucca et al., 1997a)

H2NOC

CONH2

~N~N"~ 1,3-Diazepan-2-one

Dupont-Merck (Lam et al., 1994, 1996; Nugiel et al., 1996; Rodgers et al., 1996; Wilkerson et al., 1996; Jadhav et al., 1997; Smallheer et al., 1997; Wilkerson et al., 1997; Han et al., 1998; Patel et al., 1998) Uppsala (Hulten et al., 1997)

HO~N~N~O" DMP-323 (XM-323)

(continues)

24 TABLE I

John W. Erickson and Michael A. Eissenstat

(continued) Core

Structure

H2N~Ho

Source

~'OH~NH2 ,

DMP-450 (XM-412)

H HNO O NII.N OH XV-652

HO OH 1,3-Diazepan- 2-imine

NCN

/=x

Dupont-Merck (Jadhav et al., 1998)

Ho_~N"~~4_OH ~ ~ 0 ~ IIII on)~

1,4-Diazepan-2,3-dione

Dupont-Merck (Jadhav and Man, 1996)

MeO2C~~. ~_~ ~CO2 Me N

N

(continues)

25

HIV Protease TABLE I

(continued)

Core

Structure

Source Abbott (Sham et al., 1996)

1,2,4-Triazepan-3-one

MeO

OMe

HO--NNOH A-98881 Uppsala (Backbro et al., 1997)

1,3-Diazepan-2-sulfone

N-S.N

Bayer (Wild et al., 1993)

Dibenzcycloheptanone

O BnO2CNHCH2~ ~ HO

CH2NHCO2Bn OH Gilead (Kim et al., 1996)

Thiepane-dioxide 02

x . ~

HO

OH ~ k ~

2-Pyrone

U-96988

Upjohn (Romines and Thaisrivongs, 1995; Thaisrivongs et al., 1995, 1996) W-LPD (Lunney et al., 1994; Prasad et al., 1994, 1995a,b; Tummino et al., 1994) (continues)

26

John W. Erickson and Michael A. Eissenstat

TABLE I (continued) Core

Structure

Source

OH

HO2CCH20 ~

S

(

PD-153103 Fused pyrones

OH

MeO

Upjohn (Thaisrivongs et al., 1994, 1995; Rominesand Thaisrivongs, 1995; Romines et al., 1995a,b, 1996; Skulnicket al., 1995, 1997) NHCO(CH2)2NHCO2tBu WLPD (Lunney et al., 1994; Tummino et al., 1994) N'S CN

U-103017 Dihydropyrones OH

f

f HN "S 02 PNU-140690

~

Upjohn (Thaisrivongs et al., 1996a,b;Janakiraman et al., 1998) WLPD CF3 (Taitet al'1996'1997; Tummino et al., 1996; Hagen et al., 1997)

HO OH

OH

(continues)

27

HIV Protease TABLE I

(continued) Core

Structure

Source

Fullerene

UCSF (Friedman et al., 1998)

,~,,

OH

inhibitors are essentially peptide substrate analogs in which the scissile peptide bond has been replaced by a noncleavable, transition-state analog or isostere. Many transition state-based inhibitors had been investigated that would mimic the hydrated amide presumed to represent the intermediate in amide hydrolysis catalyzed by aspartic acids (Szelke, 1985). A hydroxy group contained in nearly all HIV protease inhibitors is believed to mimic the transition state diol structure and to interact with the catalytic aspartates, residues 25 and 125. While much of the early work addressed peptide analogs with a core transition state mimetic (Dreyer et al., 1989; Norbeck, 1990; Norbeck and Kempf, 1991), it was not long before truncated analogs that removed much of the peptide character of the molecules were discovered. While much of the early investigation involved identifying suitable cores, the subsequent refinement of these structures to reduce molecular weight and peptidic character while maintaining potency became the focus of intensive efforts by many research groups. An early success story from these efforts was that of Saquinavir, which was reported by the Hoffman-La Roche group in 1990 (Roberts et al., 1990) and was eventually approved for clinical treatment of AIDS in 1996. This hydroxyethylamine-based compound contains the NCCCN core, which has provided a rich source of potent HIV protease inhibitors (Table I). A unique aspect of the structure of Saquinavir was the introduction of the decahydroisoquinoline moiety in the P I' region replacing the proline found in substrates. This substituent allows for maintenance of excellent enzyme inhibitory potency in the absence of a lipophilic core substituent normally required to make interactions with the $1' hydrophobic pocket. The oral bioavailability of this compound is low, although it did reach a mean plasma concentration above the in vitro antiviral ECgo. The poor bioavailability may be due to a combination of it being poorly absorbed and readily metabolized. These properties together with high plasma protein binding (98%) limited the

28

John W. Erickson and Michael A. Eissenstat

effectiveness of Saquinavir as a first-line therapy and provided incentive for the design of second-generation bioavailable drugs. Merck reported analogs of Saquinavir in which the decahydroisoquinoline has been replaced by a substituted piperazine and a tetrahydrothiophene derivative placed at P2 to provide analogs that are potent but more bioavailable (Kim et al., 1994, 1995a,b). Hydroxyethylene analogs that contain the N C C C C C ~ O core were developed by Merck (Lyle et al., 1991). These compounds retain a hydroxyethylamine subunit but on the P side rather than the P' side. The amine was introduced to remove peptide character as well as to provide a basic group for improved solubility. Piperazine analogs eventually proved to be optimal with the additional nitrogen providing a site for introducing a substituent to interact with the $3 subsite as well as providing a weak base for better solubility (Vacca et al., 1994; Holloway et al., 1995). At the C-terminus an aminohydroxy indane was introduced as a phenylglycine surrogate (Lyle et al., 1991). The indane group fits into the lipophilic P2 pocket and the hydroxy end formed a hydrogen bond with the NH group of Asp 29. This compound had much better oral bioavailability than Saquinavir, retained comparable potency, Ki = 0.5 nM, and eventually was approved for clinical use as Indinavir. As indicated in Table I there are many other cores that were developed into potent HIV PR inhibitors. Among the more noteworthy is the hydroxymethyl carbonyl analog (NCCC--O core) first described by Kiso and co-workers (Mimoto et al., 1992) and later developed by Japan Energy Corporation (Kageyama et al., 1993). This shortened core retained considerable potency. The main additional structural variation was the introduction of novel proline mimetics in the P' region. A variation on the hydroxyethylamine (NCCCN) core is the addition of a second nitrogen to provide a hydroxyethyl hydrazine, which also yielded potent analogs (Fassler et al., 1993; Sham et al., 1995). Another NCCCN core that became important is the hydroxyethyl urea core originally explored by Monsanto-Searle workers. This core comes from the same family as Saquinavir except that instead of being part of a basic amine the P' nitrogen is incorporated into a urea or sulfonamide moiety (Getman et al., 1993; Bryant et al., 1995; Vazquez et al., 1995). SC-52151 was taken to the clinic but failed to demonstrate antiviral activity, probably because its high protein binding decreased cellular uptake (Fischl et al., 1997). This core was eventually optimized by Vertex workers using structure-based approaches leading to a new clinical candidate, VX-478 (Amprenavir), vide infra. Much of the early work on the peptide-based inhibitors led to the realization that, unlike the case with other proteolytic enzymes such as renin, HIV PR readily tolerates dramatic departures from typical substrate side-chains in inhibitors. However, the usefulness of peptidomimetics as drug candidates has been hampered by their generally poor pharmacological properties such as oral bioavailability, metabolic stability, and pharmacokinetics.

HIV Protease

29

B. STRUCTURE-BASED INHIBITORS The crystal structure of HIV PR immediately provided a structural basis for the development of a new generation of inhibitors that did not need to rely on substrate or peptide mimicry. Virtually all of the different approaches to improving potency have incorporated some aspect of structure into their design efforts, and numerous protein crystal structures of the enzyme with inhibitors have been generated (Wlodawer and Erickson, 1993; Gait and Karn, 1995). Structure-based design has been used to identify templates for elaboration as well as to optimize the interactions found in the original peptide-based substrates and to replace peptide moieties by nonpeptide groups. Combined with medicinal chemistry and, in some cases, target-based screening efforts, these structural investigations have created a structurally diverse compendium of inhibitors that includes those such as AG-1343 (Nelfinavir), which was almost a purely structure-based design, to MK-639 (Indinavir) and VX-478 (Amprenavir), the designs of which were a blend of medicinal chemistry and structural insights. An early contribution to the structure-based design of HIV protease inhibitors was the symmetry-based inhibitors originally described by the Abbott group. They postulated that HIV protease, being a symmetrical dimer, might be inhibited by symmetrical inhibitors that were complementary to the twofold symmetry of the enzyme (Erickson et al., 1990; Kempf et al., 1990, 1991, 1993). This approach led to potent diamino diol inhibitors with the core NCCCCN. Examination of the hydroxy substituents on these compounds demonstrated that these symmetrical compounds were making unsymmetrical interactions with the enzyme, with one of the core hydroxy groups forming more hydrogen bonds to the catalytic aspartates than the other (Hosur et al., 1994). This allowed the rational removal of one of these hydroxyls with no loss in potency. As in many of the peptide based inhibitors that have been described, bioavailability was a significant problem with the Abbott compounds. The first clinical candidate, A-77003, had poor oral bioavailability. The second clinical candidate, A-80987, designed to have better solubility, was well absorbed, but had a short plasma half-life as well as high plasma protein binding. Significant exploration of substituents was done to identify a potent, bioavailable analog. One focus was to replace the readily metabolized pyridine groups. Eventually ABT-538 was identified, where the bioavailability was high despite its relatively high molecular weight (721 Da) (Graul and Castaner, 1996). Plasma protein binding remained high (99%). Further investigation of this compound revealed that its improved bioavailability was at least in part a consequence of inhibition of cytochrome P450 enzymes. While inhibiting metabolizing enzymes is generally avoided in drug candidates, in this case it allowed the compound to

30

John W. Erickson and Michael A. Eissenstat

achieve sufficient blood levels to be an effective drug clinically and it was approved as Ritonavir. An interesting sidelight of the P450 inhibitory effects of this compound is that it is being examined in combination with less bioavailable drugs such as Saquinavir and has been demonstrated to improve their bioavailability also (Kempf et al., 1997). Since both of these compounds are also substrates of the P-glycoprotein efflux system, that may also contribute to the improved bioavailability when used together (Alsenz et al., 1998; Lee et al., 1998). A problem with inhibition of P450 enzymes to improve bioavailability is that it can lead to multiple drug interactions and untoward side-effects. Later efforts in structure-based design have utilized some of the previously identified cores and used key interactions identified in inhibitor-enzyme crystal structures to design nonpeptidyl substituents that would make key energetically favorable interactions with the enzyme. One such effort was by the Vertex group, who took the hydroxyethyl urea core (NCCCN class) described previously by Monsanto-Searle workers and incorporated a sulfonamide moiety into the P' region (also reported by the Monsanto-Searle workers) and also used the 3-tetrahydrofuryloxycarbonyl substituent originally identified as a useful P2 substituent by Merck workers to generate VX-478 (Kim et al., 1995). This compound is being developed clinically as Amprenavir in a joint effort with GlaxoWellcome. It is an attractive compound in that the reduced molecular weight translates into good bioavailability while retaining significant potency (St. Clair et al., 1996). Plasma protein binding does not appear to be a major problem (Livingston et al., 1995). The Agouron group has been involved both in structure-based design to reduce the peptide nature of substrate-based analogs and in de novo design. Some of this work has been done in collaboration with Eli Lilly workers. In one approach they utilized the Lilly discovery that the P' amine of Saquinavir could be replaced by a benzamide subunit while retaining significant potency. They then set out to remove the peptidic character of the P region by using an orthobenzamide to make a hydrogen bond to the flap water. Although this inhibitor was very weak, addition of other substituents provided single-digit nanomolar enzyme inhibitory potency (AG 1284) without any amide substiments in the core and any amino acid-derived substituents in the molecule. Remarkably, this potency was achieved with only four hydrogen bonds with either protein sidechains or ordered waters (Reich et al., 1995). Several groups virtually simultaneously reported their structure-based design of benzamide P2 substituents (Kaldor et al., 1995; Kalish et al., 1995; Kempf et al., 1995, Randad et al., 1995a, 1996). The Lilly group had previously demonstrated the utility of t-butyl benzamides and the Roche group t-butyl cyclohexylcarboxamides as PI'P2' substituents as part of a nonpeptidyl hydroxyethylaryl (alkyl) core (Kaldor et al., 1994; Thomas et a|., 1994).

HIV Protease

31

The Agouron group incorporated this substituent into the P' aromatic analogs mentioned above to generate low-molecular-weight, potent inhibitors (AG 1254). However, their poor aqueous solubility and weak antiviral activity precluded their development. Introduction of these P2 substituents back into the original Saquinavir hydroxyethyl amine analog along with the additional replacement of the P1 benzyl by a phenylthiomethyl (Kaldor et al., 1995) to increase hydrophobic interactions with $1 and $3 provided Nelfinavir (NCCCN core), a potent analog with good bioavailability (Shetty et al., 1996; Rabasseda et al., 1997), which was recently approved for therapeutic use (Kaldor et al., 1997). Abbott and NCI workers independently studied the benzamide P2 substituent on the symmetrical core described above. The initial NCI paper (Randad et al., 1995a) describes the introduction of benzamide substituents at both P2 and P2' positions and the process by which the HIV protease structure was used to design the 3-hydroxy substituent. A crystal structure of this compound bound to the enzyme led to the introduction of the 2-methyl substituent to stabilize a conformation of the aromatic ring out of plane with the amide carbonyl and resulted in the same optimum substituent identified by the Agouron workers (Kalish et al., 1995). Abbott workers described similar compounds where the P and P' substituents were individually varied using both rational design and combinatorial approaches to arrive at a similar optimum substituent pattern (Kempf et al., 1995a). These substituted benzamides were also shown to be effective substituents on the Searle-Vertex core (Freskos et al., 1996). In subsequent work, the N CI group modified the aromatic substituents to incorporate an anthranilamide moiety which allows for interactions at $3 and $3' and a significant boost in potency (Randad et al., 1995b). Working initially from the hydroxyethyl amine template, Ghosh et al. designed a bis-THF P2 substituent to replace the P3/P2 spanning quinolinecarbonyl-asparagine moieties found in the earlier compound (Ghosh et al., 1994). The resulting compound was 8 • less potent in the enzyme assay but only 2• less active in the antiviral assay. An X-ray structure showed that the bis-THF moiety forms hydrogen bonds with both the Asp29 NH and the Asp30 NH backbone amides. This analog has the advantages of improved aqueous solubility, decreased logP, and decreased MW (Ghosh et al., 1994, 1996). More recently Ghosh has reported that this P2 substituent is highly effective when placed in the Monsanto-Vertex core with a Ki of 1.1 nM and an antiviral EC5o of 1.4 nM, which is an order of magnitude more effective than either the Vertex or Roche compounds (Ghosh et al., 1998). Profiling this compound against resistant mutants is in progress. The Biomega group discovered that a 2,6-dimethylphenoxyacetyl P2 substituent provided potent analogs (Tong et al., 1995). The DuPont-Merck group took a fundamentally different approach to design-

32

John W. Erickson and Michael A. Eissenstat

ing an HIV protease inhibitor. They reasoned that if one could replace the flap water by an appropriate group while retaining the interactions with the catalytic Asps, one could design potent nonpeptidic HIV PR inhibitors. The initial hits from screening of their database for these interactions were hydroquinones and cyclohexanediols with micromolar potency (Lam et al., 1994). They and others also took the approach of cyclizing the Abbott inhibitor core, with appropriate stereochemical modifications, to provide a cyclic urea. The six-membered ring pyrimidinone analogs were weakly active (Randad et al., 1994), but the sevenmembered ring analogs had subnanomolar potency, MW < 600, antiviral potency, and bioavailability in rats and dogs (Lam et al., 1994). The first of these compounds to enter clinical study was DMP-323, a bis-hydroxymethylbenzyl analog (Grubb et al., 1994). However this compound was withdrawn because of low and variable drug levels. Many crystal structures of these compounds complexed with HIV protease have been generated (Ala et al., 1998). The seven-membered ring is roughly perpendicular to the catalytic aspartates and the diol unit interacts with these residues. The carbonyl oxygen replaces the commonly found flap water molecule and accepts H-bonds from Ile50 and Ile50 r. The unsubstituted benzyl groups bind to S1 and $1 r as in the acyclics and the N-hydroxymethylbenzyl groups bind to $2 and $2 r. The four contiguous asymmetric centers are in the RSSR orientation, which contrasts with the stereochemistry seen in the acyclic analogs. Further research on these cyclic urea analogs has yielded many potent variations by both this group and others (Sham et al., 1996). The second-generation clinical candidate DMP-450 improved solubility and bioavailability by replacing the 4-hydroxymethyl substituents by 3-amino substituents. However the high (90%) plasma protein binding of this compound reduced its effective blood plasma concentration to a level where it might not be effective against mutant variants (Hodge et al., 1996). Since there is a high tolerance for variation at P2 and P2 ~, which are also synthetically more accessible, much effort has focused on these substituents (Lam et al., 1996). Nonsymmetric analogs tended to have better solubilities (Wilkerson et al., 1997). Heterocycles have been incorporated which form additional H-bonds to the Asp 30 carbonyl and NH (XV-652) (Rodgers et al., 1996; Han et al., 1998). Smaller ring-size ureas have been revisited and potency can be improved by replacing one of the P 1 benzyl groups by a P 1 phenethyl group, which allows both aromatic P1 substituents to occupy the preferred pseudoaxial positions (De Lucca et al., 1997a). A review of some of this work and that of others on cyclic inhibitors has appeared recently (De Lucca et al., 1997b). Interestingly, the interaction of the ring hydroxyls with the catalytic aspartates is less critical in the cyclic analogs since nonhydroxylated 5-, 6-, or 7-membered ring cyclic ureas retain inhibitory activity in the 100-nM range (De Lucca, 1997). The carbonyl of the urea has been replaced to generate a cyanoguanidine which also replaced the structural water molecule (Jadhav

HIV Protease

33

et al., 1998). Recent emphasis in their work has been on identifying analogs

that retain good bioavailability while remaining effective against drug-resistant mutants (Jadhav et al., 1997). The Gilead and Uppsala groups extended this work into the realm of carbohydrate-derived cyclic analogs (Kim et al., 1996; Backbro et al., 1997; Hulten et al., 1997). The Gilead group set out to design cyclic analogs that were devoid of peptide character but retained the favorable solubility and low molecular weight of some of the DuPont-Merck analogs. In the resulting thiepane dioxides a sulfonyl group is used in place of a carbonyl to replace the structural water molecule and the diol interactions with the catalytic aspartates are retained. The nitrogens of the DuPont-Merck compounds are replaced by carbons to which are attached the P2 benzyls. Benzyloxy or thiazol-4-methoxy substituents are used to generate the hydrophobic interactions with $1/$1'. The compounds were potent in the enzyme and antiviral assays and a meta-amino analog showed improved solubility and high bioavailability. The Uppsala group generated structures that were intermediate between the DuPont-Merck and Gilead compounds. D-Mannitol provided the required stereochemistry at all of the asymmetric centers. A key to using this starting material efficiently was the use of phenoxymethyl as the P1/PI' substituent. The nitrogens required for urea formation were brought in by double azide displacement of two of the hydroxy groups. Not surprisingly, the RSSR stereochemistry was required. Sulfamide (1,3-diazepan-2-sulfone) analogs were also potent. Surprisingly the crystal structure of a sulfamide showed that the presumed P2' benzyl group actually binds in the $1' pocket and the (P1 ') phenoxymethyl group binds in the $2' pocket. This was proposed to be due to the difference in ring geometry between the cyclic urea and the sulfamide.

C. NONPEPTIDE INHIBITORS FROM SCREENING Two groups have been pursuing a different cyclic nonpeptide template for the development of HIV protease inhibitors. Reviews of these efforts have appeared (Lee et al., 1996). Both Parke-Davis and Upjohn identified 4-hydroxypyrone derivatives as weak HIV protease inhibitors during mass screening of their chemical inventories. The Parke-Davis group identified coumarin and pyrone derivatives with Ki's in the micromolar range (Tummino et al., 1994). The low molecular weights, nonpeptide nature, and high solubility of these compounds encouraged extensive SAR studies. The crystal structure of the initial 4-hydroxy coumarin lead showed that the hydroxy group was H-bonded to the catalytic Asps (Lunney et al., 1994). The rest of the molecule existed in two different binding modes, both of which had the lactone oxygens interacting with the Ile50 NH group in the flap. Because the coumarin system did not allow

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John W. Erickson and Michael A. Eissenstat

efficient occupancy of the $2 subsite, work focused on the pyrone analogs, which showed similar binding interactions with the catalytic aspartates and the flap (Prasad et al., 1994). A highly potent (Ki = 3 nM) analog which only spanned the $1-$2' subsites was generated by introducing a 2-o-t-butylphenylthio substituent to occupy $1' and $2' and a 6-phenyl substituent to occupy $1 (Prasad et al., 1995a,b). Dihydropyrone analogs were generated since they gave additional flexibility for substitution that would provide favorable interactions (Hamilton et al., 1996; Tait et al., 1996, 1997). However, while this variation allowed for improvement in enzyme inhibitory potency to the single-digit nanomolar range, it did not translate into potent antiviral activity. It was found that polar substituents had to be introduced into the 3-arylthio group and the 6-phenylethyl group before submicromolar antiviral activity could be observed (Hagen et al., 1997). As anticipated from their structures, these compounds showed very high oral bioavailabilities. The Upjohn series has been developed along a somewhat parallel path. High throughput screening of their chemical database uncovered the coumarin derivative warfarin as a weak HIV protease inhibitor. They rapidly identified a pyrone analog with aralkyl substituents at the 3- and 6-positions which had a Ki of 36 nM against the protease, an ECs0 of 3/zM in the antiviral assay, and high oral bioavailability. This low-molecular-weight (362 Da), easily synthesized (three steps) compound, U-96988, was advanced to the clinic. Another series of compounds which have been explored by Upjohn are 5,6-cycloalkylpyrones, which also have greater flexibility than the coumarins. The cyclooctyl analog was significantly more potent with the eight-membered ring effectively folding into the $1' pocket. A potent analog containing an a-cyclopropylbenzyl substituent at C-3 was identified (K~ = 15 nM) with high bioavailability, but poor antiviral activity (Romines and Thaisrivongs, 1995; Romines et al., 1995a). Substitution of the benzyl substituent at the meta-position by a p-cyanobenzenesulfonamideimproved enzyme potency to K~ = 0.8 nM and antiviral activity to ICs0 = 1.5/zM. This compound (U-103017) had high bioavailability and was advanced to the clinic (Skulnick et al., 1997). A similar variation allowed for the preparation of potent coumarin analogs, which, however, lacked antiviral activity. A similar variation in the pyrone series showed similar potency. The X-ray structure showed that the sulfonamide oxygens formed bifurcated H-bonds with the Asp30 OH and NH groups. The imidazole NH group is H-bonded to the Asp29 NH group (Thaisrivongs et al., 1996). Potent dihydropyrones were also identified (Thaisrivongs et al., 1996b). The compound PNU-140690 has been shown to be effective against Ritonavir-resistant mutants in cell culture (Lee et al., 1996; Poppe et al., 1997). The parallels between these two independent design efforts are striking. In both cases, the core 4-hydroxy-2-pyranone ring system was discovered from high throughput enzyme-based screening. In each case, the potency of the initial lead compounds was too low to possess measurable antiviral activity. Fur-

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ther optimization was guided by X-ray crystal structure analysis combined with modeling to identify novel binding modes and key positions of the molecules that could be used to incorporate substituents that would improve potency, retain bioavailability, and inhibit resistant mutants.

D. IRREVERSIBLE INHIBITORS Irreversible enzyme inhibitors are often ignored as therapeutic targets because of toxicity and bioavailability concerns. A number of weak irreversible inhibitors of HIV protease have been described and crystal structures of the resulting alkylated enzyme have been generated (Yu et al., 1996, and references therein). More recently a Korean group has described a series of potent irreversible HIV protease inhibitors where the reactive moiety is an epoxide functionality (Park et al., 1995; Lee et al., 1996; Choy et al., 1997). It was expected that the epoxide would bind in the catalytic site with one aspartate activating the epoxide via protonation while the other (as the carboxylate anion) would nucleophilically attack an epoxide carbon. Key to the reactivity of these compounds was the presence of only the P1 benzyl group (NCCCCN core). The presence of the P 1' benzyl group commonly found in the reversible inhibitors destroys activity in the irreversible series. These compounds have subnanomolar apparent Ki s in the protease assay. Noteworthy is the good bioavailability observed with LB-71350 (NCCCCCC~O core), suggesting that the inherent reactivity of the epoxide is only unveiled once it is bound in the enzyme pocket (Jeong et al., 1997). We have generated a crystal structure of one of these compounds after reaction with HIV protease, which verifies the expected reaction mode and suggests a unique mode of binding in the P' region (T. N. Bhat, unpublished data). Irreversible inhibition of the enzyme may be an approach that proves useful in attacking protease mutants.

E. PHARMACOKINETICSOF H I V PROTEASE INHIBITORS Unlike the case for the majority of RT inhibitors, most HIV PR inhibitors have serious pharmacokinetic limitations. Poor oral absorption, serum protein binding, liver enzyme metabolism, and other factors can all but eliminate the antiviral benefits of many potent protease inhibitors. Currently approved protease inhibitors need to be taken often and in large quantities to maintain effective antiviral concentrations in the blood. Of the currently available antiviral drugs for AIDS, protease inhibitors are among the most effective, but are costly and require difficult treatment regimens. As a result, the failure or drop-out rate of

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John W. Erickson and Michael A. Eissenstat

patients on protease inhibitor therapy tends to be relatively high, the compliance of treatment with protease inhibitors is likely to be poor, and the development of resistance to these highly effective drugs is a growing problem (Deeks et al., 1997).

V. DRUG RESISTANCE Although many promising new anti-HIV drugs have been developed, their effectiveness has been hampered by the emergence of drug-resistant variants. Mutant viruses emerge in the presence of antiviral agents whenever the balance of mutant virus replication is favorable, i.e., the mutant provides a selective advantage to the virus in the presence of the drug. Biological selection of HIV mutants is particularly favorable owing to the nature of HIV infection, which is chronic, persistent, and characterized by a high steady-state rate of replication. Pharmacodynamic studies using potent HIV PR inhibitors have revealed that the combined half-life of plasma virus and virus-producing cells in the body is on the order of 2 days or less, with new virus being produced at a rate of 108-109 virions per day (Ho et a|., 1995; Wei et al., 1995). These conditions, coupled with the high error rate of HIV-1 reverse transcriptase, less than 1 in 10,000 bases, favor rapid mutation and selection of drug-resistant virus (Coffin, 1995). Clinical resistance to every newly introduced anti-HIV agent has become the rule and resistance to PR inhibitors is no exception (Erickson, 1995; Richman, 1996). Recent reports of the selection and transmission ofmultidrugresistant HIV strains that contain multiple PR and RT mutations in the pol gene and also in cleavage site sequences in the gag gene have raised new alarms in the AIDS community. An understanding of the biological and structural mechanisms of resistance to HIV PR inhibitors is necessary in order to predict optimal salvage therapies; indeed, the full implications of drug resistance for disease outcome have not yet been realized. A great deal of progress has been made at unraveling the structural, biochemical, and virologic mechanisms of resistance to PR and RT inhibitors in light of the three-dimensional atomic structures of these proteins (Richman, 1996). An important goal of these studies is to gain insights that may lead to new strategies to combat resistance and to eventually develop paradigms for structure-based strategies to combat resistance to antiinfectious disease agents.

A. MECHANISMS Over 57 mutations have been observed in at least 27 positions in the 99-residue HIV PR monomer in response to drug selection pressure (Hammond et al.,

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1997) (Fig. 7). Many of these mutations were first observed in vitro and presaged the emergence of mutants in the clinical setting in vivo, although the actual number and order of appearance of multiple mutations varied (Condra et al., 1995; Molla et al., 1996; Ives et al., 1997). They can be classified as active site vs nonactive site mutations according to whether they occur inside or outside the inhibitor binding subsites. A variety of resistance mechanisms to HIV PR inhibitors have been proposed based on our understanding of the structural biochemistry of the PR and on the nature of inhibitor binding to the enzyme (Erickson, 1995). Mutations of specificity-determining residues that would directly interfere with inhibitor binding and lead to loss of potency constitute an obvious mechanism for resistance to HIV PR inhibitors. Active site mutations are necessary but often are not sufficient for high-level resistance in the clinical setting (Condra et al., 1995). An explanation for this observation comes from biochemical studies that reveal a negative impact of many resistance-conferring active site mutations on enzyme activity (Sardana et al., 1994; Condra et al., 1995; Gulnik et al., 1995), suggesting that such mutations result in suboptimal virus. A second mechanism of resistance involves nonactive site mutations that indirectly alter the active site architecture via longrange structural perturbations. Mutations that enhance enzyme catalysis in the presence of inhibitors could constitute a third mechanism. Finally, mutations that affect dimer stability, cleavage site mutations that lead to altered processing kinetics by mutant enzymes, and "regulatory" mutations elsewhere in the genome that lead to improved viral growth in the presence of PR inhibitors comprise additional resistance pathways. Any mutation that influences the binding of a specific inhibitor can be expected to have an effect on substrate cleavage kinetics as well as perhaps on substrate recognition. For this reason, combinations of two or more mutations in HIV PR may lead to a variety of additive, synergistic, or compensatory effects, depending on which property is being measured.

B. ACTIVE SITE MUTANTS The first-described resistance mutation for HIV PR was a single substitution of a valine residue by alanine at position 82 (V82A mutation) that was selected using a symmetric diol inhibitor (Otto et al., 1993). Since then, resistance mutations have been observed in each of the specificity pockets, $3, $2, S 1 and, by symmetry, S 1 ', $2', and $3 '. However, only a subset of all residues that constitute a particular subsite mutates in response to a particular drug. The structural effects of mutations on drug binding have been modeled using the crystal structures of the appropriate wild-type enzyme/inhibitor complexes and used to rationalize the effects of specific mutations on drug binding (Otto et al., 1993; Ho

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John W. Erickson and Michael A. Eissenstat

et al., 1994; Kaplan et al., 1994; Markowitz et al., 1995; Baldwin et al., 1995a). Most of the subsite mutations, like I84V and V82A, -I, or -F, affect hydrophobic and van der Waals' interactions and can be considered to be "packing" mutants, somewhat analogous to hydrophobic mutations in a protein (Markowitz et al., 1995; Baldwin et al., 1995a,b). Crystallographic analyses of HIV PR/inhibitor complexes show that most of the surface of an inhibitor and its immediate protein environment are solvent inaccessible. Some mutations, such as V82I, are more effective when combined with a second active site mutation, such as V32I, owing to synergistic effects on enzyme activity (Kaplan et al., 1994). Other mutations can affect electrostatic contacts. The effect of the R8Q mutation on binding to A-77003 was shown to be due to loss of a chargeinduced dipole interaction between the guanidinium side-chain of the enzyme and the pyridine ring of the inhibitor (Ho et al., 1994). While this mutation resulted in a dramatic loss of inhibition, it also produced a virus with severely impaired growth kinetics. A possible explanation for this defect is the symmetric loss of the intersubunit salt bridge between Arg8 and Asp129, which may decrease the stability of the enzyme. Despite the demonstration of strong crossresistance of the R8Q PR to Ritonavir, Indinavir, and Saquinavir (Gulnik et al., 1995), this mutation has not yet been observed clinically. Thus, the design of inhibitors that selectively interact with Arg8 appears to be a useful strategy for the development of "resistance-repellent" drugs (Erickson, 1995; Mo et al., 1996). While numerous crystal structures of wild-type HIV PR/inhibitor complexes have been published, crystal structures of mutant HIV PR/inhibitor complexes have appeared with less frequency in the literature. The structure determination of the V82A mutant of HIV PR complexed to the C2 symmetrybased inhibitor A-77003 revealed unexpected backbone changes that resulted in the repacking of enzyme and inhibitor atoms in the $1/$1' and $3/$3' subsites (Baldwin et al., 1995b). The structure of the V82I mutant with DMP-323, a cyclic urea-based inhibitor, exhibited a loss of interaction between the CG1 methyl group of valine and the inhibitor despite the larger bulk of the mutant isoleucine side-chain (Chang et al., 1994). This was because the energetically favored side-chain rotamer for isoleucine resulted in a repositioning of the CD 1 methyl away from the P 1 group of the inhibitor. In both examples, the observed structural changes were unexpected based on modeling studies. The protein residue Val82 resides in a structurally flexible loop that can exhibit a variety of different conformations in response to different conditions (Erickson, 1993). This flexibility is consistent with the fact that Val82 can be replaced by both larger (Ile and Phe) and smaller (Ala) residues in viable mutant enzymes. Crystal structures of cyclic ureas complexed with V82F, I84V, and V82F/I84V mutants provide further support for the flexibility of this region and also demon-

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strate that the conformation of the 82F mutant side-chain depends on the nature of the side-chain at position 84 (I or V) (Ala et al., 1997). The structure of Indinavir complexed to the cross-resistant multiple mutant M46I/L63P/ V82T/I84V demonstrated little discernible change from the wild-type enzyme complex although the mutant showed a 70-fold (---2.5 kcal/mol) increase in Ki (Chen et al., 1995). These studies point out the pitfalls inherent in reliance on pure modeling approaches and underscore the need for continued experimental studies of inhibitors complexed with resistant mutants of HIV PR.

C.

NON-ACTIVE SITE MUTANTS

While the precise structural mechanism of drug resistance can often be pinpointed for active site mutations that directly affect inhibitor binding, the evaluation of non-active site mutants is more challenging. Some mutations might act in concert with active site mutations by compensating for a functional deficit caused by the latter. For example, the R8Q mutation is found almost exclusively in combination with one or more mutations outside the active site region, such as M46I (Ho et al., 1994). Mutations of Met46 to Ile, Leu, or Phe are often found in the presence or absence of other active site mutations, such as V82I, -A, or -F and I84V. The residue Met46 is in the flap of HIV PR and molecular dynamics simulations on flap movement have shown that the M46I mutant exhibits a markedly different dynamical behavior than the wild-type enzyme (Collins et al., 1995) and presumably exhibits altered enzyme kinetics. A role for Met46 in polyprotein substrate recognition is also possible. Other non-active site mutations may indirectly alter the structure of the active site region alone or in combination with one or more active site mutations. Modeling studies of a hextuple mutant selected using KNI-272 (B. Anderson and H. Mitsuya, personal communication) reveal that mutations far from the active site, such as A71V (Fig. 7, see color plate), may influence inhibitor or substrate binding via a concerted "domino" effect (T. N. Bhat, unpublished data). A similar mechanism may be operative for the Saquinavir-resistant L90M mutant. The side-chain of this residue can affect the conformation of the adjacent active site loop that contains the catalytic aspartic acids. Most of the non-active site mutations result in a larger hydrophobic side-chain which can be expected to perturb packing interactions within the enzyme core. This perturbation may be transferred to the active site where the internal strain energy in the protein may be relieved through alterations in the interaction energy with the inhibitor. In some cases, the introduction of drug-selected non-active site mutations alone does not lead to a measurable reduction in drug binding, in contrast to the case for active site mutations (Gulnik et al., 1995). However, the fact that these

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John W. Erickson and Michael A. Eissenstat

mutations are only observed in the presence of drug means that they must provide the mutant virus with a competitive growth advantage over wild-type HIV. At least one engineered HIV PR mutant, G48Y, exhibited a greater catalytic efficiency than the wild-type enzyme toward artificial peptide substrates (Lin et al., 1995). However, this mutant has not been observed either in vitro or in vivo in the presence or absence of inhibitors.

D. CLEAVAGESITE MUTANTS AND SUBSTRATE SPECIFICITY Active site mutations can strongly affect catalytic efficiency of HIV PR (Gulnik et al., 1995), but the magnitude of the effect depends on the substrate sequence (Ridky et al., 1998). The addition of one or more non-active site mutations may compensate for a catalytically defective active site mutation (Gulnik, 1995; Schock, 1996). These conclusions are based on enzymology studies with recombinant HIV PR mutants. Recent longitudinal studies of individual drugtreated patients demonstrate an ordered accumulation of mutations in which one or two active site mutations usually occur early and are followed by numerous nonactive site mutations (Molla et al., 1996; Zhang et al., 1997). Thus, the clinical evolution of drug resistance to protein inhibitors seems to qualitatively mirror expectations based on the enzymology studies. Since active site mutations may be expected to alter the rate of one or more cleavages that must occur during viral maturation, one may imagine that compensating mutations in the cleavage sites on the Gag or Gag-Pol polyproteins might result in better substrates for particular mutant enzymes. Recent studies identified a Leu449Phe mutation in the p 1/p6 Gag polyprotein cleavage site that can synergize with the I84V PR mutation to produce a virus with 350- to 1500-fold decreased sensitivity to substrate-based inhibitors Bila-1906 and Bila-2185 (Doyon et al., 1996). The mutation altered the pl/p6 cleavage site from PheLeu to Phe-Phe. A synthetic peptide containing the mutant Phe-Phe cleavage site was cleaved at higher catalytic efficiency by the I84V HIV PR mutant than the corresponding peptide with the wild-type sequence (Lamarre et al., 1994). Salzman and co-workers subsequently identified mutations at the NC/pl Gag cleavage site in breakthrough resistant virus isolated from patients on indinavir therapy (Zhang et al., 1997). This is an important finding since it confirms the possibility for this drug resistance mechanism to be operative in the clinical setting. Several groups have followed the lead of these investigators and have confirmed the presence of cleavage site mutations in clinical isolates from multidrug-resistant HIV in preliminary reports (Larder et al., 1998; Mammano et al., 1998).

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VI. F U T U R E C H A L L E N G E S I N H I V P R O T E A S E INHIBITOR DESIGN

A. PREDICTION OF RESISTANCE PATHWAYS: DEFINING BIOCHEMICAL FITNESS FOR VARIANT PROTEASES A key issue in combating resistance is to first define the most important resistance mutations for a particular inhibitor. While there is no guarantee that in vitro selection studies will accurately mirror in vivo results, a recent study using a panel of over a dozen recombinant PR mutants suggests that those mutations that were selected in vivo by a particular inhibitor displayed the highest enzyme "vitality," or fitness, defined as the ratio of (K~ • kcat/Km)mutant (Ki • kcat/Km)wildtype' for a given inhibitor (Gulnik et al., 1995). A number of studies indicate that cross-resistance is a problem for most clinically useful PR inhibitors, Saquinavir being a notable exception (Sardana et al., 1994; Condra et al., 1995; Gulnik et al., 1995; Wilson et al., 1997). However, this is to be expected since all the inhibitors were highly optimized to bind the wild-type enzyme. Mutations have been found in every subsite of HIV PR. However, not every subsite residue has been found to mutate for a particular drug. Further, only a limited number of the total possible single-step mutations that could occur have actually been found at each variant position. These results suggest that there are constraints that limit the potentially available resistance pathways. Besides the usual factors that limit viable mutations in enzymes, such as proper folding, stability, and catalytic function, HIV PR has several additional and unique constraints. The homodimeric nature of the enzyme means that every mutation at the genetic level will result in a double mutant at the enzyme level (unless heterodimer formation occurs in vivo). The requirement for recognition and cleavage of at least nine specific polyprotein cleavage sites should limit mutations to ones that will not diminish processing below some tolerable threshold. Enzymology and virus infectivity studies with recombinant HIV PR mutants independently suggested that this threshold may be between 5 and 10% of wild-type activity (Babe et al., 1995; Gulnik et al., 1995; McPhee et al., 1996). The relative cleavage rates for certain Gag polyprotein processing sites, such as p2-CA, have also been shown to be important for ordered assembly and maturation of infectious virus (Pettit et al., 1994; Weigers et al., 1998). Finally, the fact that some subsite

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amino acids, such as Arg8, also participate in the dimer interface suggests that some subsite mutants may adversely affect dimer stability and reduce inhibitor potency through thermodynamic linkage of inhibitor binding and dimer formation (Xie et al., 1998). It is interesting to note that many of the resistance mutations in HIV-1 PR correspond to the wild-type residues in HIV-2 or in other retroviral proteases at structurally equivalent locations. Thus, there may be a limited number of solutions to the PR sequence/substrate specificity combinatorial puzzle for retroviruses, and mutational strategies for resistance tend to evolve toward one or more of them.

B. IMPLICATIONS FOR INHIBITOR DESIGN There are two major challenges that lie ahead for HIV PR inhibitor design. One concerns the issue of pharmacokinetics. As potent and bioavailable as these inhibitors are, they need to be even more potent and more bioavailable to maximize their effectiveness and usefulness. The daily requirements for PR inhibitors is massive at upward of 800 mg/kg two to three times/day. Side-effects can be troublesome, if not debilitating, for most of these compounds. Indinavir therapy leads to painful reoccurrence of kidney stones in about 20% of individuals. Ritonavir has serious drug interaction issues since it is a highly potent inhibitor of a major liver enzyme, cytochrome P450 3A4, involved in drug metabolism. A syndrome of peripheral lipodystrophy (fat wasting), hyperlipidemia, and insulin resistance is emerging as a frequent side-effect of long-term PR inhibitor therapy (Carr et al., 1998). Finally, these drugs tend to be costly to make. Coupled with the massive dosages required for maximal viral suppression to be maintained, the economics of PR inhibitor therapy is not a trivial consideration. The solution lies in the design of more potent, less costly, and more bioavailable inhibitors. This in turn raises the question of what is the theoretical limit of potency that can be achieved for HIV PR inhibitors and how close are we to this limit? A related question is can we design inhibitors with a better potency/MW ratio and thereby achieve significant improvements in bioavailability and ease of synthesis? The second major challenge confronting the field of HIV PR inhibitors is the problem of how to effectively combat drug resistance. Given the unusual constraints on HIV PR and our understanding of inhibitor binding and resistance at an atomic level of detail, it should be possible at a minimum to design inhibitors that target different mutants. These inhibitors could then be used together in multidrug combination therapy approaches. It may even be possible to design inhibitors that target multiple mutants simultaneously. The potency of Saquinavir against multiple active site mutants suggests that this may be feasible. In certain instances, these second-line drugs might prompt the selection

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of wild-type revertants that can then be treated with the original drug. Classical sequential therapy approaches have been unsuccessful in controlling infectious diseases and have given way to multidrug treatment. However, one can even envision a therapeutic strategy for HIV PR inhibitors that could apply specific, structure-guided selection pressure to influence clinical outcome (Erickson, 1995). Such strategies would represent a fundamental departure from currently held concepts of infectious disease treatment and will require a great deal of experimental virology assuming that the appropriate inhibitors can be designed in the first place.

VII. S U M M A R Y A N D C O N C L U S I O N S Human immunodeficiency virus proteases have proven to be valuable instructional targets for the design of mechanism- and structure-based drugs and for providing novel strategies for designing antiviral therapeutics. The introduction of PR inhibitors to the armamentarium of AIDS drugs has had a profoundly successful impact on the AIDS community where these drugs are prolonging life and providing new hope for the long-term maintenance of relatively good health until such time as a more effective cure or vaccine becomes available. However, the success of PR inhibitor therapy is a qualified one. A growing number of individuals are developing multidrug-resistant strains of HIV and there is emerging evidence that these strains can be readily transmitted. At present, there are no effective treatment options available for these patients. This brings us to the brink of a new forefront in antiviral research that requires novel approaches to deal with the problem of drug resistance. This problem poses different challenges than we faced in the design of the first-line drugs, as it forces us to think about selection pressure mechanisms in addition to the usual issues of potency, pharmacology, safety, and mechanism of drug action. Unfortunately, those very features that contribute to the specificity and efficacy of PR inhibitors also provide the virus with strategies to develop drug resistance. The resistance picture for HIV PR inhibitors is not limited to this class of compounds; cross-resistance to both the nucleoside and non-nucleoside HIV RT inhibitors is common. The experience obtained with HIV antiviral therapy should be a warning to those targeting other viruses. The rapid replication rate of HIV in vivo coupled with the long duration of viral infection favors the emergence of resistant mutants to virtually any targeted antiviral agent. Computer simulation studies show that a mutant virus with a selective advantage of only 5% (i.e., a replication rate 1.05 x wild-type virus) will propagate to become the dominant genotype within --~100 virus generations (Coffin, 1995). Human immunodeficiency virus undergoes 100 replication cycles in several months. These same considerations imply that those viral mutants that only arise under

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drug pressure and arise early should be less robust than their wild-type progenitors. Thus, effective strategies to combat resistance should be ones that can limit the selection of mutational pathways to a small number of mutants and not drive the virus far from wild type. A somewhat more optimistic picture of resistance can be painted with viruses that cause acute infections that resolve within a few weeks, since they may have less of a tendency to develop dominant resistant strains than HIV. However, treatment of HIV infection has taught us that, under the appropriate selection pressure, rapid and robust resistance is an ever-present danger. Finally, with the growing number of potential resistance mutations, improved approaches to structure-based drug design and the development of quantitative methods of modeling and binding affinity prediction take on added significance. Meanwhile, it is important to continue to search for combinations of drugs with complementary resistance profiles and to explore new treatment modalities such as structure-based approaches that can be used to exert specific selection pressures on HIV in the hopes that at least a stalemate, if not an end game, strategy can be found to control the progression of AIDS. Lessons learned with HIV PR may provide valuable strategies for dealing effectively with other retroviral infections, such as HIV-2 and HTLV-I, as well as with important animal diseases caused by retroviruses, through targeting of retroviral proteases.

ACKNOWLEDGMENTS The authors thank Ms. Christine Ray for help with the preparation of this chapter. This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. NO1-CO-56000.

REFERENCES Abdel-Meguid, S. S. (1993). Inhibitors of aspartyl proteinases. Med. Res. Rev. 13(6), 731-778. Abdel-Meguid, S. S., Zhao, B., Murthy, K. H., Winborne, E., Choi, J. K., DesJarlais, R. L., Minnich, M. D., Culp,J. S., Debouck, C., and Tomaszek, T. A.,Jr. (1993a). Inhibition of human immunodeficiency virus-1 protease by a C2-symmetric phosphinate: Synthesis and crystallographic analysis. Biochemistry 32(31), 7972-7980. Ala, P. J., Huston, E. E., Klabe, R. M., McCabe, D. D., Duke, J. L., Rizzo, C. J., Korant, B. D., DeLoskey, R. J., Lam, P. Y., Hodge, C. N., and Chang, C. H. (1997). Molecular basis of HIV-1 protease drug resistance: structural analysis of mutant proteases complexed with cyclic urea inhibitors. Biochemistry 36(7), 1573-1580. Ala, R., DeLoskey, R., Huston, E., Jadhav, P., Lain, P., Eyermann, C., Hodge, C., Schadt, M., Lewandowski, F., Weber, P., McCabe, D., Duke, J., and Chang, C. (1998). Molecular recognition of cyclic urea HIV-1 protease inhibitors.J. Biol. Chem. 273(20), 12325-12331. Alsenz, J., Steffen, H., and Alex, R. (1998). Active apical secretory emux of the HIV protease inhibitors saquinavir and ritonavir in Caco-2 cell monolayers. Pharm. Res. 15,423-428.

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Romines, K. R., Watenpaugh, K. D., Howe, W. J., Tomich, P. K., Lovasz, K. D., Morris, J. K., Janakiraman, M. N., Lynn, J. C., Horng, M. M., and Chong, K. T. (1995a). Structure-based design of nonpeptidic HIV protease inhibitors from a cyclooctylpyranone lead structure. J. Med. Chem. 38(22), 4463-4473. Romines, K. R., Watenpaugh, K. D., Tomich, P. K., Howe, W. J., Morris, J. K., Lovasz, K. D., Mulichak, A. M., Finzel, B. C., Lynn, J. C., and Horng, M. M. (1995b). Use of medium-sized cycloalkyl rings to enhance secondary binding: Discovery of a new class of human immunodeficiency virus (HIV) protease inhibitors.J. Med. Chem. 38(11), 1884-1891. Rutenberg, E. E., McPhee, F., Kaplan, A. P., Gallion, S. L., Hogan, J. C., Jr., Craik, C. S., and Stroud, R. M. (1996). A new class of HIV-1 protease inhibitor: The crystallographic structure, inhibition and chemical synthesis of an aminimide peptide isostere. Bioorg. Med. Chem. 4(9), 1545-1558. Sardana, V. V., Schlabach, A. J., Graham, P., Bush, B. L., Condra, J. H., Culberson, J. C., Gotlib, L., Graham, D. J., Kohl, N. E., LaFemina, R. L., Schneider, C. L., Wolanski, B. S., Wolfgang, J. A., and Emini, E. A. (1994). Human immunodeficiency virus type I protease inhibitors: Evaluation of resistance engendered by amino acid substitutions in the enzyme's substrate binding site. Biochemistry 33, 2004-2010. Schechter, I., and Berger, A. (1967). On the size of the active site in proteases. I. Papain. Biochem. Biophys. Res. Commun. 27(2), 157-162. Scholz, D., Billich, A., Charpiot, B., Ettmayer, P., Lehr, P., Rosenwirth, B., Schreiner, E., and Gstach, H. (1994). Inhibitors of HIV-1 proteinase containing 2-heterosubstituted 4-amino-3-hydroxy5-phenylpentanoic acid: Synthesis, enzyme inhibition, and antiviral activity. J. Med. Chem. 37, 3079-3089. Seelmeier, S., Schmidt, H., Turk, V., and vonder Helm, K. (1988). Human immunodeficiencyvirus has an aspartic-type protease that can be inhibited by pepstatin A. Pro. Natl. Acad. Sci. USA 85, 6612-6616. Sham, H. L., Betebenner, D. A., Zhao, C., Wideburg, N. E., Saldivar, A., Kempf, D. J., Plattner, J. J., and Norbeck, D. W. (1993). Facile synthesis of potent HIV-1 protease inhibitors containing a novel pseudo-symmetric dipeptide isostere.J. Chem. Soc. Chem. Commun. 13, 1052-1053. Sham, H. L., Zhao, C., Marsh, K. C., Betebenner, D. A., Lin, S., McDonald, E., Vasavanonda, S., Wideburg, N., Saldivar, A., and Robins, T. (1995). Potent inhibitors of the HIV-1 protease with good oral bioavailabilities. Biochem. Biophys. Res. Commun. 211 (1), 159-165. Sham, H. L., Zhao, C., Marsh, K. C., Betebenner, D. A., Lin, S., Rosenbrook, W., Jr., Herrin, T., Li, L., Madigan, D., Vasavanonda, S., Molla, A., Saldivar, A., McDonald, E., Wideburg, N. E., Kempf, D., Norbeck, D. W., and Plattner, J. J. (1996). Novel azacyclic ureas that are potent inhibitors of HIV-1 protease. Biochem. Biophys. Res. Commun. 225(2), 436-440. Shetty, B. V., Kosa, M. B., Khalil, D. A., and Webber, S. (1996). Preclinical pharmacokinetics and distribution to tissue of AG1343, an inhibitor of human immunodeficiency virus type i protease. Antimicrob. Agents Chemother. 40(1), 110 -114. Shoeman, R. L., Sachse, B., Honer, E., Mothes, E., Kaufmann, M., and Traub, P. (1993). Cleavage of human and mouse cytoskeletal and sarcomeric proteins by human immunodeficiency virus type i protease: Actin, desmin, myosin, and tropomyosin. Am. J. Pathol. 142,221-230. Skulnick, H. I., Johnson, P. D., Aristoff, P. A., Morris, J. K., Lovasz, K. D., Howe, W. J., Watenpaugh, K. D., Janakiraman, M. N., Anderson, D. J., Reischer, R. J., Schwartz, T. M., Banitt, L. S., Tomich, P. K., Lynn, J. C., Horng, M. M., Chong, K. T., Hinshaw, R. R., Dolak, L. A., Seest, E. P., Schwende, F. J., Rush, B. D., Howard, G. M., Toth, L. N., Wilkinson, K. R., and Romines, K. R. (1997). Structure-based design of nonpeptidic HIV protease inhibitors: the sulfonamidesubstituted cyclooctylpyranones. J. Med. Chem. 40(7), 1149-1164. Skulnick, H. I., Johnson, P. D., Howe, W. J., Tomich, P. K., Chong, K. T., Watenpaugh, K. D., Janakiraman, M. N., Dolak, L. A., McGrath, J. P., and Lynn, J. C. (1995). Structure-based design of

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Proteases of the Hepatitis C Virus ANDREA URBANI, RAFFAELE DE FRANCESCO, AND CHRISTIAN STEINKUHLER Instituto di Ricerche di Biologia Molecolare (IRBM) "P. Angeletti," 00040 Pomezia, Rome, Italy

I. II. III. IV. V.

Introduction Genomic Organization The NS2-NS3 Protease The NS3 Protease Conclusions References

I. I N T R O D U C T I O N After the development of serological tests for the hepatitis A and B viruses in the 1970s it became clear that an additional agent accounted for approximately 90% of transfusion-associated hepatitis (non-A non-B hepatitis, NANBH) (Houghton, 1996). Initial experiments indicated that the causative agent of NANBH probably was a small, enveloped virus that could be transmitted to chimpanzees. Its very low titer in infected sera made conventional attempts aimed at its identification impossible. In 1989 Houghton and co-workers at Chiron succeeded in identifying an NANBH-specific cDNA clone from an immunoscreening of a cDNA expression library obtained from an infected chimpanzee plasma pool using a panel of presumptively infected human sera as a specific anti-NANBH immunoglobulin source (Choo et al., 1989). The cloned cDNA was shown to be derived from a single RNA molecule of 9.5 kb with homologies to the genome of Flavi- and Pestiviruses (Miller and Purcell, 1990; Francki et al., 1991). The novel agent, hence termed hepatitis C virus

Proteases of Infectious Agents Copyright 9 1999 by Academic Press. All rights of reproduction in any form reserved.

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(HCV), was classified within the Flavivirideae family. Diagnostic tests for antiHCV antibodies developed thereafter proved that HCV was indeed the predominant cause of NANBH (Kuo et al., 1989). The prevalence of the infection varies between 0.01 and 2% according to the geographic region with peaks as high as 14% (Mast and Alter, 1993; Cuthbert, 1994; Houghton, 1996). It is estimated that about 0.5-1.5% of the total world's population is infected with HCV. The main route of transmission is parenteral with high incidence among intravenous drug users and recipients of blood products and renal dialysis patients before implementation of screening regimens. Other risk factors are tattooing and needle stick injuries. Sexual and congenital transmissions appear less frequently (Alter, 1995). The incidence of new infections has greatly diminished as a consequence of the development of reliable diagnostic assays and an effective screening of blood or blood products. Nevertheless, between 20 and 50% of HCV cases are not associated to classic risk factors. These "sporadic" infections correlate with a low socioeconomic background which may be associated with enhanced contact with risk groups such as intravenous drug users. Infection with HCV often occurs without overt clinical symptoms, with only 10% of infected individuals developing an acute illness. About 50% of the infections, however, lead to chronic hepatitis, with elevated serum transaminases, persistence of HCV RNA, and histologic liver lesions. Liver cirrhosis, characterized by an overgrowth of connective tissue replacing the damaged hepatocytes, develops in 20% of the cases within 20 years after the infection (MacDonnell and Lucey, 1995; Houghton, 1996). At this stage, the disease may further progress into liver failure. Furthermore, chronic HCV infection has been associated with hepatocellular carcinoma (Saito et al., 1990; Shimotohno, 1993). At present, ce-interferon is the only licensed treatment for chronic HCV infection in the UK and in the U.S. The efficacy of this therapy is, however, rather low: the response rate to ce-interferon treatment is about 50%. Roughly half of these responders relapse after the end of the treatment, resulting in only 25% of sustained response with disappearance of HCV-RNA from liver tissue, serum, and peripheral blood mononuclear cells (Davies et al., 1989; Alberti, 1995; Blair et al., 1996). The efficacy of ce-interferon appears to be enhanced in a combination therapy with ribavirin (DiBisceglie et al., 1992; Kakumu et al., 1993; Reichard et al., 1993; Koskinas et al., 1995). Even though a very significant improvement of sustained response over monotherapy has been recently reported for the interferon-ribavirin combination therapy, this treatment results in less than 50% of sustained responders (McHutchison et al., 1998; Davies et al., 1998). Furthermore, ce-interferon therapy has a number of sideeffects such as flu-like symptoms, irritability, fatigue, depression, anorexia, nausea, rashes, alopecia, thrombocytopenia, and leukopenia. Additional sideeffects may arise during the combination therapy with ribavirin. In view of the

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lack of vaccines against HCV there is an urgent need for an efficacious treatment of the disease by an effective antiviral drug. This necessity has boosted research on the biology of HCV with the primary focus being to identify possible targets for pharmaceutical intervention.

II. G E N O M I C O R G A N I Z A T I O N The investigation of HCV biology was facilitated by the homology of its genome structure to that of Flavi- and Pestiviruses. As observed in these two genera, HCV has a single-stranded RNA genome of 9.5 kb with positive polarity containing a single, large open reading frame (ORF). This ORF encodes a polyprotein of 3010-3033 amino acids. The ORF is flanked by 5' and 3' untranslated regions (UTR). The 5' UTR harbors a very well-conserved sequence that is thought to play a role in translation initiation through a mechanism of internal ribosomal entry (Tsukiyama-Koharak et al., 1992; Wang et al., 1993; Fukushi et al., 1994). The 3' UTR contains a poly (U) stretch flanked by an upstream variable region of 2 8 - 4 2 nucleotides and downstream by a highly conserved 98-nucleotide sequence predicted to form a stem-and-loop structure (Tanaka et al., 1995, 1996; Kolykhalov et al., 1996). Owing to the lack of an efficient in vitro propagation system of HCV our knowledge about the fate of the translation product of the viral genome is based on transient transfection or in vitro translation of portions of the viral genome (Hijikata et al., 1991; 1993a; Grakoui et al., 1993a; Selby et al., 1993; Tomei et al., 1993; Lin et al., 1994a; Mizushima et al., 1994a,b). These studies have shown that, upon its synthesis, the polyprotein precursor is co- and posttranslationally processed into at least 10 individual structural and nonstructural mature viral proteins. These gene products are arranged as shown in Fig. 1, following the order C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B. The structural proteins C, El, E2, and p7 are located at the N-terminus of the polyprotein, followed by the nonstructural proteins at its C-terminus. The core protein (C), located at the amino terminus of the polyprotein, is considered to be the viral capsid protein. It is very basic and was shown to bind to RNA (Santolini et al., 1994). The proteins E1 and E2 are the viral envelope glycoproteins, whereas the function of p7 is unknown. The mature structural proteins C, El, and E2 are released from the nascent polypeptide chain through cotranslational cleavage by host cell signal peptidases (Hijikata et al., 1991; Grakoui et al., 1993a; Lin et al., 1994a; Mizushima et al., 1994a,b). In contrast, generation of the free C- and N-termini of p7 appears to be a posttranslational event as judged by the occurrence of E l - p 7 and p7-NS2 precursors after in vitro translation of E2-NS2 precursor polyproteins (Lin et al., 1994a; Mizushima et al., 1994a,b). All cleavages downstream of NS2 are mediated by virally

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FIGURE 1 Organization of the HCV polyprotein. Cleavage sites for signal peptidase (*), NS2/ NS3 protease (A), and NS3 serine protease (X) are indicated.

encoded proteases. The mature C-terminus of NS2 is generated by an intramolecular cleavage catalyzed by a protease encoded within NS2 and the N-terminal third of the NS3 protein (Grakoui et al., 1993c; Hijikata et al., 1993b; Santolini et al., 1995). The N-terminal --~180 amino acids of NS3 also form a chymotrypsinlike serine protease domain that is responsible for all the cleavages downstream of NS3, i.e., at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5ANS5B junctions (Bartenschlager et al., 1993; Eckart et al., 1993; Grakoui et al., 1993b; Hijikata et al., 1993b; Tomei et al., 1993; D'Souza et al., 1994; Manabe et al., 1994). The C-terminal two-third of the NS3 protein shows conserved sequence motifs that are characteristic of RNA helicases (Gorbalenya et al., 1989, 1990; Kim et al., 1997). Deletion experiments have shown that the isolated domains of NS3 retain their enzymatic activities and various forms of recombinant proteins encompassing the C-terminal domain of NS3 have been shown to possess RNA-stimulated NTPase activity (Suzich et al., 1993; Gwack et al., 1995; Preugschat et al., 1996) as well as RNA-helicase activity (Kim et al., 1995; Jin and Peterson, 1995; Tai et al., 1996; Gwack et al., 1996; Hong et al., 1996). Recently, the truncated protease and helicase domains of NS3 were crystallized and their three-dimensional structure was solved (Kim et al., 1996; Love et al., 1996; Yao et al., 1997; Yan et al., 1998). The NS4A protein was shown to be a cofactor of the NS3 protease activity (Failla et al., 1994; Bartenschlager et al., 1994; Lin et al., 1994b; Tanji et al., 1995a) and to stimulate the hyperphos-

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65

phorylation of NS5A (Tanji et al., 1995b; Asabe et al., 1997). Although the function of the NS5A protein is unknown there is accumulating evidence that this protein may be involved in the mechanism of viral resistance to interferon therapy (Herion and Hoofnagle, 1997). In fact, sequence comparisons between c~-interferon-resistant and -sensitive HCV isolates suggested a correlation between interferon response and mutations in a discrete region of NS5A (Enomoto et al., 1995, 1996; Chayama et al., 1997; Kurosaki et al., 1997). Furthermore, a recent study has shown that NS5A is a potent inhibitor of PKR, an interferon-induced protein kinase (Gale et al., 1997). NS5B was demonstrated to be the viral RNA-dependent RNA polymerase (Behrens et al., 1996; Yuan et al., 1997; Lohmann et al., 1997). No specific function has been so far assigned to the NS4B protein. Since the generation of the mature nonstructural HCV proteins, including the viral polymerase, relies on cleavages mediated by the virally encoded NS2-NS3 and NS3 proteins, specific inhibition of their proteolytic activities is presently regarded as a promising strategy to combat HCV infection (Bartenschlager, 1997; Neddermann et al., 1997). In the following, the progress made toward the understanding of the structure and the mechanism of action of the two HCV proteases is summarized.

III. T H E N S 2 - N S 3 P R O T E A S E The mature N-terminus of NS2 was reported to be generated by a signalasemediated cleavage (Mizushima et al., 1994b). Conversely, it was concluded that cleavage at the NS2-NS3 junction had to be mediated by a virally encoded protease since processing at this site could be observed in mammalian ceils as well as in Escherichia coli and plant and animal cell-free translation systems in the absence of microsomal membranes (Bartenschlager et al., 1993; Grakoui et al., 1993c; Hijikata et al., 1993b; Komoda et al., 1994a). Radiosequencing of the mature cleavage products showed that processing at the NS2-NS3 junction occurs between Leu1026 and Ala1027, within the sequence GWRLL-API (Grakoui et al., 1993c). Cleavage at the 2/3 site is remarkably resistant to single amino acid substitutions, the only dramatically inhibiting mutations being those likely to cause conformational alterations, simultaneous deletion of the residues flanking the cleavage site, or their concomitant substitution with alanine residues (Reed et al., 1995). Although not essential, membranes were shown to enhance cleavage efficiency at the NS2-NS3 junction in a strainspecific way (Santolini et al., 1995) and membrane activation could be mimicked by detergent micelles (Pieroni et al., 1997). In vitro translation studies showed that the NS2-NS3 precursor is targeted to the ER membrane through interaction with the signal recognition particle receptor and NS2 derived from

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the NS2-NS3 cleavage was demonstrated to be a transmembrane protein, having its C-terminus translocated into the ER lumen and at least part of its N-terminus exposed to the cytosol (Santolini et al., 1995). An active NS3 serine protease is not required for processing of the NS2-NS3 junction since mutations of the residues that constitute the catalytic triad of the NS3 serine protease affect all cleavages downstream of NS3, but do not influence the processing at the NS2-NS3 cleavage site (Hijikata et al., 1993b; Bartenschlager et al., 1993; Grakoui et al., 1993c; Tomei et al., 1993). A deletion mutagenesis analysis made to map the minimum domain required for efficient processing at the NS2NS3 site indicated an N-terminal boundary between residues 898 and 923 within NS2, whereas a sharp drop in cleavage efficiency was observed upon Cterminal truncations beyond residue 1207 within NS3 (Grakoui et al., 1993c; Hijikata et al., 1993b; Reed et al., 1995; Santolini et al., 1995). These findings indicate that the presence of the NS3 protease domain, but not its serine protease activity, is specifically required for processing at the NS2-NS3 site. In fact, the NS3 sequence could not be substituted by other parts of the HCV polyprotein without inactivating the NS2-NS3 protease (Santolini et al., 1995). It is remarkable that the NS3 serine protease and the 2/3 protease overlap, with the N-terminus of the NS3 protein contributing to two different enzymatic functions. As there is no obvious sequence homology between the NS2-NS3 protease and other known cellular or viral enzymes the nature of this enzyme was investigated using classic protease inhibitors. Thiol-reactive agents such as iodacetamide, N-ethylmaleimide or N-tosyl-L-phenylalauine Chloromethylketone were found to inhibit the enzyme, which was also shown to be redox sensitive (Pieroni et al., 1997). Also, metal chelators such as EDTA or phenanthroline abolished cleavage and subsequent addition of zinc or cadmium was able to restore activity (Hijikata et al., 1993b; Pieroni et al., 1997). Based on these findings, Hijikata and co-workers (1993b) proposed that NS2-NS3 might be a zinc-dependent metalloprotease. In order to identify possible zinc ligands they mutagenized all conserved histidine, cysteine, and glutamic acid residues within the minimum core of the NS2-NS3 protease and identified His952 and Cys993, which are localized within NS2 as essential for proteolytic activity. Although these residues were originally proposed to participate in the coordination of the zinc ion, the finding of a structural zinc binding site in the NS3 protease domain (De Francesco et al., 1996; Kim et al., 1996; Love et al., 1996; Yan et al., 1998) suggests that zinc could in fact be required for the NS2-NS3 protease activity because it stabilizes the fold of NS3. The residues His1175, Cys1123, Cys1125, and Cys1171 were identified as the zinc ligands in NS3. Interestingly, mutation of these cysteine residues into alanine decreased both the NS3 serine protease activity and processing at the 2/3 site (Hijikata et al., 1993b). In contrast to these findings, Reed et al. (1995) reported that a trun-

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cated precursor protein spanning residues 827-1137 of the HCV polyprotein still possesses some NS2-NS3 cleavage activity, even though the truncation has eliminated the zinc ligating Cys1171 and His1175 in NS3. It would be interesting to investigate whether the activity of this truncated protein is still influenced by zinc. Several hypotheses about the cleavage mechanism of the 2/3 junction are compatible with the experimental findings at this point. 1. There is one zinc ion bound in the NS3 protease domain that promotes its proper folding. The cleavage is catalyzed by a cysteine protease having His952 and Cys993 as a catalytic diad. In accord with this view, Gorbalenya and Snijder (1996) have recently classified NS2-NS3 as a cysteine protease. 2. The zinc ion bound in NS3 participates in the catalysis. Usually, the metal coordination sphere in zinc-dependent hydrolases involves nitrogen and oxygen and seldom sulfur ligands as observed in the NS3 zinc binding site (Vallee and Auld, 1990a,b), which has the characteristics of a structural, hydrolytically inactive or poorly active zinc site. Even though in the structure of the NS3 protease domain the metal binding site is quite close to the N-terminus of the enzyme (Kim et al., 1996; Yan et al., 1998; Wu et al., 1998). His1175, which is coordinated to the zinc via a bridging water molecule, is shielding the metal ion from the bulk solvent. However, both X-ray crystallography (Love et al., 1996) and NMR studies (Urbani et al., 1998) have shown that this histidine residue is endowed with a considerable flexibility, compatible with a switch of the metal binding site between an "open" and a "closed" conformation. It could be speculated that a facile movement of His1175, possibly leading to the exposure of a metal-activated water molecule, might play some role in the proteolysis of the 2/3 junction. 3. There are two zinc ions present in NS2-NS3, one being ligated to His952 and Cys993 and having a catalytic function. In this case, a dimerization of the 2/3 precursor should be conceived in order to provide a stable coordination to the second zinc ion. The answer to the question of which mechanism is operative in the cleavage of the 2/3 junction would be facilitated by a trans-cleavage assay using purified components. Reed et al. (1995) investigated this possibility. They concluded that bimolecular cleavage is only possible if the substrate polypeptide contributes a functional domain to the formation of an active protease. Precursors that contain a 2/3 site that is cleavable in trans are capable of supplying either the N-terminal domain of NS3 or a functional NS2 region to form the NS2-NS3 protease. This behavior suggests dimer formation to occur as a requisite for cleavage of the 2/3 junction. Attempts at demonstrating this interaction by immunoprecipitation failed, indicating that if dimer formation occurs the affinity

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between the single subunits must be rather low. A further indication for possible dimerization of 2/3 precursors comes from the observation that autocleavage of the 2/3 site could be inhibited by the addition of polypeptides capable of participating in bimolecular cleavage reactions, i.e., those precursors having either a functional NS2 portion or an intact NS3 region (Reed et al., 1995). This latter observation indicates that cleavage at the 2/3 site can potentially be inhibited in trans. Whether this inhibition requires protein-protein interactions involving a large surface area or is also amenable to low-molecular-weight compounds will hopefully be addressed by future studies aimed at the elucidation of this intriguing aspect of HCV polyprotein processing.

IV. T H E N S 3 P R O T E A S E The N-terminal portion of the NS3 protein was predicted to contain a serine protease domain as judged from conserved sequence patterns and by homology to Flavi- and Pestiviruses (Miller and Purcell, 1990; Francki et al., 1991; Rice, 1996). Within this region, residues His1083, Aspll07, and Ser1165 constitute the catalytic triad of the enzyme and mutagenesis of either residue was found to abolish proteolytic activity (Bartenschlager et al., 1993; Eckart et al., 1993; Grakoui et al., 1993b; Tomei et al., 1993; Manabe et al., 1994; Hijikata et al., 1993b). Transient transfection and in vitro transcription/translation experiments have been extensively employed as tools for the characterization of the role of NS3 in the maturation of the viral polyprotein. Its activity was required for the processing of all junctions downstream of NS3, i.e., at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B boundaries (Bartenschlager et al., 1993; Grakoui et al., 1993b; Hijikata et al., 1993b; Tomei et al., 1993). There is a temporal hierarchy with which these sites are processed by the enzyme (Fig. 2). Several lines of evidence such as insensitivity to dilution, lack of detectable NS3-NS4A precursors and failure to observe transcleavage at this site suggested that processing at the NS3-NS4A boundary is an intramolecular reaction, which precedes all other cleavages. The remaining cleavage sites were found to be processed in an intermolecular fashion following the preferred order NS5A-NS5B>NS4A-NS4B>NS4B-NS5A (Fig. 2) (Bartenschlager et al., 1994; Failla et al., 1995; kin et al., 1994b; Tanji et al., 1994a). Based on the known structures of trypsinlike serine proteases as well as on the conservation pattern of protease sequences among different HCV strains, a homology model of the specificity pocket of the NS3 protease was built (Pizzi et al., 1994). According to this model, the presence of a phenylalanine side-chain in the pocket was predicted to determine the requirement for small, hydrophobic residues in the P1 positions of NS3 substrates, with a preference for cysteine residues. Radiosequencing of the N-termini of NS3 cleavage products has confirmed this

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FIGURE 2

Kinetics of HCV polyprotein processing by the NS3 serine protease.

prediction (Grakoui et al., 1993b; Pizzi et al., 1994), yielding the NS3 substrate consensus sequence D/E-X-X-X-X-Cys/Thr~Ser/Ala (Table I). Cleavage was demonstrated to occur as predicted after a cysteine residue in all trans cleavage sites, whereas the intramolecular site between NS3 and NS4A was shown to differ from this consensus, having a threonine residue in its P 1 position. Other features are a conserved negatively charged residue in the P6 positions and a serine or alanine residue in the P 1' positions.

TABLE I

Sequences of the NS3-Dependent Cleavage Sites

Cleavage site NS3/4A (cis)

Sequence

Position

DLEVVT

STWV

1658

NS4A/4B

DEMEEC

ASHL

1706

NS4B/5A

DCSTPC

SGSW

1967

NS5A/5B

EDWCC

SMSY

2414

Consensus

D E

S A

C T

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Urbani et al.

A. THE N S 3 - N S 4 A INTERACTION Using a transient expression system, cleavage of an NS4B-NS5B precursor protein by the NS3 protease supplied in trans from another plasmid was found to occur only at the NS5A-NS5B site, whereas efficient cleavage at all sites was observed with a precursor harboring also the NS4A protein (Bartenschlager et al., 1994; Failla et al., 1994; Lin et al., 1994b; Tanji et al., 1995a). This observation led to the discovery that NS3 is necessary but not sufficient for the efficient processing of the HCV polyprotein and that the NS4A protein is required as a cofactor of the NS3 serine protease, the active protease being a heterodimer consisting of both NS3 and NS4A. The conclusion drawn from these findings was that NS4A is a functional analog of Flavivirus NS2B and Pestivirus p l0 proteins (Failla et al., 1994). Heterodimer formation in cells can be accomplished by providing NS4A either in cis or in trans and can be demonstrated by coimmunoprecipitation of the two polypeptides, suggesting the formation of a very tight complex (Hijikata et al., 1993a; Bartenschlager et al., 1995b; Failla et al., 1995; Lin et al., 1995; Satoh et al., 1995). This complex formation is absolutely required for the processing of the NS4B-NS5A and NS3-NS4A junctions, whereas cleavage at the NS5A-NS5B site could be observed also in the absence of the NS4A protein. Besides acting as a cofactor of the serine protease, NS4A has also been shown to target NS3 to membranes and to increase its metabolic stability (Tanji et al., 1995a). Deletion mutagenesis showed that the N-terminal 22 residues of NS3 are involved in cofactor binding (Failla et al., 1995; Satoh et al., 1995; Koch et al., 1996) N-terminal deletions in NS3 leading to both a decrease in cleavage efficiency of the NS4B-NS5A site and to an impairment in complex formation, as detected by immunoprecipitation experiments. A quite complicated picture concerning the generation of a mature NS3 N-terminus engaged in a complex with NS4A emerges from these observations. As outlined above, cleavage at the 2/3 site is mediated by the NS2-NS3 protease. It is at present not clear what the exact temporal sequence of these events is, but it is likely that processing at the 2/3 site precedes NS4A binding to the N-terminus of NS3. On the other hand, cleavage at the intramolecular NS3NS4A site was demonstrated to rely on the previous activation of the NS3 protease by its cofactor (Failla et al., 1994), implying that this processing event occurs posttranslationally after intramolecular complex formation between NS4 and the N-terminal region of NS3. The likely sequence of events therefore is: (1) cleavage of the 2/3 site and insertion of NS2 into the ER membrane, (2) intramolecular complex formation between NS4A and the mature Nterminus of NS3, and (3) intramolecular cleavage of the NS3-NS4A junction and generation of the activated, heterodimeric NS3-NS4A protease bound to the ER membrane. It remains to be determined whether interference with a

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FIGURE 3 Hydropathy plot of the NS4A protein. The amino acid sequence of NS4A and a secondary structure prediction are shown (B = ~-strand; H - helix; T = turn). The central region involved in NS3 activation is highlighted in the plot and shown in boldface in the sequence.

correct sequence of events might impair either 2/3 processing itself or a functional NS3-NS4A heterodimer formation. While NS3 constructs extending into NS2 were found to be activated by NS4A, implying correct binding of the cofactor to occur even without processing of the 2/3 site (Failla et al., 1994), D'Souza and co-workers (1994) reported that constructs deficient in NS2-NS3 protease activity also showed an impaired processing of the NS3-dependent cleavage sites that could be restored upon deletion of NS2 sequences. These latter findings suggest that a correct processing in this region may be a prerequisite for the activation of the NS3 protease by its cofactor. Further investigations are needed to define the role of efficient processing at the 2/3 site in the formation of the NS3-NS4A complex. The protein NS4A is composed of 54 residues. According to hydropathy plots and secondary structure algorithms it can be subdivided into three portions (Fig. 3): residues 1-34 are highly hydrophobic with the first 20 residues predicted to have a high propensity of forming an a-helix, whereas residues 2 0 - 3 4 are assumed to preferentially form fl-strands. The remaining 20 C-terminal residues of NS4A are hydrophilic with preference for adopting a helical conformation. Deletion mutagenesis experiments have shown that residues 2 0 - 3 4 corresponding to the extended, hydrophobic region of NS4A are sufficient for eliciting full activation of the NS3 protease (Lin et al., 1995; Shimizu et al., 1996; Tomei et al., 1996; Koch et al., 1996; Butkiewicz et al., 1996). Also peptides encompassing this region were shown to bind to the enzyme and to activate it (Lin et al., 1995; Shimizu et al., 1996; Steink~ihler et al., 1996a,b; Tomei et al., 1996; Bianchi et al., 1997). Many publications have reported deletion mutagenesis experiments of the NS3 protein that mapped the minimum protease domain to the - 2 0 0 N-terminal residues (Bartenschlager et al., 1994; Failla et al., 1995; Tanji et al.,

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1994b; Han et al., 1995; Lin et al., 1994b; SteinkCthler, 1996a,b). Several pieces of evidence furthermore suggested that both basal and NS4A-stimulated protease activity of a truncated protein encompassing this region of NS3 were indistinguishable from data obtained with a full-length 70-kDa NS3 protein. This has prompted many groups to adopt a "minimalist" approach, focusing on the characterization of a system composed of a 20-kDa NS3 protease domain and a synthetic 14-mer peptide as an NS4A cofactor mimic. Purification of the truncated enzyme using several different heterologous expression systems has been reported (Suzuki et al., 1995; Shoji et al., 1995; Mori et al., 1996; Steinki~hler et al., 1996a,b; Markland et al., 1997; Vishnuvardhan et al., 1997) and the effort has culminated in the crystallization of the free NS3 protease domain and the complex with a cofactor peptide (Kim et al., 1996; Love et al., 1996; Yan et al., 1998). Recently, the functional independence of protease and helicase domains has been questioned by a report by Morgenstern et al. (1997), who observed polynucleotide modulation of the protease activity of the full-length NS3-NS4A complex but not of the truncated NS3 protease domain in the presence of an NS4A peptide. These conclusions are based on rather indirect findings and should be substantiated by a more rigorous quantitative study using peptidic substrates. Such studies could be easily performed since several reports have now appeared on expression and purification procedures of both the full-length NS3 protein and of the NS3-NS4A complex (D'Souza et al., 1995; Kakiuchi et al., 1995; Hong et al., 1996; Hamarake et al., 1996; Gallinari et al., 1998; Sali et al., 1998). The native, noncovalent complex between NS3 and NS4A was shown to be formed by in vivo cleavage of an NS3-NS4A precursor during protein expression and to be stable during purification procedures. The topology and the three-dimensional structure of the NS3 protease domain complexed with an NS4A peptide are shown in Figs. 4 and 5. Analysis of the X-ray structure revealed that the NS3 protease folds in a canonical chymotrypsinlike fold, consisting of two j3barrel-like domains, each containing a Greek key motif (Fig. 4). The C-terminal domain contains a six-stranded ~-barrel common to most members of the chymotrypsin family of serine proteases, which ends with a structurally conserved helix. The residues of the catalytic triad (His1083, Asp1107, and Ser1165) are located in a groove at the interface of the two domains (Fig. 5, see color plate). The N-terminal domain contains eight j3-strands, including one strand contributed by NS4A (Kim et al., 1996, Yah et al., 1998). The central region of NS4A, spanning residues 21-34, is embedded into the core of the NS3 protease domain with a total of 2400A 2 of surface area buried by the interaction between the two molecules. The Nterminus of NS3 that interacts with this region of NS4A has a peculiar ~-cr-~ fold with the j3-strand contributed by the cofactor intercalating into this structure. In the structure without NS4A, this N-terminus interacts with neighboring molecules binding to hydrophobic surface patches (Love et al., 1996).

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FIGURE 4 Secondarystructure topology of the NS3 protease domain-NS4A peptide complex. The c~-helicesare marked as c~1-3;/t-strands are marked A0-F2. The strand contributed by the cofactor is shown in black. Greek key motifs are shaded. Numeration starts with residue 1026 of the HCV polyprotein.

These interactions are likely to be peculiar features of the crystallized enzyme and in solution the N-terminal region of NS3 is probably disordered in the absence of NS4A, thus providing an explanation for the enhanced metabolic stability of the NS3-NS4A complex with respect to the uncomplexed enzyme (Tanji et al., 1995a). Complex formation between the truncated NS3 protease and an NS4A peptide spanning residues 21-34 was shown to go along with changes in the protein near-UV CD spectrum and in its tryptophan fluorescence spectrum (Bianchi et al., 1997). These spectroscopic changes were interpreted in terms of changes in the environment of Trp 1111, which is engaged in the interaction with Va123 of the cofactor peptide and have allowed calculation of complex dissociation constants in the low micromolar range and a complex half-life of 3.5 min (Bianchi et al., 1997). Therefore, the complex with the peptide analog of NS4A appears to be significantly less stable than what can be extrapolated for the native complex with the full-length cofactor. Systematic deletion experiments in transfected cells showed that, while the central domain of NS4A

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is important in the activation of the protease, truncations affecting the N-terminal hydrophobic sequence of NS4A impair the coimmunoprecipitation of NS3 and N-S4A (Bartenschlager et al., 1995b; Lin et al., 1995; Tanji et al., 1995a; Koch et al., 1996). In the light of these findings either "loose" or "tight" complexes can be formed, depending on the integrity of the N-terminus of NS4A. Since this region of NS4A is likely to be responsible for the membrane anchoring of NS3-NS4A complexes, tight complex formation probably involves membrane association. This process may serve other functions than protease activation alone, such as membrane targeting of the helicase and subsequent formation of a membrane-associated replication complex. The proximity, shown by the X-ray crystal structure, of the N-terminus of the protease domain to the C-terminus of the activator peptide has suggested that both molecules may be linked within a single polypeptide chain (Pasquo et al., 1998; Taremi et al., 1998; Dimasi et al., 1998). The single-chain proteases were shown to be constitutively activated molecules and indistinguishable in all enzymatic and physicochemical properties from the NS3 protease complexed with its cofactor peptide when analyzed under identical conditions. The single-chain design has thus led to a tightening of the NS3-cofactor peptide interaction even in the absence of the N-terminal portion of NS4A. Despite the wealth of structural information now available relatively little is known about the mechanism by which NS4A activates the enzymatic functions of the NS3 protease. The NS3 protease is active also in the absence of its cofactor, albeit with greatly reduced catalytic efficiency. Active site titrations performed in the absence of cofactor yielded 94% of catalytically active enzyme molecules (Urbani et al., 1997), indicating the presence of a homogeneous enzyme population with reduced specific activity. In the crystal structure obtained in the absence of the NS4A peptide the catalytic Aspll07, which is expected to provide charge stabilization to His1083 after deprotonation of Serl165, is oriented away from His1083, and forms an ion pair with Argll81 (Love et al., 1996). Instead, a correct orientation of Asp1107 is observed in the structure obtained in the presence of NS4A (Kim et al., 1996). Nevertheless, relatively minor readjustments would suffice to correctly position the sidechain of Asp1107 in the absence of the cofactor, suggesting that also in this case a classic catalytic triad configuration will probably exist during catalysis (Love et al., 1996). This hypothesis is in line with the finding that complex formation with NS4A does not alter the pK value of His1083, as determined both by activity titration and by NMR (Landro et al., 1997; Urbani et al., 1998). The kinetic consequences of complex formation depend on both the assay conditions and the substrate peptide used. Thus, either an enhancement of kcat alone or a simultaneous decrease in Km and a concomitant increase in kcat values are both observed (Shimizu et al., 1996; Steink~ihler et al., 1996a,b; Bianchi et al., 1997; Landro et al., 1997; Urbani et al., 1997). These findings are

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suggestive of an activation of the catalytic machinery and of altered binding modes experienced by the substrate in the NS3-NS4A complex. In a recent steady-state kinetic analysis, Landro and co-workers (1997) propose a kinetic scheme according to which an ordered, sequential binding of NS4A and substrate occur. They further reported evidence for the influence of NS4A on binding of inhibitors to prime-side subsites, especially to $1' and $4', suggesting that the correct formation of these subsites is influenced by the cofactor. This is in line with the crystal structure data, which show that NS4A binding occurs in the N-terminal ~-barrel, which is predicted to accommodate the prime-side residues of the substrate. It is still not clear whether interference with NS4A binding is a viable strategy for the development of protease inhibitors. Although the contact surface between the two molecules is quite extended, Kim et al. (1996), reasoning that there are unique, highly conserved features in this interaction, suggested that small, predominantly hydrophobic molecules may be designed that could compete with NS4A binding and activation.

B. SUBSTRATE SPECIFICITY OF THE NS3 PROTEASE The NS3 cleavage sites have the consensus sequence D/E-X-X-X-X-Cys/ThrSer/Ala (Table I), with cleavage occurring after cysteine in all trans cleavage sites (i.e., NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B), or after threonine in the intramolecular cleavage site between NS3 and NS4A. Preference for cysteine residues in the P1 positions of NS3 substrates was rationalized on the basis of the peculiar structure of the S1 pocket of the enzyme. This pocket is hydrophobic and shallow, being occluded by the aromatic ring of Phe1180 (Pizzi et al., 1994; Kim et al., 1996; Love et al., 1996; Yan et al., 1997). Furthermore, the sulfhydryl group of cysteine has been shown to engage in favorable interactions with the aromatic ring system of phenylalanine. Mutagenesis of residues flanking the S1 pocket was used to engineer proteases with altered substrate specificities (Failla et al., 1996; Koch and Bartenschlager, 1997), confirming the role of Phe1180 and a minor importance of Ala1183 in determining the selectivity of the NS3 protease for substrates harboring cysteine as a P 1 residue. It is noteworthy that threonine, the residue found in the P1 position of the intramolecular cleavage site between NS3 and NS4A, cannot be optimally fitted into the S1 pocket and was shown to decrease cleavage efficiency when introduced into the P1 positions of the other cleavage sites (Bartenschlager et al., 1995a; Kolykhalov et al., 1994; Komoda et al., 1994b). Komoda and co-workers (1994b) were able to demonstrate trans cleavage of a chimeric protein harboring the NS3/NS4A cleavage site expressed in Escherichia coli when the wildtype threonine residue was replaced by cysteine, suggesting that threonine is

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suboptimal also in the context of the NS3-NS4A junction. Using transient expression in eukaryotic cells or in vitro translation of an NS2-NS4 precursor, the NS3-NS4A cleavage was shown to be remarkably tolerant to mutations in the consensus sequence (Bartenschlager et al., 1995a; Leinbach et al., 1994; Kolykhalov et al., 1994). This has led to the conclusion that processing at the cis cleavage site is determined primarily by polypi'otein folding (Bartenschlager et al., 1995a). In any case, the reason for the counterselection of an optimal P 1 residue in the NS3-NS4A junction is still not understood. Susceptibility to mutations of the trans cleavage sites depends on the sequence context with a gradient of increasing sensitivity following the order of NS4A-NS4BNS4A-NS4B>NS4B-NS5a), which was taken as evidence for primary structure being a major determinant for trans cleavage efficiency (Landro et al., 1997; Steinkahler et al., 1996b). Also, the extent of cleavage activation by NS4A, determined using peptide substrates, paralleled the hierarchy observed during polyprotein processing with the NS4B-NS5A junction being absolutely dependent on NS4A and the NS5ANS5B site being efficiently processed also in the absence of the cofactor. Catalytic efficiencies, expressed as kcat/Km values, obtained with peptide substrates are rather low and vary between 40 and 20,000 M -1 sec -1, depending on the substrate sequence. These values can be improved using ester substrates in which the P1 cysteine residue was replaced by 2-amino-butyric acid (Bianchi et al., 1996). In particular, incorporation of an ester linkage as scissile bond in the context of peptides harboring P' residues yielded very efficient substrates. This principle was used to generate internally quenched depsipeptide sub-

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strates with kcat/Kmvalues of 345,000 M -1 sec -1 that permit continuous monitoring of enzymatic activity (Taliani et al., 1996). Alanine scanning experiments performed on peptide substrates based either on the NS4A-NS4B (Urbani et al., 1997) or on the NS5A-NS5B (Zhang et al., 1997) cleavage sites came to the conclusion that besides P1 also P6, P3, and P4' residues contribute to efficient substrate recognition by the NS3 protease. In the context of peptide substrates, a more pronounced sensitivity of the NS3 protease to P 1 substitutions was observed than using polyprotein substrates in transient transfection experiments (Landro et al., 1997; Urbani et al., 1997; Zhang et al., 1997). The P1 substitutions in the context of an NS4A-NS4Bbased substrate gave the following order of decreasing efficiencies: cysteine > homocysteine > allyglycine >2-amino-butyric acid > threonine > norvaline > valine. Serine, alanine, glycine, or leucine in the P 1 position yielded uncleavable substrates (Urbani et al., 1997). Similar results were obtained introducing modifications in the P1 position of substrates based on the sequence of the NS5A-NS5B junction (Landro et al., 1997; Zhang et al., 1997). These findings have to be compared with the results from mutagenesis experiments of the P 1 residues in the polyprotein NS4A-NS4B junction, where only Arg and Asp resulted in complete abolishment of cleavage (Kolykhalov et al., 1994), while efficient processing was still observed with residues such as glycine, serine, or leucine that were incompatible with peptide cleavage. These differences may reflect an increased activity of the NS3 protease on polyprotein substrates that render it less discriminative against suboptimal P1 residues. In peptide substrates, all cysteine substitutes so far reported were found to decrease cleavage efficiency by at least one order of magnitude, suggesting that it will be difficult to eliminate the P 1 cysteine residue in peptide-based inhibitors ofNS3 protease without incurring a substantial loss in potency. It was concluded that ground-state binding of peptide substrates to the NS3 protease is mediated by multiple, weak interactions involving distal residues spanning at least from P6 through P4', whereas the efficiency with which the bound substrate will proceed through the transition state is strongly influenced by the nature of the residue in the P1 position (Urbani et al., 1997). This requirement for relatively long peptide substrates and the apparent lack of strong specific interactions with distal subsites can be rationalized by analyzing the structure of the enzyme. Apart from the P 1 pocket, the surface of the enzyme involved in substrate recognition is relatively flat and featureless. All major loops, that in other serine proteases contact the P2, P3, and P4 moieties of substrates, are absent in NS3 (Kim et al., 1996; Love et al., 1996; Yan et al., 1998). Two positively charged residues, Arg1187 and Lys1191, have been invoked to engage interactions with the conserved negatively charged residue in the P6 position of NS3 substrates (Love et al., 1996). Possibly, substrates are anchored to the active site via the side-chain of the charged P6 residue and

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through main-chain interactions between P5 and P2, with P'-site residues, especially P 1' and P4', also contributing to ground-state binding (Landro et al., 1997). This peculiar substrate recognition mechanism, requiring an extended interaction network, renders the development of low-molecular-weight inhibitors of the NS3 protease a formidable challenge.

C. THE METAL BINDING SITE OF THE N S 3 PROTEASE A homology model of the NS3 protease domain based on the known structures of serine proteases and on the sequence similarities between HCV isolates and the recently described HCV-related viruses GBV-A, GBV-B, and HGV (Simons et al., 1995; Muerhoff et al., 1995; Ohba et al., 1996) first predicted the presence of a metal binding site in the NS3 protease (De Francesco et al., 1996). This prediction was made on the basis of a clustering in space of three cysteines and one histidine residue in the model and was subsequently demonstrated by atomic absorption spectroscopy of the native enzyme and electronic spectroscopy of the Co(II) and Cd(II) derivatives of the NS3 protease (De Francesco et al., 1996; Stempniak et al., 1997). Zinc was shown to be essential for the structural integrity of the protein, its removal leading to unfolding and aggregation of the enzyme. Mutagenesis experiments showed that mutations affecting any of the three cysteine residues predicted to be involved in metal chelation resulted in an impaired NS3 protease activity (Hijikata et al., 1993b). Later, X-ray crystallography confirmed the assignment of the metal ligands predicted by the model. The zinc ion is in fact tetrahedrally coordinated by Cys1123, Cys1125, Cys1171, and through a water molecule by His1175, which shields the metal from bulk solvent (Fig. 6) (Kim et al., 1996; Love et al., 1996; Yan et al., 1998). The indirect coordination role of His1175 is consistent with the relatively weak effects of mutations in this position. A recent NMR study suggested that not water itself, but rather an OH- is the bridging ligand between the metal and His1175 (Urbani et al., 1998). This study also showed that His1175 is endowed with a significant flexibility, transiently exposing the metal to bulk solvent in a pH-dependent fashion. This flexibility is also evident from the analysis of the crystal structures: in the absence of NS4A, coordination by His1175 is observed only in two of the three monomers in the asymmetric unit. In the third monomer, His1175 is 4 A away from the zinc and does not participate in metal chelation. In contrast, a homogeneous situation is observed in the presence of NS4A, with His1175 always pointing toward the metal in both published crystal structures (Kim et al., 1996; Yan et al., 1998). A spectroscopic study using a Co(II)-substituted protein found that NS4A binding is indeed accompanied by changes in the coordination geometry of the metal binding site

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FIGURE 6 Three-dimensionalstructure of the zinc binding site of the NS3 protease.

(Urbani et al., 1998). Interestingly, changes in the coordination geometry were also observed with small, anionic ligands and these changes went along with a modest enhancement of the enzymatic activity of the NS3 protease (Urbani et al., 1998). The results are suggestive of an influence of the coordination geometry of the metal binding site on the conformation of the active site. A possible explanation for these findings resides in the topological location of the zinc ion in the NS3 protease. The metal ligating residues are located in a long loop connecting the two fl-barrel domains of the protease and in a short hairpin loop in the second domain. Since the residues of the catalytic triad are distributed between the two domains, the relative orientation of the two ~-barrels is expected to affect enzymatic activity. Extracellular serine proteases often contain disulfide bridges that are believed to serve the purpose of stabilizing the relative positions of the two domains. Since disulfide bridges are not stable in the reducing intracellular milieu the metal binding site of the NS3 protease might serve as a reductant-stable surrogate of an S-S bridge. In this context, it is interesting to note that in picornavirus 3C proteases, which have neither disulfides nor a structural zinc, stability in this region may be provided by the N-terminal c~-helix that packs against the interbarrel loop (Love et al., 1996).

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In contrast, picornavirus 2A proteases contain two sequence motifs, Cys-XCys and Cys-X-His, that are located in a region which is topologically similar to the one harboring the metal binding site of HCV NS3 protease. Picornavirus 2A protease has indeed been found to rely on zinc for the formation of a native structure (Voss et al., 1995). This is even more remarkable since 2A proteases belong structurally to the serine protease family but utilize cysteine as nucleophile, implying that the metal binding site is more conserved than the catalytic site.

D.

INHIBITORS OF N S 3 PROTEASE

Since the definition of the role of the NS3 protease in HCV polyprotein processing a considerable effort has been devoted in the development of expression systems, purification protocols, and in vitro activity assays for this enzyme. These efforts have proven successful despite the poor solubility and low catalytic efficiency of the enzyme and the intrinsic difficulties in assay design arising from the necessity to supply a cofactor in order to get a reasonable substrate turnover. Inhibition studies have shown that NS3 is only modestly inactivated by classic serine protease inhibitors such as chloromethylketones or phenylmethyl sylfonylfluoride (Bouffard et al., 1995; Hahm et al., 1995; Lin and Rice, 1995; Steink~ihler et al., 1996a; Sudo et al., 1996; Shoji et al., 1995; Mori et al., 1996; Markland et al., 1997). It is, however, very sensitive to inhibition by copper ions (Han et al., 1995; Stempniak et al., 1997), which might inactivate the enzyme by catalyzing the oxidation of exposed cysteine residues. The assay systems that have been developed are presently used to screen for selective NS3 protease inhibitors. Some reports on the identification of such compounds have appeared in the literature and are briefly reviewed below. 1. Macromolecular Inhibitors Quite a few reports about macromolecular inhibitors of NS3 protease activity have appeared. Using phage display technology, Martin and co-workers (1997) report the affinity selection of a "camelized" variable domain antibody fragment that inhibited NS3 protease in a competitive way (Ki -- 150 nM). The same strategy was used in the search for inhibitors by sampling repertoires of a minimized antibodylike molecule ("minibody") and of human pancreatic secretory trypsin inhibitor (HPSTI) (Dimasi et al., 1997). A low-micromolar, noncompetitive minibody inhibitor and a competitive HPSTI NS3 protease inhibitor (Ki = 360 nM) were selected. A design approach using an NS3-directed macromolecular inhibitor has been recently reported by Martin et al. (1998). In this approach the active site binding loop of eglin C was engineered to in-

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corporate NS3 P5-P4' consensus residues without affecting the integrity of the eglin scaffold. Competitive inhibitors with potencies in the low nanomolar range were obtained. The interaction between NS3 and the designed eglin inhibitors was characterized in detail and compared to the inhibition of other serine proteases by the parent eglin C molecule. The complexes of a series of serine proteases with wild-type eglin C are characterized by slow off-rates and long half-lives. In contrast, inhibition of NS3 by designed eglin inhibitors was characterized by very fast association and dissociation processes, possibly reflecting the peculiar, poorly structured substrate recognition region of the HCV serine protease. Using a different approach, Kumar and co-workers (1997) selected from a pool of random RNA molecules (aptamers) that inhibited the NS3 protease in a competitive way (K~ = 3/.LM). All these macromolecular inhibitors may prove useful as tools for cocrystallization with the enzyme and as a starting point for structure-based drug design. 2. Nonpeptide Inhibitors Chu and co-workers (1996) report the isolation of a phenanthrenequinone during a natural product screen which inhibited the protease activity in an in vitro translation assay. A series of thiazolidine, benzamide, and benzanilide derivatives were identiffed in an HPLC-based assay using an NS3-NS4A complex fused to maltose binding protein at the N-terminus of NS3 and a peptide substrate based on the sequence of the NS5A-NS5B cleavage site (Sudo et al., 1997a,b; Kakiuchi et al., 1998). The compounds were found to inhibit the protease in a noncompetitive manner and showed poor selectivity. 3. Peptide Inhibitors Landro and co-workers (1997) first reported on the development of peptidebased inhibitors of the NS3 protease. A competitive hexapeptide aldehyde, having the sequence EDVVAbuV-CHO, inhibited the NS3 protease in an NS4Aindependent fashion (Ki = 50/xM). In their substrate specificity studies, Landro et al. (1997) have identified PI' substitutions such as proline, tetrahydroisoquinoline-3-carboxylic acid, or pipecolinic acid that abolished cleavage but retained a high affinity of the corresponding peptides for the protease. The decapeptide EDWLCTicNleSY was a competitive inhibitor of the protease with Ki = 340 nM in the presence of NS4A and K~ = 28/xM in the absence of the cofactor, suggesting that NS4A influences binding of inhibitors extending into the P' side, but does not affect the affinity of ligands that are confined to the P side. Two groups have reported that the NS3 protease undergoes significant

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inhibition by its N-terminal hexamer cleavage products (SteinkOhler et al., 1998; Llinas-Brunet et al., 1998a). The Ki values of the hexamer products arising from hydrolysis of the NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B junctions turned out to be up to 1 order of magnitude lower than the K,, values of the corresponding substrate peptides. The physiological significance of this pronounced product inhibition is unknown at present. Interestingly, the Nterminal hexapeptide product of the intramolecular cleavage site between NS3 and NS4A was shown not to inhibit the protease up to a concentration of 500 /xM. Since this is the only cleavage site containing a suboptimal P1 threonine residue it has been argued that the counterselection of an optimal P 1 residue at this junction has occurred in order to avoid premature inhibition of the enzyme by the first intramolecular cleavage product. Based on the observation of product inhibition, two groups (Ingallinella et al., 1998; Llinas-Brunet et al., 1998a,b) have sequentially optimized the sequences of the natural hexapeptide products to set up structure-activity relationships. These studies have shown that the main contribution to the binding energy derives from the P1 amino acid through both its side-chain and its ce-carboxylic acid function. Molecular modeling, pH-dependence studies, and site-directed mutagenesis suggested that the P1 a-carboxylic acid is anchored in the active site via interactions with the protonated E-N of the catalytic His1083, the backbone amide groups of Ser1165 and Gly1163 (forming the oxyanion hole of the enzyme) and with the ~/-N of the conserved Lys1162 (SteinkOhler et al., 1998). Deletion of the acidic residue in the P6 position led to a > 10-fold drop in potency, whereas an acidic residue is not strictly required in the P5 position. In this position amino acids with D chirality are also well accepted and even preferred. In the P4 position a preference for large, hydrophobic residues was found, whereas either Glu or the hydrophobic Val or teu were best accepted in P3. Both negatively charged and hydrophobic residues were favored in the P2 position. A fully optimized hexapeptide that inhibited the protease with IC~o = 1.5 nM was reported (Ingallinella et al., 1998). Llinas-Brunet and co-workers (1998b) have explored the possibility of substituting the P1 ce-carboxylic acid of hexapeptide inhibitors with activated carbonyl groups. This was done in the context of hexapeptides containing a suboptimal but chemically inert P 1 norvaline residue. Trifluoromethylketones, pentafluoroethylketones, and ce-ketoamides were synthesized and showed only a modest improvement in potency with respect to peptide acids. The best compound, a hexapeptide ce-ketoamide (IC~0 = 0.64/zM), showed poor selectivity. Whereas replacement of the P 1 ce-carboxylic acid with a primary amide resulted in a complete loss of activity, methyl- and benzylamides showed inhibition in the low micromolar range. However, these compounds were equally potent against different serine proteases.

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V. C O N C L U S I O N S Only 4 years elapsed between the first description of the enzymatic activity of the NS3 protease in transfected cells and the determination of its threedimensional structure by X-ray crystallography. On the way from the characterization of the cleavage products by immunoprecipitation to the atomic resolution of the active site of the enzyme a great deal of information has also been gained on functional aspects of substrate recognition and on the catalytic mechanism. The news was often rather sobering: the requirement of extended interactions spanning at least 10 residues in the substrate, the need for a cysteine as optimum P1 residue and the absence of P2, P3, and P4 binding loops make the development of potent and selective NS3 protease inhibitors a challenging project. The same effort devoted by the scientific community to unravel the biology and enzymology of the NS3 protease will have to converge into the discovery of inhibitors. If this can be accomplished, one should be optimistic about the fact that efficacious anti-HCV therapeutics will be developed, with the ultimate goal to cure a potentially life-threatening disease that affects 100 million individuals worldwide.

ACKNOWLEDGMENTS We thank Youwei Yan, Sanjeev Munshi, Zhongguo Chen, and Lawrence Kuo for letting us use the coordinates of the NS3-NS4A complex prior to publication. Special thanks to Giovanni Migliaccio and Licia Tomei for helpful discussions and critical reading of the manuscript and to Uwe Koch for the preparation of Figs. 5 and 6.

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Human Herpesvirus Proteases XIAYANG QIU AND SHERIN S. ABDEL-MEGUID Department of Structural Biology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406

I. II. III. IV. V. VI. VII. VIII. IX.

Introduction Role of the Protease in the Virus Life Cycle Primary Structures Enzymatic Activity Three-Dimensional Structures Substrate Specificity Mechanism of Action Inhibitors Ligand Binding References

I. I N T R O D U C T I O N Human herpesviruses are responsible for a variety of diseases from subclinical infections to fatal diseases in the immunocompromised and immunosuppressed; clinical manifestations of the diseases they cause are very different (Table I). They are divided into three subfamilies designated or,/3, and 3' (Table I). The a-subfamily includes herpes simplex viruses types I and 2 (HSV-1 and HSV-2) and varicella-zoster virus (VZV); the/3 group includes cytomegalovirus (CMV) and human herpesviruses 6 and 7 (HHV-6 and HHV-7); and the 5' group includes Epstein-Barr virus (EBV) and human herpesvirus 8 (HHV-8) also known as Kaposi's sarcoma-associated herpesvirus. These viruses vary greatly in their biological properties and remain latent in a specific set of cells (Table I). The a-subfamily replicates relatively fast, destroys infected cells efficiently, and establishes latency primarily in sensory ganglia (thus called neurotropic). The/3-subfamily replicates slowly, often causes enlargement of the infected cells, and can be latent in secretory glands, lymphoreticular cells, and other Proteases of Infectious Agents Copyright 9 1999 by Academic Press. All rights of reproduction in any form reserved.

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tissues. The y-subfamily is specific for either B- or T-lymphocytes (lymphotropic) and exists at either a latent or lytic stage but does not produce infectious progeny. Viruses of the a-subfamily are among those causing serious diseases. The herpes simplex viruses were the first of the human herpesviruses to be discovered and are among the most extensively investigated of all viruses. Herpes simplex virus type 1 is the virus responsible mainly for herpes labialis (cold sores), while HSV-2 causes genital herpes. The latter disease is of increasing public health importance. The recurrent nature of the infection, its differing clinical manifestations, and complications such as aseptic meningitis and neonatal infection, are of great concern to patients and health care providers. Varicella-zoster virus is responsible for chickenpox, shingles, and postherpetic neuralgia. Primary exposure to VZV results in chickenpox, reactivation of the virus following a period of latency gives rise to shingles, and postherpetic neuralgia is probably the result of nerve damage during the active replication phase of shingles. Cytomegalovirus is a ubiquitous opportunistic pathogen that can result in life-threatening infections in congenitally infected infants, immunocompromised individuals, and immunosuppressed transplant patients. Both HHV-6 and HHV-7 have been associated with the childhood diseases such as roseola. Epstein-Barr virus could cause mononucleosis, and HHV-8 has recently been linked to the development of Kaposi's sarcoma.

II. R O L E OF THE P R O T E A S E IN T H E V I R U S LIFE C Y C L E Herpesviruses are enveloped double-stranded DNA viruses that share a common pathway of assembly. The DNA is packaged into an icosahedral capsid in the nucleus of infected cells. The icosahedral capsid is surrounded by an amorphous material referred to as the tegument and enclosed within a lipid envelope of cellular origin that is acquired while the virus buds from the infected cell. Packaging of the viral DNA requires processing of an assembly protein precursor designated ICP35 in HSV-1. The processed ICP35 appears to form an inner scaffold that supports the proper assembly of the capsid. It is found in the capsid prior to DNA packaging, but is absent in the mature virions. The precursor ICP35 is processed through removal of 25 amino acid residues from its C-terminus by a virally encoded 635-amino-acid serine protease that contains the assembly protein at its C-terminus (Fig. 1). This site of cleavage is known as the maturation (M) site. The protease is also capable of catalyzing its own cleavage at the release (R) site to produce a 247-residue N-terminal domain that has full catalytic activity (Fig. 1).

Human Herpesvirus Proteases

97

FIGURE 1 HSV-1protease (UL26gene product) and its substrate (UL26.5geneproduct). Maturation (M-site) and release (R-site) cleavagesare indicated.

Liu and Roizman (1991) and Welch et al. (1991) were the first to report the identification of these serine proteases from herpesviruses; the former identified the protease from HSV-1 and the latter from CMV. These two enzymes are the most studied of all human herpesvirus proteases. Gao et al. (1994) showed, using a null mutant virus, that the HSV-1 protease is essential for capsid formation and production of infectious virus, making it an attractive target for therapeutic intervention. This and other studies showing that herpesvirus proteases are essential for the virus life cycle have been summarized in recent reviews by Holwerda (1997) and Gibson and Hall (1997).

III. P R I M A R Y S T R U C T U R E S The full-length proteases of the various human herpesviruses range from 512 amino acids in HHV-7 to 708 amino acids in CMV. The N-terminal (catalytic) domains range from 226 amino acids in HHV-7 to 256 amino acids in CMV, indicating that most of the variability in the size of these proteases is in the C-terminal (assembly protein) portion. The catalytic domains show significant sequence homology within each subfamily of herpesvirus, but only limited homology between the different subfamilies (Fig. 2 and Table II). For example, the amino acid sequence of the HSV-1 protease catalytic domain is 91 and 50% identical to that of HSV-2 and VZV, respectively, while it is only 26% identical to that of CMV protease. Extensive homology searches against all known sequence databases revealed little homology to any other protein. Liu and Roizman (1992) showed that the HSV-1 protease was inhibited by

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H u m a n Herpesvirus Proteases TABLE II

Amino Acid Sequence Identities between H u m a n Herpesvirus Proteases a

(%)

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HSV-2

VZV

CMV

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HHV-8

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some serine protease inhibitors, but not by inhibitors of metallo-, acidic, or thiol proteases. This was surprising, given the absence in herpesvirus proteases of the conserved G-X-S/C-G-G motif for chymotrypsinlike and G-T-S-M/A for subtilisinlike proteases, and an early clue that these enzymes could be a novel class of serine proteases.

IV. E N Z Y M A T I C A C T I V I T Y All human herpesvirus proteases cleave a peptide bond between an alanine and a serine, with CMV protease being the most extensively characterized in terms of its enzymatic activity (Burck et al., 1994; Pinko et al., 1995; Liang et al., 1998). The purified wild-type CMV protease shows a clip between Ala143 and Ala144. This site of cleavage is referred to as the internal (I) site. The CMV protease is the only member of the family known to show this I-site clip; all others have been purified as a single chain. A number of mutations to ablate cleavage at the I-site have been engineered (Pinko et al., 1995; Qiu et al., 1996), with the resulting single-site mutant having nearly identical catalytic activity as the wild-type (clipped) enzyme (Table III). This suggests that the immediate vicinity of the I-site is involved neither in catalysis nor substrate binding, as was confirmed later from the crystal structure of CMV protease (the I-site being part of a disordered loop on the surface of the enzyme). The CMV protease differs in its catalytic activity toward the R- and M-sites. The turnover rate of the M-site (GVVNA$SCRLA) cleavage is an order of magnitude faster than that of the R-site (SYVKA$SVSPE), while having similar Km values. Moreover, the hydrolysis of the R-site peptide has an optimal pH of about 7, while the hydrolysis of the M-site peptide has a biphasic pH dependence with optima of approximately 7 and 9, probably due to the protonation

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Human Herpesvirus Proteases

101

of the arginines and the lysine in the peptide substrate (Burck et al., 1994). Unlike CMV protease, HSV-1 protease does not have a preference for the Mover the R-site. Its pH optimal is approximately 8.0, and it is about 10 times slower than CMV protease (DiIanni et al., 1993; Darke et al., 1994; Hall and Darke, 1995). The catalytic activity of the herpesvirus proteases is influenced significantly by the nature and concentration of cosolvents. For CMV protease, the maximal specific activity is increased by about 10-fold in the presence of 30% glycerol (Margosiak et al., 1996). Similar enhancement in catalytic activity has also been reported for the HSV-1 protease (Hall and Darke, 1995). The most dramatic cosolvent effect is observed with sodium citrate. The CMV protease catalytic efficiency increases by 290-fold (kcat/Kmof 1.24 min-1/zM -1) in the presence of 0.8 M citrate over that in the absence of the cosolvent (0.0044 min-1/zM-~). Similarly, HSV-1 protease has a kcaJKm of 0.25 min-1/zM -~ (kca~4 min -~, K~ 16/zM) in 0.8 M citrate, compared to the k c J K ~ value of 0.0003 min-1/zM -1 in the absence of citrate, an increase of 800-fold in catalytic efficiency. The cosolvent effects led to the discovery that both CMV and HSV-1 proteases are active as homodimers (Margosiak et al., 1996; Schmidt and Darke, 1997). For CMV protease, the dissociation constant is 6.6 or 0.55/zM in 10 or 20% of glycerol, respectively (Darke et al., 1996). For HSV-1 protease, they are 0.96 or 0.23/zM in 20% glycerol and 0.2 or 0.5 M citrate, respectively (Schmidt and Darke, 1997). The dimer association is rather weak, probably an artifact of working with only the catalytic domain. The cosolvent effects on herpesvirus protease activity are likely due to their ability to enhance dimerization and stabilize the conformation of active dimers. Another important observation is the apparent low catalytic efficiency of herpesvirus protease when peptide substrates were used (Table III). Using authentic M-site derived peptides in 50% glycerol (Burck et al., 1994), CMV protease catalytic efficiency is about 104 worse than that of chymotrypsin and about 30 times worse than that of HIV protease. The apparent low efficiencies of the herpesvirus proteases may be an important property of their biological functions in the well-orchestrated events during capsid maturation (Babe and Craik, 1997). Although data support the view that herpesvirus proteases are less active than other viral proteases, this may just be an artifact of working with only the catalytic domains. In fact, when the assembly protein is used as substrate, the K~ is enhanced by over 100-fold (with similar kcat) e v e n in the absence of glycerol (Burke et al., 1994). It is also known that HSV-1 protease, when coexpressed with ICP35, exhibits greater catalytic efficiency (Deckman et al., 1992). This suggests that substrate binding may involve interactions beyond the catalytic domain, i.e., with the C-terminal domain in the full-length enzyme.

102

XiayangQiu and Sherin S. Abdel-Meguid

V. THREE-DIMENSIONAL STRUCTURES A. NOVEL FOLD The crystal structure of CMV protease has been determined by several groups (Qiu et al., 1996; Shieh et al., 1996; Tong et al., 1996; Chen et al., 1996), followed by those of VZV, HSV-1, and HSV-2 proteases (Qiu et al., 1997; Hoog et al., 1997). Unlike the structures of classic serine proteases having two distinct fl-barrel domains, the CMV protease is a single-domain protein. Its overall fold can be described as a seven-stranded orthogonally packed fl-barrel core surrounded by seven to eight c~-helices and connecting loops (Fig. 3, see color plate). The core/~-barrel is mostly antiparallel, except for strands B2 and B6, which are parallel. To our knowledge, the herpesvirus proteases three-dimensional fold has not been observed in any other protein. The overall fold of the four herpesvirus proteases with known threedimensional structures is very similar. As expected, the structures of the three a-herpesvirus (VZV, HSV-1, and HSV-2) proteases are nearly identical, and despite limited sequence identity (Table II), the overall fold of the a-herpesvirus proteases is similar to that of the fl-herpesvirus CMV protease (Qiu et al., 1997, Hoog et al., 1997). Superposition of the 197 pairs of equivalent a-carbon atoms of HSV-2 and VZV proteases (Fig. 4) gives an rms deviation of 0.9 A, while superposition of 142 pairs of those of VZV and CMV protease (Fig. 4) gives 1.3 A. The core fl-barrels of the VZV and CMV proteases superimpose even better (Fig. 4), with rms differences between the 52 pairs of c~-carbon atoms

FIGURE 4 Superimpositionof a-carbon atoms of VZV and CMVproteases (left), and VZV and HSV-2proteases (right). The VZVprotease structures are in light and thick lines and CMVprotease or HSV-2protease structure is in dark and thin lines.

HumanHerpesvirusProteases

103

being only 0.7 A. As anticipated, most of the significant structural differences between CMV and VZV protease are in the loops (Qiu et al., 1997). Moreover, an additional loop containing a small or-helix (referred to as the AA loop) has been observed in the VZV protease structure (Fig. 3) but not in the CMV protease. The corresponding segment in the CMV protease was either totally or partially disordered in apo-CMV protease structures.

B. NOVEL CATALYTIC TRIAD Unlike all previously known serine proteases having a catalytic triad comprised of a serine, a histidine, and an aspartic acid (Perona and Craik, 1995), the herpesvirus protease's catalytic triad consists of a serine and two histidines. The catalytic serine of HSV-1 protease was identified by DFP-modification experiments (DiIanni et al., 1994). Site-directed mutagenesis was able to determine the catalytic serine and histidine of cytomegalovirus protease (Welch et al., 1993), but failed to identify the third member of the catalytic triad. It was only after the determination of the crystal structure of the CMV protease (Qiu et al., 1996; Shieh et al., 1996; Tong et al., 1996; Chen et al., 1996) that the novel SerHis-His catalytic triad for the herpesvirus proteases was identified. In CMV protease, the catalytic triad residues are Ser132, His63, and His157. In this chapter, we use CMV protease numbering (Qiu et al., 1997; Fig. 2) to describe all herpesvirus protease residues. This should eliminate confusion and help to standardize numbering of catalytic triad residues as has been done with the chymotrypsin family of serine proteases. The active site of CMV protease is situated in the only region of the core /3-barrel that is not sheltered by helices and flanking loops (Fig. 3). The active site region is very shallow with the catalytic residues exposed to solvent. This shallowness is not unreasonable considering the P 1-P 1' residues (Ala-Ser) are small. Residues of the triad are absolutely conserved among all human herpesvirus proteases (Fig. 2) and superimpose almost perfectly on the Ser195His57-Asp102 catalytic triad of trypsin (Fig. 5; Qiu et al., 1996; Qiu et al., 1997; Hoog et al., 1997). When the second histidine (His157) is mutated to an alanine, the CMV protease is nearly inactive (Welch et al., 1993) and the HSV-1 protease is completely inactive (Liu and Roizman, 1992). The residual activity in CMV H157A mutant protease is reminiscent to that seen in classical serine proteases when the catalytic aspartate is mutated (Perona and Craik, 1995).

C.

DIMER INTERFACE

As indicated above, it has been shown that CMV and HSV-1 proteases are active as homodimers (Margosiak et al., 1996; Darke et al., 1996; Schmidt and Darke,

104

XiayangQiu and Sherin S. Abdel-Meguid

FIGURE 5 Stereoviewof the superimposition of catalytic triad residues of CMVprotease (light) and trypsin (dark).

1997). A dimer interface related by a twofold crystallographic axis has been first identified in the structure of CMV protease (Qiu et al., 1996; Fig. 6). The most distinct structural element in the CMV dimer interface is helix A6, with the two A6 helices almost parallel and A6 of each monomer interacting with helices A1, A2, A3, and A6 of the other (Qiu et al., 1996). Subsequently, similar dimer interfaces have also been found in the structures of cr-herpesvirus proteases (Qiu et al., 1997; Hoog et al., 1997; Fig. 6). The interface area between the two monomers of these proteases is about 1300 A 2. This is in general agreement with the reported (Margosiak et al., 1996; Darke et al., 1996; Schmidt and Darke, 1997) micromolar dissociation constants of herpesvirus proteases and suggests that the crystallographically observed dimer is indeed the biologically active dimer. Although the herpesvirus proteases are active as homodimers, each monomer has a well-defined active site containing all the residues necessary for catalytic activity. The two active sites are on opposite sides of the dimer (Fig. 6, see color plate). Since the dimer interface is distal to the catalytic triad (Fig. 6), dimerization must only influence enzymatic activity indirectly. We had speculated (Qiu et al., 1996) that this occurs by stabilization of the conformation of helix A6. This helix is part of a shallow groove running across the catalytic site. One side of the groove is relatively wide and deep. It is defined by the end of helix A6, the end of strand B6, His63, and the highly conserved Gly164Arg165-Arg166 segment. This side of the groove has been proposed (Qiu et al., 1997) to be the S' subsite. In the absence of dimer formation, helix A6, the core of the dimer interface, could move toward the active site to block substrate access, thus rendering the enzyme inactive or much less active. Although dimer formation appears to be a common feature for all herpesvirus proteases, there are notable differences between the dimer interfaces

105

Human Herpesvirus Proteases

of cr- and ~8-herpesvirus proteases. The two A6 helices of the dimer are almost parallel in the CMV protease structure but show an approximately 30 ~ twist in the VZV and HSV protease structures (Qiu et al., 1997; Hoog et al., 1997; Fig. 6). Helix A2 adopts a very different conformation in VZV protease comparing to that of CMV protease. It is interesting to note that the amino acid residues of helices at the dimer interface are less conserved than those of the /3-strands, with the least conserved being those of helix A2 (Fig. 2). These differences further support the notion that the dimer interfaces do not directly contribute to catalysis, but do it indirectly.

VI. S U B S T R A T E S P E C I F I C I T Y As indicated above, human herpes protease precursor molecules undergo autoproteolytic cleavage at two sites (R and M), and they all cleave between an alanine and a serine. Studies with both CMV (Sardana et al., 1994) and HSV-1 (DiIanni et al., 1993; McCann et al., 1994) proteases have identified the P 4 - P I ' residues of the M-site as highly conserved between the two proteases and as having sequence-specific interactions with the S and S' subsites of the protease. However, inspection of all sequences at the R- and M-sites (Table IV) shows a consensus of (V,L,I)-X-A$S, where X is a hydrophilic residue. This recognition sequence is unique among known serine proteases.

TABLE IV Recognition Sites of Several Human Herpesvirus Proteases HSV-1

GALVNA*S SAAHVDV HTYLQA*SEKFKMWG

M R

HSV-2

GALVNA*S SAAHVNV HTYLQA*SEKFKIWG

M R

VNAVEA*S

M

VZV

SKAPL

IQ

HVYLQA*STGYGLAR

R

CMV

AGVVNA*SCRLATAS ESYVKA*SVSPEARA

M R

HHV-6

PSI LNA*SLAPETVN CTY I KA*SEPPVE

I I

M R

HHV-7

PSVVNA*SLTPGQDR STY I KA*SENLTANN

M R

EBV

KKLVQA*SASGVAQS ESYLKA*SDAPDLQK

M R

HHV-8

SNRLEA*S SRS SPKS PVYLKA*SQFPAGIQ

M R

106

XiayangQiu and Sherin S. Abdel-Meguid

Despite sharing the same core M-site sequence (VNA$S), HSV-1 protease does not cleave at the CMV protease M-site; however, CMV protease does cleave at the corresponding HSV-1 site (Welch et al., 1995). Likewise, the EBV protease can process both EBV and CMV assembly proteins, but CMV protease cannot process the EBV assembly protein (Donaghy and Jupp, 1995). The high selectivity of the HSV-1 protease is consistent with studies using peptide substrates. The smallest peptide mimic of the CMV protease M-site that is cleaved by that protease is P4-P4 r (Sardana et al., 1994), whereas 13 residues from P5-P8 ~ are required for cleavage by HSV-1 protease (DiIanni, 1993). This is surprising given the high sequence homology of residues lining the active site cavity of the two enzymes (Hoog et al., 1997). Therefore, it was suggested that HSV-1 protease has a more extended substrate binding pocket and that differences in substrate specificity between the two enzymes result from differences in loop conformations around the active site cavity (Hoog et al., 1997). These loops show low sequence homology and are of differing lengths.

VII. M E C H A N I S M O F A C T I O N

A. CATALYTIC MECHANISM The herpesvirus protease structures suggest a novel Ser-His-His catalytic triad, while all other known serine proteases carry a Ser-His-Asp triad. There are two common models for the mechanism of serine proteases (Perona and Craik, 1995). In the "two-proton transfer" model, the negatively charged aspartic acid accepts a second proton to become uncharged during the transition state. In the second model, the most important role for the Asp is the groundstate stabilization of the required tautomer and rotamer of the catalytic histidine. In the crystal structures of herpesvirus protease, His63 forms hydrogen bonds with both Ser132 and His157 (Fig. 5). At the optimal pH (7 to 8) of these enzymes and with the two histidines mostly exposed to solvent, it is most probable that they are neutral. Thus, it is unlikely that in the transition state His157 would accept the complete transfer of the proton it shares with His63, unless it could transfer a proton to a negatively charged residue. On the other hand, His157 can assume the role of properly orienting His63 for catalysis. Therefore, the existence of an active Ser-His-His triad seems to support the second model. An aspartic acid (Asp65) was found in the CMV protease structure near His157, suggesting the possibility of a catalytic tetrad composed of Ser132, His63, His157, and Asp65 (Qiu et al., 1996). The likelihood of a catalytic tetrad was, however, diminished because Asp65 of the CMV protease is not conserved among all human herpes proteases; for example, it is a lysine in the VZV pro-

Human Herpesvirus Proteases

10 7

tease and an alanine in the HSV-1 and HSV-2 proteases (Fig. 2). Moreover, the activity of the D65A mutant CMV protease is reduced by only 35% (Liang et al., unpublished data). Another important element of serine protease catalysis is the existence of an oxyanion hole to stabilize the negatively charged oxygen of the substrate in the transition state. Overlays of the catalytic triad of any of the herpesvirus protease structures with that of trypsin suggested that the highly conserved Arg165 and Arg166 are involved in stabilization of the oxyanion intermediate. Such overlays resulted in superimposition of the Arg165 backbone atoms with those of Gly193 of trypsin. The latter is known to stabilize the oxyanion intermediate through a hydrogen bond with its backbone NH. The Ser195 of trypsin is also known to stabilize the enzyme active site oxyanion intermediate through a hydrogen bond with its backbone NH. The equivalent residue in the herpes proteases is absent. Instead, a water molecule (Fig. 7) held by the sidechain of Arg166 in the viral proteases was proposed to form a hydrogen bond with the oxyanion (Qiu et al., 1996; 1997; Hoog et al., 1997). The roles of Arg165 and Arg166 in catalysis are further supported by the fact that both residues are absolutely conserved among all herpes proteases (Fig. 2) and the fact that the CMV R165A mutant protease still has 30% of the wild-type activity while the R166A mutant is about four orders of magnitude less active (Liang et al., 1998).

B. MODE OF PROTEOLYTIC PROCESSING There is no direct experimental evidence to support dimer formation of fulllength herpesvirus proteases. However, the extent and the intricate nature of the dimer interface of the catalytic domain suggest that the full-length proteases will also form dimers. Although greater enzymatic activity of the catalytic domain is attained with the influence of cosolvents that are thought to enhance dimer formation, the full-length protease is quite active in the absence of cosolvents. This implies that dimer formation may be further stabilized by the assembly protein or the C-terminal portion of the full-length protease. Little is known about autoprocessing of the herpes proteases. Inspection of the crystal structure shows the active site and the C-terminus are on opposite sides within a protease monomer (Fig. 6). Within a dimer, the C-terminus of one monomer is on the same side as the active site of the other monomer and they are connected by a well-defined groove (Fig. 6). However, not only are they 29 A apart, but also considerable conformational change must occur in order for the C-terminus to position itself properly and assume the correct orientation in