The Herpesvirus Proteases As Targets for Antiviral Chemotherapy

Antiviral Chemistry & Chemotherapy 11:1–22

Review The herpesvirus proteases as targets for antiviral chemotherapy

Lloyd Waxman and Paul L Darke*

Department of Antiviral Research, Merck Research Laboratories, West Point, PA 19486, USA

Corresponding author: Tel: +1 215 652 7533 ; Fax: +1 215 652 6452; E-mail: [email protected]

Viruses of the family Herpesviridae are respon- and catalytic properties of the herpesvirus pro- sible for a diverse set of human diseases. The teases lead to common considerations for this available treatments are largely ineffective, group of proteases in the early phases of with the exception of a few drugs for treatment inhibitor discovery. In general, classical serine of herpes simplex virus (HSV) infections. For protease inhibitors that react with active site several members of this DNA virus family, residues do not readily inactivate the her- advances have been made recently in the bio- pesvirus proteases. There has been progress chemistry and structural biology of the essen- however, with activated carbonyls that exploit tial viral protease, revealing common features the selective nucleophilicity of the active site that may be possible to exploit in the develop- serine. In addition, screening of chemical ment of a new class of anti-herpesvirus agents. libraries has yielded novel structures as starting The herpesvirus proteases have been identified points for drug development. Recent crystal as belonging to a unique class of serine pro- structures of the herpesvirus proteases now tease, with a Ser-His-His catalytic triad. A new, allow more direct interpretation of ligand struc- single domain protein fold has been deter- ture–activity relationships. This review first mined by X-ray crystallography for the proteas- describes basic functional aspects of her- es of at least three different herpesviruses. Also pesvirus protease biology and enzymology. unique for serine proteases, dimerization has Then we discuss inhibitors identified to date been shown to be required for activity of the and the prospects for their future development. cytomegalovirus and HSV proteases. The dimerization requirement seriously impacts methods Keywords: herpesvirus; cytomegalovirus; her- needed for productive, functional analysis and pes simplex virus; serine protease; proteinase; inhibitor discovery. The conserved functional inhibitor; dimerization; drug design

Introduction

The Herpesviridae family of viruses includes herpes sim- brought on by activation of latent HSV-2 affect millions plex virus types 1 (HSV-1) and HSV-2, human worldwide (Corey et al., 1983; Whitley, 1996). cytomegalovirus (HCMV), varicella-zoster virus Neurotropic VZV, responsible for chickenpox, some- (VZV), Epstein–Barr virus (EBV) and human her- times re-emerges decades later as shingles (Arvin, pesvirus 6 (HHV-6), HHV-7 and HHV-8, also known 1996). In addition, a variety of malignancies have been as Kaposi’s sarcoma related herpesvirus (KSHV). associated with certain herpesviruses, including EBV Herpesvirus infections in humans cause a variety of mal- (Rickinson & Kieff, 1996) and most recently, KHSV adies, ranging in severity from the occasional coldsore (Levy, 1997). An excellent, comprehensive overview of brought on by HSV-1 to the fatal complications of human herpesvirus biology is available in Fields’ Virology HCMV infection in immunocompromised or immuno- (Fields et al., 1996). suppressed patients (Britt & Alford, 1996). These large, The genomes of herpesviruses are double-stranded double-stranded DNA viruses vary greatly in biological DNA circles of approximately 150 kilobases, encoding at properties, with diverse cell tropisms and immunologi- least a dozen enzymes. As chemotherapeutic targets, cal responses. A common feature of the group is long- enzymes encoded by the virus are more appealing than term latent infection, with periods of recurring viral host enzymes or receptors because complete selective replication. For example, recurring genital lesions inhibition of the viral target is less likely to have side

effects on the patient. However, not all virally-encoded Table 1. Herpesvirus proteases expressed for in vitro enzymes are essential for herpesvirus replication in cell characterization culture (Roizman & Sears, 1996), raising doubt about Virus Host cell type References the ultimate utility of inhibiting certain enzymes in infected humans. The essential nature of the herpesvirus HSV-1 Escherichia coli Liu & Roizman (1993) protease for viral replication was implicated by the dis- Apeler et al (1997) Weinheimer et al (1993) covery of a temperature sensitive mutant HSV with a HSV-2 E. coli Hoog et al. (1997) mutation in the protease coding region (Preston et al., HCMV E. coli Baum et al. (1993) 1983). More recently, detailed analyses of protease func- Burck et al. (1994) tion in replication have appeared, including the preven- LaFemina et al. (1996) tion of HSV-1 replication through directed mutation of Tomasselli et al. (1998) the protease coding region or cleavage sites, establishing Simian CMV Human, insect Welch et al. (1993) the essential nature of protease catalytic activity in the Hall & Gibson (1996) viral life cycle (Gao et al., 1994; Matusick-Kumar et al., VZV E. coli Qiu et al. (1997) 1995). EBV E. coli Donaghy & Jupp ( 1995) The principal biochemical findings for the her- HHV-6 E. coli Tigue et al. (1996) pesvirus proteases, which impact inhibitor discovery, HHV-8 E. coli Unal et al. (1997) have been obtained with cloned versions of HSV and HCMV enzymes. Additional herpesvirus proteases these late proteins enter the nucleus for new capsid from VZV, EBV and the more recently discovered her- assembly and subsequent DNA packaging. Newly repli- pesviruses have been cloned and are being characterized, cated DNA is cleaved to unit length and transported as listed in Table 1. The general similarities found for through pores in the preformed capsid (Figure 1). HSV and HCMV proteases allow discussion of the her- The process of capsid assembly occurs in several pesvirus proteases as a group in consideration of the stages. The protein components of an early form of the inhibitor discovery process. There have been reviews capsid, known as B capsid, assemble in the nucleus into prior to this one regarding protease biochemistry stable enclosures. A more mature form of the capsid into (Gibson, 1996) and its potential as a target for which the DNA genome has been packaged is referred chemotherapy (Flynn et al., 1997; Holwerda, 1997). to as C capsid (Gibson & Roizman, 1972, 1974). B capsids are quite stable and can be assembled from their Herpesvirus protease catalysis in viral protein components in a heterologous insect expression replication system (Thomsen et al., 1994). A remarkable 3-dimensional picture of the overall HSV-1 capsid structural Generation of the viral nucleocapsid organization based upon electron microscopy has been Herpesviruses are enveloped viruses, wherein the DNA presented by Schrag et al. (1989). genome is packaged within an inner ‘nucleocapsid’ Within the B capsid is found the most abundant pro- structure. Most of what is known regarding capsid con- tease substrate, a nucleocapsid-associated assembly pro- struction has been determined with HSV-1. The virus tein known as ‘ICP35’ for HSV and generally referred to particle consists of four parts: (1) the membrane-like as ‘assembly protein’. Assembly protein is absent from outer envelope; (2) an amorphous tegument between the the more mature C capsid, and is thus thought of as a envelope and nucleocapsid; (3) a stable assemblage of scaffolding assisting in the correct assembly of the other proteins forming the nucleocapsid; and (4) the core con- protein components, analogous to the scaffolding pro- tained within the nucleocapsid, which consists primari- tein used in bacteriophage T4 assembly (Casjens & ly of the DNA genome. Cellular entry of infectious virus King, 1975). During or following the formation of B involves attachment to a cell surface receptor and subse- capsid, assembly protein is cleaved by the viral protease quent fusion of the outer viral envelope with the cell at a single site about 50 amino acids from the C-termi- membrane, as depicted in Figure 1. The DNA-contain- nus. Thus the viral protease must localize to the capsid ing capsid is released to the cytoplasm. Genomic DNA inside the nucleus before expressing its activity without the surrounding capsid structure is then trans- (Robertson et al., 1996). In addition to cleavage of the ported through the cell nuclear membrane into the assembly protein, the protease activity releases the pro- nucleus, wherein transcription and genomic replication tease catalytic domain from its precursor. Following occur. A series of seven proteins are synthesized for the DNA packaging and C capsid formation, additional construction of the new nucleocapsids for progeny viral proteins are added to the outer portion of the viruses late in the infection cycle. Following translation nucleocapsid and the nucleocapsid leaves the nucleus.

Figure 1. The basic steps in herpesvirus replication

(a)

(b)

(a) Viral entry and protein synthesis. The enveloped virus binds to the cell surface and upon fusion with the cellular membrane releases the nucleocapsid to the cytoplasm. Without entry of the capsid, the viral genome is transferred to the nucleus, where transcription takes place. Genes transcribed late in the replication cycle code for the protein components that form new capsids. (b) Capsid assembly. Protein components of new capsids are transported to the nucleus along with the assembly protein and the protease precursor. The assembly protein is thought to provide a scaffold for correct capsid construction. Upon completion of the basic capsid structure, the assembly protein is proteolytically cleaved by the viral protease and leaves the capsid prior to, or during, DNA packaging. If proteolysis is prevented, DNA packaging does not occur (Gao et al., 1994).

Antiviral Chemistry & Chemotherapy 11:1 3 L Waxman & PL Darke

Figure 2. Processing of a herpesvirus precursor and replication or if they have additional functions. substrate This interesting relationship between protease and substrate means that the protease precursor contains all of the amino acids of its substrate, and is also a substrate (Figure 2). There are two primary cleavage sites, often referred to as the R site, for release of the mature protease, and M site, for maturation of the substrate

assembly protein. Cleavage at the R site to release the mature protease means that the mature enzyme has

one of the products of the reaction as its C terminus. The other product is a longer version of the assembly protein, which may have functions in capsid assembly distinct from the independently synthesized assembly protein (Matusick-Kumar et al., 1995; Robertson et al., 1996). The precursor form of HCMV protease has been demonstrated to have intermolecular (‘trans’) cleavage activity (Welch et al., 1993; Jones et al., 1994). In the case of HSV, the precursor catalytic activity is sufficient to support viral replication if complemented by the appropriate proteins, so that the precursor should not be thought of as a zymogen (Matusick- Kumar et al., 1995; see Essential nature of protease catalysis, below). The precursor form of the enzyme is apparently the initiating catalyst for all subsequent Both the protease precursor and the protease substrate mRNA are processing because its expression in heterologous sys- transcribed from the same gene segment, with the substrate being the shorter product (Liu & Roizman 1991a; Welch et al., 1991). tems produces correct processing, but only when the Details shown here are for HCMV. The mature 256 amino acid (aa) protease catalytic domain is active. protease is released from its precursor. In the case of HCMV, the protease undergoes further cleavage, producing fragments that For the HCMV protease there is an additional cleav- remain associated as an active enzyme (Hall & Gibson, 1996). age site within the mature 256 amino acid protease, which has been observed in heterologous expression Subsequently, tegument and envelope are added to com- (Baum et al., 1993; Burck et al.,1994). This internal plete the viral particle, which leaves the cell. cleavage (termed I site) has not been observed with mature proteases from other viruses and it is not clear if Protease forms and substrates it occurs during HCMV replication (Tigue et al., 1996). Synthesis of a herpesvirus protease originates from a The resultant two chain form of the HCMV protease is gene encoding a precursor form, and the gene contains active (Holwerda et al.,1994). It retains substrate speci- within it nested genes encoding the substrate assembly ficity similar to the 256 amino acid mature form, and protein. All of these shorter nested gene open reading can be assembled from separately expressed halves of the frames are in-frame with the protease precursor and end protein (Hall & Gibson, 1996). at the same 3′-terminus. Thus, proteins from nested In summary, there are multiple forms of catalytically gene expression lack the N-terminal residues of the pro- competent herpesvirus proteases that occur in vivo tease wherein essential catalytic amino acids are located because of the precursor nature of the protease synthe- (Liu & Roizman, 1991a,b, 1992). In the case of HSV, sis. All herpesvirus protease enzymology and inhibition there is one nested gene (UL26.5) within the protease studies to date have been performed with mature forms precursor gene (UL26) that is independently transcribed of approximately 250 amino acids. to its own shorter mRNA and ultimately produces the more abundant assembly protein substrate (Liu & Essential nature of protease catalysis Roizman, 1991a). An example of this arrangement and A temperature sensitive HSV-1, defective in DNA some resultant proteins is illustrated in Figure 2 for encapsidation, was identified as having a lesion in the N- HCMV. Herpesviruses vary in the number of nested terminal region of the UL26 gene (Preston et al., 1983), genes, with as many as eight found for VZV (Welch et a region later shown to be the protease catalytic domain al., 1991). For the nested genes found in herpesviruses (Liu & Roizman, 1991a). More recently, a series of stud- other than HSV, it is not known if they are essential for ies with mutant viruses have identified the essential

Table 2. Herpesvirus protease cleavage site sequences protease has been studied in any depth. This section Virus ↓ Site reviews the substrate requirements of the herpesvirus HSV TYLQASEKFK R site proteases while emphasizing recent progress as it relates ALVNASSAAH M site to the development of assays useful for screening and CMV SYVKASVSPE R site mechanistic studies. GVVNASCRLA M site DDVEAATSLS I site Substrate specificity and kinetic parameters

Well conserved amino acids are shown in bold. As discussed above, the herpesvirus proteases are synthe- The sequences shown are taken from Welch et al. (1993). sized as precursors that undergo autoproteolytic cleavages at the M and R sites (reviewed by Gibson & Hall, 1997). nature of protease catalysis for viral replication. By devel- The naturally occurring consensus cleavage sequence for oping stable cell lines transfected with the HSV-1 pro- both of these sites is (V,L,I)-X-A↓S, where X is a polar tease precursor and variants of it, Gao and co-workers amino acid. Additionally, the I site, apparently unique to have been able to genetically complement virus mutants the HCMV protease, is V-E-A↓A. Examples of sub- that are unable to replicate alone, allowing the identifica- strate sequences are shown in Table 2 for the HSV and tion of numerous essential functions of the protease gene HCMV proteases. The pattern of conservation among all product. HSV-1, which does not express the 635 amino of the herpesvirus protease cleavage sites suggests essen- acid protease precursor, but does express the 329 amino tial recognition elements for cleavage susceptibility in the acid assembly protein, is not capable of replication. This P3, P1 and P1′ residues, and less dependence upon amino protease deficient virus can be complemented by the full- acid side chain identity on the C-terminal side of P1′. length protease precursor, restoring virus replication. The Synthetic peptides representing the naturally cleaved 247 amino acid protease catalytic domain alone is not protein sequences are hydrolysed appropriately between sufficient to complement however, indicating an essen- the P1 Ala and P1′ Ser and have been employed in the tial function for the released, C terminal portion of the quantification of cleavage kinetics with the recombinant precursor, which contains 59 amino acids at the N ter- enzymes (DiIanni et al., 1993; Darke et al., 1994; Burck minus of the assembly protein (Gao et al. 1994). et al., 1994; Holwerda et al., 1994; Sardana et al., 1994; HSV-1 with a mutation in the cleavage site (R site) is Stevens et al., 1994; O’Boyle et al., 1995). The pH opti- unable to release the catalytic domain, but is still able to mum for cleavage is between 7 and 8 (Burck et al., process the assembly protein ICP35, demonstrating the 1994). In the case of HSV-1 protease, with naturally catalytic activity of the precursor. Nonetheless, this occurring amino acids, efficient peptide cleavage mutant must also be complemented by the protease pre- requires 5–8 residues on both sides of the scissile bond cursor protein in order to replicate. In this experiment, (DiIanni et al., 1993; Darke et al., 1994). For HCMV the complementing protease precursor was made cat- and HHV-6 proteases, 4 amino acids are required on alytically inactive through mutation of the active site, each side (Sardana et al., 1994; Tigue & Kay, 1998). In but it is processed via the active precursor from the the case of the HCMV protease, R site peptides are six mutant virus, generating the essential C-terminal prod- to 10-fold poorer as substrates than those based upon uct (Matusick-Kumar et al.,1995). This system was the M site, while the cleavage of the I site peptide mim- used to demonstrate that proteolytic processing occurs ics is 50-fold less efficient than M site based substrates within the B capsid (Robertson et al., 1996). (Sloan et al., 1997). In contrast, the protease from HSV- 1 cleaves M and R site-derived peptide substrates with Herpesvirus protease sequence specificity comparable efficiency (DiIanni et al., 1993) and the and assays HHV-6 protease hydrolyses peptides based upon the R site two to threefold better than an M site peptide Characterization of the sequence specificity of the her- (Tigue & Kay, 1998). pesvirus proteases is fundamental to optimization of The catalytic efficiency of these enzymes with pep- synthetic substrates for assay development and mecha- tide substrates is low in comparison to other serine pro- nistic studies. Minimal substrate requirements also proteases. For example, early reports for kcat/Km values for vide a starting point for the design of inhibitors which the HSV-1 and HCMV proteases of 38 M-1s-1 and 404 are frequently peptide-based or incorporate functionali- M-1s-1,respectively (DiIanni et al., 1993, Darke et al., ties that mimic amino acid side chains that can be 1994; Sardana et al., 1994; LaFemina et al.,1996), are accommodated by the protease. Although all her- orders of magnitude lower than those reported for the pesvirus proteases recognize the same general amino serine proteases chymotrypsin and trypsin (107 M-1s-1) acid sequence motifs, only the specificity of the HCMV (Valenzuela & Bender, 1971; Higgins et al.,1983).

Antiviral Chemistry & Chemotherapy 11:1 5 L Waxman & PL Darke

Table 3. Substrates of herpesvirus proteases

-1 -1 -1 Substrate Conditions* Km (mM) kcat (min ) kcat/Km (M s ) Reference CMV protease GVVNA↓S-Abu-RLA 20% glycerol 0.0198 2.195 1843 Waxman, unpublished RWGVVNA↓S-Abu-RLA 20% glycerol 0.0120 1.619 2245 Waxman, unpublished DABCYL-RGVVNA↓SSRLA-EDANS 20% glycerol 0.094 22.7 4000 Darke et al. (1996) ↓ Abz-GVVNA SSRLAY(NO2) 25% glycerol 0.125 35 4667 Margosiak et al. (1996) ↓ Abz-VVNA SSRLY(NO2)R 0.5 M Na2SO4 0.76 12.0 260 Bonneau et al. (1998) ↓ Abz-Tbg-Tbg-Asn(NMe2)A SSRLY(NO2)R 0.5 M Na2SO4 0.0032 3.06 15940 Bonneau et al. (1998) ↓ Ac-Tbg-Tbg-Asn(NMe2)A AMC 0.5 M Na2SO4 0.0132 2.1 2650 Bonneau et al. (1998) GVVNA↓AMC 20% glycerol 0.236 0.546 38.5 Waxman, unpublished GVVNA↓pNA 20% glycerol 0.131 0.301 38.3 Waxman, unpublished SYVKA↓pNA 20% glycerol 1.953 0.578 4.7 Waxman, unpublished GVVNA↓Sbzl 20% glycerol 0.1585 152.5 16033 Waxman, unpublished SYVKA↓Sbzl 20% glycerol 0.125 115.1 15291 Waxman, unpublished ↓ RWGVVNA NH2 20% glycerol 0.417 0.633 25.3 Waxman, unpublished Assembly protein precursor 0% glycerol 0.003 13.3 73300 Pinko et al. (1995) HSV-1 protease HTYLQA↓SEKFKMW-amide 0.8 M citrate 0.016 0.4 4176 Hall & Darke (1995) HTYLQA↓SEKFKMW-amide 0.2 M citrate 1.32 3 40 Hall & Darke (1995) LVLA↓pNA 20% glycerol 2.0 20 100 O’Boyle et al. (1997) 0.15 M acetate

*Conditions list the solvent components likely to affect dimerization and kinetic parameters. Abbreviations: Abu, L-α-aminobutyric acid; Abz, o-aminobenzoyl; Tbg, t-butylglycine; AMC, 7-amino-4-methylcoumarin; pNA, p-nitroanilide;

Sbzl, -SCH2C6H5 or thiobenzyl ester.

These initial indications of low activity are attributable aliphatic side chain for efficient cleavage, and substitu- in part to dissociation of the active dimer to the inactive tion with Gly eliminates cleavage (Sardana et al.,1994). monomer in in vitro assays. Correction for monomer- Interestingly, the troublesome tendency of the mature ization gives maximal specificity constants in the order HCMV protease to cleave itself in purified in vitro of 104 M-1s-1 (Darke et al., 1996; Margosiak et al., 1996; preparations is stopped by mutation of the P3 Val-143 Schmidt & Darke, 1997), closer to the 105 M-1s-1 found to Gly or Ala at the I site, availing a stable, homogenous, for other viral proteases such as the human immonode- fully active enzyme (Holwerda et al., 1994; LaFemina et ficiency virus (HIV) protease and hepatitis C virus NS3 al.,1996). A further branching from the β carbon of the protease (Meek et al., 1994; Yan et al.,1998). The P3 Val side chain to t-butylglycine (Tbg) markedly low- impact of an inactive monomer/active dimer equilibri- ers the Km for peptide substrates, and the additional um on inhibitor discovery and kinetics is discussed binding given with Tbg has been exploited in both sub- below in ‘Activation by dimerization’. strate and inhibitor design (Bonneau et al., 1998; Synthetic peptides have been used to explore essential Ogilvie et al., 1997) (Table 3). The enzyme S3 pocket is features of herpesvirus protease substrate recognition. As defined in the inhibited structure deduced by Tong et al. suggested by the pattern of conservation in the natural (1998), with the P3 side chain extending into a large substrate sequences, residues at positions P3, P1 and P1′ cavity. Surprisingly, the cavity has a hydrophilic compo- prove most sensitive to substitution. The P1 Ala may be nent, with the guanidino group of Arg-166 near the substituted with the smaller Gly, resulting in an eightfold back of the pocket. It remains to be determined how to drop in kcat/Km (Sardana et al., 1994), but larger side reconcile the marked preference for P3 hydrophobic chains, including the modest methyl-to-ethyl substitu- residues in both substrates and inhibitors with the tion of Ala to aminobutyric acid (Handa et al. 1995), hydrophilic character of the S3 binding pocket. eliminate peptide substrate cleavage. These observations The requirements for the P2 residue in an efficient for P1 are readily understandable in light of the recently substrate are less well defined, and are context specific. reported structure of a substrate analogue bound to the In the context of the M site sequence of HCMV, Asn HCMV protease, wherein the P1 Ala side chain is seen can be substituted with Gln, Glu or Lys with an order directed into a small enzyme pocket (Tong et al., 1998). of magnitude loss in cleavage efficiency, while P2 Gly is The P3 residue (Val, Leu or Ile) needs a branched 30-fold worse than the native sequence. Bonneau et al.

(1998) have combined a useful N-dimethyl-asparagine of detection, a rapidly cleaved substrate offers ease of use

[Asn(NMe2)] substitution at P2 with the aforemen- with nanomolar concentration of enzyme that is desir- tioned P3 Tbg to generate the best peptide substrate able for analysis of potent inhibitors, which is frequent- reported to date (Table 3). Interestingly, the preference ly an issue with the slow turnover viral proteases. A of HCMV protease for M site over R site-derived pep- simple modification to a peptide substrate to give a 10- tides may largely be due to the P2 residue, since a switch fold increase in sensitivity is to append a Trp residue at of the P2 residues, naturally Asn at the M site and Lys one end, exploiting the intrinsic fluorescence of Trp at the R site, reverses the relative susceptibility of these (Hall & Darke, 1995). Numerous additional improve- peptides to cleavage (LaPlante et al., 1998). ments have been made with fluorogenic and chro- With the exception of P1′ Ser, specificity for mogenic substrates for the HCMV protease, and the residues is poor on the C-terminal side of the cleaved techniques described below should be directly applicable bond, as shown in Table 4. The alcohol functionality of to the other herpesvirus proteases. P1′ Ser does not appear critical because Ser may be Internally quenched fluorogenic substrates offer sig- substituted with the smaller Ala or Gly, but not Thr or nificant advantages for kinetic studies and high volume larger side chains (Sardana et al., 1994; Handa et al., screening of complex mixtures, and are increasingly 1995). As shown in Table 4, a variety of amino acid popular because the general design is applicable to any substitutions in P2′,P3′ or P4′ do not drastically affect class of protease, as well as allowing continuous fluoro- the observed specificity constants. A substitution for metric monitoring of reaction progress. These substrates the naturally occurring P2′ Cys of the HCMV R site are peptides with a fluorophore (energy donor) on one sequence with Ser or isosteric α-aminobutyric acid has side of the scissile bond and a chromophore (energy frequently been used to avoid the tendency of Cys to acceptor) on the other side of the scissile bond, resulting oxidize (Tables 3 and 4). The lack of specificity for in radiationless resonance energy transfer that quenches residues P2′ to P4′ in peptide substrates is mirrored in the fluorescence of the donor. Cleavage of the peptide cleavage site mutagenesis data for the protein sub- between the two chromophores relieves the quenching, strates, when assessed in an expression system generating a fluorescence signal. Efficient quenching (McCann et al., 1994). Despite the requirement for up and a low background signal for a donor–acceptor pair to four residues at the C-terminal to the cleaved bond is obtained with good spectral overlap of the donor for efficient cleavage of peptides containing natural emission spectrum and the acceptor absorption spec- amino acids, it is not clear in the existing crystal struc- trum. Two effective donor–acceptor pairs that have been tures where these P′ residues bind to the enzyme (Chen incorporated into peptide substrates for the herpesvirus et al., 1996; Hoog et al., 1997; Tong et al., 1996; Shieh proteases are the donor 5-[(2′-aminoethyl)-amino]- et al., 1996; Qiu et al., 1996, 1997). naphthalene-1-sulphonic acid (EDANS) quenched The P2′ to P4′ residues in a substrate can be dis- with [4-4′-dimethylaminophenazo]benzoic acid (DAB- pensed with entirely if loss of binding is compensated CYL), or the donor o-aminobenzoic acid (Abz) for by using an optimized N-terminal sequence quenched with 3-nitrotyrosine [Y(NO2)] (Handa et al, (Bonneau et al., 1998). Thus, the specificity constant for 1995; Pinko et al.,1995). Thus, the commercially avail-

HCMV cleavage of Ac-Tbg-Tbg-Asn(NMe2)- able HCMV protease substrate, DABCYL- Ala↓aminomethylcoumarin is comparable to longer RGVVNA↓SSRLA-EDANS (Bachem Biosciences, substrates containing P2′ to P5′ residues and the natur- Philadelphia, PA, USA) (Holskin et al.,1995), has been al GVVNA N-terminal sequence (Table 3). For HSV-1, used for both enzymology (Darke et al., 1996) (Table 3) an optimized N-terminal sequence was identified using and high-throughput screening in microtitre plate for- substrate phage display. This led to the synthesis of a p- mat. Similarly, the related substrate, Abz-GVVNA↓SS- ↓ nitroanilide substrate, LVLA p-nitroanilide having RLAY(NO2) (Table 3) provides adequate sensitivity to superior kinetic properties to those described previously detect nanomolar levels of the HCMV protease (Pinko (O’Boyle et al., 1997) (Table 3). et al., 1995; Margosiak et al.,1996). Fluorogenic substrates using this donor-acceptor pair are also described Substrates for screening and kinetics for HHV-6 protease (Tigue et al.,1996). Holwerda While standard peptide HPLC methods with UV (1997) has tabulated a list of herpesvirus protease sub- detection are suitable for the definition of essential substrates and has suggested that modified peptide substrate requirements, such as in the studies cited above, strates have a lower Km than their unaltered peptide the most useful substrates for inhibitor screening or backbones because of enhanced binding mediated by enzyme kinetics give rise to products that are either flu- the additional reporter groups. More significantly, the ↓ orescent or coloured. Combined with a high sensitivity substrate Abz-VVNA SSRLY(NO2)R can be modified

Antiviral Chemistry & Chemotherapy 11:1 7 L Waxman & PL Darke

Table 4. Effect of amino acid substitutions on HCMV protease peptide substrates, C-terminal side of cleavage

µ -1 -1 -1 Substrate sequence Km ( M) kcat (min )kcat/Km (M s ) Naturally occurring sequence RWGVVNA↓Ser-Cys-Arg-Leu-Ala 9.73 1.559 2670 RWGVVNA↓Ser-Abu-Arg-Leu-Ala 11.07 1.475 2220 Other sequences tested RWGVVNA↓Ala-Abu-Arg-Leu-Ala 1.51 0.266 2920 RWGVVNA↓Gly-Abu-Arg-Leu-Ala 15.92 1.695 1770 RWGVVNA↓Hse-Abu-Arg-Leu-Ala 8.08 0.184 380 RWGVVNA↓Tyr-Abu-Arg-Leu-Ala 12.64 0.035 46 RWGVVNA↓Thr-Abu-Arg-Leu-Ala 25.94 0.066 43 RWGVVNA↓Ser-Nle-Arg-Leu-Ala 9.05 2.285 4210 RWGVVNA↓Ser-Abu-Arg-Leu-Ala 11.07 1.475 2220 RWGVVNA↓Ser-Tyr-Arg-Leu-Ala 1.03 0.124 2010 RWGVVNA↓Ser-Val-Arg-Leu-Ala 33.47 1.765 880 RWGVVNA↓Ser-Abu-Dab-Leu-Ala 7.34 1.294 2940 RWGVVNA↓Ser-Abu-Ser-Leu-Ala 19.62 1.180 1000 RWGVVNA↓Ser-Abu-Arg-Nle-Ala 9.14 1.672 3050 RWGVVNA↓Ser-Abu-Arg-Pro-Ala 2.91 0.236 1350

The effects of substitutions for the naturally occurring amino acids on the C-terminal side of the cleavage site (M site) for CMV protease were examined (L Waxman, unpublished observations). The amino acid changed from the starting sequence shown at the top is in bold. Note that the first two amino acids, Arg and Trp, are not part of the natural cleavage site and were added to aid solubility (Arg) and detection of products (Trp). These additions at P6 and P7 are known to have no effect upon CMV protease hydrolysis rates. Note also that the second sequence listed substitutes Abu for the problematic Cys, a substitution used throughout the remaining peptides. The reaction conditions used minimized monomerization effects (Darke et al., 1996). Abbreviations used are: Abu,L-α-aminobutyric acid; Nle, norleucine; Hse, homoserine; Dab, diaminobutyric acid. in the P4-P2 residues with the optimized sequence, t- released AMC product has a bright fluorescence, pro- butylglycine-t-butylglycine-dimethyl asparagine [Tbg- viding sensitive product detection.

Tbg-Asn(NMe2)], to give a substrate with a kcat Protease hydrolysis of thioesters is often more rapid 240-fold greater and a kcat/Km 60-fold greater than that than with the corresponding nitroanilide or AMC of the equivalent non-optimized substrate (Bonneau et amide substrates due to the high reactivity of thioester al., 1998) (Table 3). bonds. Km values for thioester hydrolysis by serine pro- Optimization of the N-terminal side of a peptide teases are also often lower than those for amide sub- substrate allows hydrolysis of a suitable leaving group strates (Harper et al.,1984). Thus thiobenzyl ester from the C-terminus by the HCMV protease, as in Ac- peptides based upon the P5–P1 M and R site sequences ↓ Tbg-Tbg-Asn(NMe2)-Ala AMC (Bonneau et al., are rapidly hydrolysed by the CMV protease (Table 3). 1998) (Table 3) (See ‘Substrate specificity and kinetic As with other serine proteases, the kcat is 200–500-fold parameters’). In this regard, the herpesvirus proteases greater than for amide substrates, but the Km values are resemble the classic serine proteases such as elastase; only slightly affected. Thioester hydrolysis rates are amides of ammonia or p-nitroaniline as well as often spectrophotometrically measured in a continuous thioesters are substrates (Table 3). For the amides, it is assay by reaction of the released thiol with 4,4′-dithio- interesting to note the differences in specificity con- bis(pyridine) or 5,5′-dithiobis(2-nitrobenzoic acid) pre- stants observed. HCMV protease releases ammonia sent in the reaction mixture (Castillo et al., 1979). from a C-terminal amidated peptide, albeit at a rate two Unlike most serine proteases, however, HCMV protease orders of magnitude less than the longer substrate (com- contains sulphydryl groups reactive with these thiol ↓ ↓ pare RWGVVNA NH2 to RWGVVNA S-Abu- reagents (see ‘Inhibitors of herpesvirus proteases’). RLA, Table 3). The ammonia, pNA and AMC amides Nonetheless, the thioesters can be used in end-point of GVVNA are all similarly poor substrates. assays in which the thiol reagent is added at termination Optimization of the peptide portion with Ac-Tbg-Tbg- of the enzymatic reaction, or the reaction products can ↓ Asn(NMe2)-Ala AMC raises kcat/Km 100-fold and be analysed by HPLC. In consideration of the large brings Km and kcat parameters into useful ranges (Table increases in specificity constants for optimized 3). This is particularly noteworthy considering that sequences, together with the increases for the non-opti- AMC amide substrates are non-fluorescent and the mized thioesters shown in Table 3, we hypothesize here

that a thioester substrate with an optimized sequence not easily mimicked with peptides. Alternatively, con- might allow rapid enough acylation of the active site formational constraints upon the cleaved region may serine for determination of individual rate constants for provide an entropic advantage for protein substrate some of the steps in the catalytic cycle. binding over the freely flexible peptides. More extensive Scintillation proximity assay (SPA) technology has investigation of the protein substrate protease interac- been applied to develop an assay for the HCMV pro- tions via kinetics and X-ray structures will aid in defin- tease that may be suitable for the simultaneous process- ition of the factors contributing to enhanced binding. ing of many samples, as in inhibitor screening (Baum et al., 1996a). Following the cleavage reaction, remaining Catalytic mechanism biotinylated substrate, which is radiolabelled at the C- Abundant evidence from mutagenesis, chemical modifi- terminus, is bound to streptavidin-coated SPA beads to cation and X-ray crystallography studies show that the generate the signal. Cleavage by the protease results in a herpesvirus proteases are serine proteases, with an active decrease in the signal, which is prevented by the pres- site triad of Ser-His-His. Sequence alignments and ence of a protease inhibitor. mutagenesis of candidate residues identified Ser118 of simian CMV protease as essential for activity (corre- Protein substrates sponding to Ser132 and Ser129 of HCMV and HSV-1 The sequence specificity of herpesvirus protease cleav- proteases, respectively), suggesting an active site role age of its natural protein substrates generally parallels (Welch et al., 1993). The characteristic reaction of diiso- the specificity observed using peptides. Thus the co- propylfluorophosphate with the nucleophilic serine of expression of HSV-1 or HCMV proteases with their serine proteases was used to identify the HSV-1 pro- cognate substrate proteins demonstrates the necessity of tease as a serine protease, with modification of Ser129 the P3 to P1′ conserved sequence of (V/L)-X-A↓S (Liu (DiIanni et al.,1994). Similarly, Ser132 of HCMV pro- & Roizman, 1993; Welch et al., 1993; McCann et al., tease is so modified (Stevens et al.,1994). The formation 1994; Jones et al., 1994; Godefroy & Guenet, 1995). of a covalent bond from Ser132 to an active site trapping Although co-expression of protease and substrate in carbonyl can now be seen in a complex of a ketoamide intact cells does not allow the quantitative analysis pos- with the HCMV protease (Tong et al.,1998). sible with in vitro peptide cleavage, HCMV protease The three dimensional structures of the herpesvirus studies demonstrate that the order of the cleavages is M proteases show that, in addition to the active site serine, site followed by the R site and then the I site, in both conserved His63 and His148 (HCMV numbering) are plasmid-transfected (Welch et al., 1993) and virus- in the active site, with His63 positioned for hydrogen infected cells ( Jones et al.,1994). bonding to the nucleophilic serine (Chen et al., 1996; As defined by co-expression of enzyme with protein Qiu et al., 1996, 1997; Shieh et al., 1996; Tong et al., substrates, an R site requirement for a conserved Tyr at 1996; Hoog et al.,1997). The more distant His148 is P4 is now known to be one of enzymatic activity. That positioned similarly to the active site aspartate of is, the catalytic domain of the HCMV precursor con- trypsin, and may perform a similar function. In the case tains this Tyr and requires it for trans activity as well as of trypsin, mutation of the active site Asp102 to 4 for autocleavage of the precursor (Welch et al, 1993). asparagine reduces kcat by a factor of 10 , and a base Similarly, HHV-6 protease precursor processing is pre- functionality for the aspartate anion has been suggested vented with the analogous Tyr227Ala mutation, and the to facilitate catalysis (Craik et al.,1987). While His148 mature form of the Tyr227Ala HHV-6 protease is inac- is positioned to potentially provide a function similar to tive against peptide substrates (Tigue & Kay, 1998). trypsin’s Asp102, it is interesting to note that the trypsin Interestingly, the mature enzyme C-terminus is distant Asp102Asn mutant still has an activity for peptide from the active site in the crystal structure, suggesting cleavage close to that of herpesvirus proteases (Unal et that the ‘autoprocessing’ R site cleavage is an intermole- al., 1997). Viral growth studies with reconstructed cular reaction (Tong et al.,1998). HSV-1 have shown that changes in the absolutely con- The in vitro kinetics of protein substrate cleavage is served His148 of the protease alter or abolish the abili- only described for the case of mature HCMV protease ty of the virus to replicate in culture, such that a range cleavage of its assembly protein (Pinko et al.,1995). The of viral phenotypes is observed depending upon the specificity constant of 73300 M-1s-1 is higher than amino acid substituted (Register & Shafer, 1997). The observed with peptide substrates; primarily owing to the mutation His148Glu, which would be expected to bet- µ low Km of 3 M (Table 3). It may be that binding of ter resemble the catalytic machinery of trypsin, still per- natural protein substrates to the enzyme involves mits replication, although not nearly as well as wild-type regions not contiguous with the cleavage site, and hence virus. The simian CMV protease residue homologous to

Antiviral Chemistry & Chemotherapy 11:1 9 L Waxman & PL Darke

Table 5. Dimerization parameters for herpesvirus protease

µ Protease Kd ( M) Temperature pH Solution components* Method of analysis Reference CMV 6.6 30°C 7.5 10% glycerol Enzyme kinetics Darke et al. (1996) 0.55 30°C 7.5 20% glycerol Enzyme kinetics 0.54† 30°C 7.5 20% glycerol Size exclusion chromatography CMV 59 20°C 7.5 Sedimentation Cole (1996) equilibrium 17 20°C 7.5 Sedimentation velocity 5.7 20°C 7.5 20% glycerol Sedimentation equilibrium CMV 8 25°C 7.2 2% DMSO Enzyme kinetics Margosiak et al. (1996) 0.0019 25°C 7.2 25% glycerol, Enzyme kinetics 2% DMSO HSV-1 0.964 15°C 7.5 20% glycerol, Enzyme kinetics Schmidt & Darke (1997) 0.2 M citrate 0.225† 15°C 7.5 20% glycerol, Enzyme kinetics 0.5 M citrate

*Solution components listed are those either known or potentially able to affect the Kd. †Application of the size exclusion chromatography method used with CMV protease to the determination of Kd for HSV protease was attempted and found to be inadequate, due to the more rapid exchange rate of monomers and dimers in the case of HSV-1 protease.

HSV-1 protease His148 is His142. Mutagenesis of this mutagenesis of Arg165 has only a modest effect, sug- His142 to either Ala or Gln drastically reduces but does gesting a lesser role for Arg165 (Liang et al.,1998). not eliminate enzyme activity (Welch et al., 1993), as judged from studies employing co-expression with sub- Activation by dimerization strate. Thus, the third member of the catalytic triad is Early reports of peptide cleavage activities of the mature not essential for activity and definition of its precise role HCMV and HSV proteases describe turnover rates that in catalysis awaits more detailed in vitro kinetic analysis. are quite low. Initial assays required hours of incubation As a serine protease, herpesvirus protease catalysis and sensitive detection techniques (DiIanni et al., 1993, proceeds in two chemical steps, with initial cleavage of Burck et al.,1994). Both the HSV and HCMV proteas- the scissile amide bond by nucleophilic attack of serine es are now known to require dimerization for significant upon the carbonyl of the amide, generating an acyl- activity, and have dissociation constants (Kd) in the enzyme ester intermediate and the C-terminal cleavage micromolar range, as shown in Table 5. Activity in a product. Subsequent water hydrolysis of the intermedi- typical assay is increased by the inclusion of high con- ate regenerates free enzyme and the N-terminal cleav- centrations of antichaeotropes, or water structure-form- age product. No details of the kinetics or equilibria of ing cosolvents, such as glycerol or citrate (Burck et al., the chemical steps have been presented. No reports of 1994; Hall & Darke, 1995; Yamanaka et al., 1995). The the ester hydrolysis expected of serine proteases have principal effect of these activators is to shift the appeared, although we have found that peptides of the monomer-dimer equilibrium toward the active, dimeric appropriate sequence with a thioester replacement of the form of the enzyme (Cole, 1996; Darke et al., 1996; scissile amide are substrates for the HCMV protease Margosiak et al., 1996; Schmidt & Darke, 1997). (Table 3). This facile esterolysis may allow definition of Dimers have been observed in the structures determined acyl-enzyme hydrolysis or product release rates. by X-ray crystallography for the VZV, HSV-2 and An ‘oxyanion hole’ is found in serine proteases, HCMV proteases (Qiu et al., 1996, 1997; Shieh et al., wherein a developing negative charge on the scissile 1996; Tong et al., 1996; Hoog et al.,1997). amide carbonyl is stabilized during nucleophilic attack. The Kd values reported for the dimerization of the For the HCMV protease, the stabilization has been pro- HCMV protease range from 1.9 nM to 57 µM, posed to be mediated by Arg165 (Tong et al., 1998) or depending upon solvent conditions (Table 5). For the µ both Arg165 and Arg166 (Qiu et al., 1996), which are HCMV protease the Kd is about 1 M in the presence appropriately positioned in the active site. Mutagenesis of 20% glycerol at pH 7.5. There is approximate agree- of Arg166 nearly eliminates catalytic activity, while ment of a functional Kd measurement using enzyme

kinetics at 30°C (0.55 µM) with the thermodynamical- must be made for proper quantitative evaluation of ly rigorous sedimentation equilibrium result at 20°C inhibitors or during the study of other structure–activi- µ (5.7 M). Direct comparison of Kd values across the ty relationships, such as in mutagenesis studies. For different studies shown in Table 5 are potentially mis- example, prior to reports that dimerization is needed for leading because of differing solvent conditions activity, a mutation of Glu122 in HCMV protease was employed; our experience with the HSV-1 protease found to be inactivating, leading to the suggestion that serves as an example. The HSV-1 protease Kd values this conserved residue is the herpesvirus protease coun- found using kinetics, 0.964 µM and 0.225 µM in 0.2 terpart of Asp in the classic Ser-His-Asp catalytic triad and 0.5 M citrate, respectively (Table 5), are close to of serine proteases (Cox et al., 1995). It has since been µ those reported for the HCMV protease (0.55 M) found that a Glu122Ala mutation raises the Kd of determined with similar methodology, in the absence of dimerization by two orders of magnitude. By using con- citrate. Yet our attempts to obtain a Kd for the HSV-1 ditions that promote dimerization, substantial catalytic protease with size-exclusion chromatography under activity of the Glu122Ala mutant can be demonstrated conditions previously successful for the HCMV pro- (Darke, unpublished results). More recent mutagenesis tease failed due to the instability of the HSV-1 protease studies have taken this analytical complication into con- dimer. Only the combined effects of lower temperature sideration, employing assays wherein the concentrated, (5°C) and the addition of 0.4 M citrate stabilized the dimeric enzyme is diluted into an assay for initiation of HSV protease dimers during chromatography to the the reaction and the assay duration is short enough to extent that they were observable. Even then size exclu- ensure that monomerization has not occurred to a sig- sion chromatography only gives only a qualitative indi- nificant degree. In this way, Arg166 of the HCMV pro- cation of dimerization because exchange between the tease has been identified as essential for catalytic activity monomeric and dimeric forms still occurs on the time (Liang et al.,1998). scale of minutes (Schmidt & Darke, 1997). We con- If monomer–dimer re-equilibration occurs during an clude that the HSV protease dimers are kinetically and assay of a competitive inhibitor, an erroneously high thermodynamically much less stable than dimers of IC50 or Ki value may result. This potential for obtaining HCMV protease. erroneous Ki values has been reviewed in the context of It may be that dimerization is a regulatory mecha- the HIV protease in some detail (Darke, 1994). Briefly, nism whereby activity is only realized when it is needed, in a system where an enzyme monomer is inactive, does after the protease has been concentrated within the not bind the inhibitor and is in equilibrium with the nascent capsid. In measurements of catalytic activity the active dimer that does bind an inhibitor, the monomer micromolar Kd can be a nuisance, in that nanomolar acts as a buffer to the loss of active dimers. Addition of enzyme concentrations of enzyme are usually desired. the inhibitor that binds to the dimeric state shifts the Yet for the virus, the protease is concentrated at its site equilibrium, with monomers dimerizing to partially off- of action, within the nascent nucleocapsid. It seems rea- set the inhibition event. HCMV protease peptide sonable that in vivo, without glycerol or inhibitors that are related in sequence to a substrate have antichaeotropes, but with the aid of molecular crowding, been shown to exhibit this behavior in assays at 30°C. a high micromolar Kd may be just right for turning on For the herpesvirus proteases, kinetic stabilization of activity at the appropriate time. dimers by use of low temperature, brief assays or high The activation of viral proteases through protein asso- concentrations of antichaeotropes are ways to avoid the ciation is common and found in diverse viruses, such as complication (Liang et al., 1998; Darke & Kuo, 1998). hepatitis C virus (NS3 protease, Lin & Rice, 1995), ade- Since Ki is not a function of enzyme concentration in an novirus (Mangel et al., 1993; Webster et al., 1993) and assay, a simple diagnostic method for detecting a prob- HIV (Navia et al., 1989). The importance of viral regu- lem with monomer–dimer re-equilibration during lation of proteolytic activity through subunit association kinetic analyses is to determine if the Ki varies with the was demonstrated for the HIV-1 protease (Krausslich, enzyme concentration used. 1991). Although HIV and its protease are otherwise unrelated to HSV, this example illustrates that the tim- Inhibitors and inhibition of herpesvirus ing of proteolysis can be critical in viral assembly. The proteases premature dimerization and activation of the HIV protease prevented viral particle formation, while attenua- A detailed picture of enzyme catalysis for the serine pro- tion of the ‘pre-dimerized’ enzyme with an inhibitor teases has emerged over decades of investigation, result- restored viral particle formation (Krausslich, 1992). ing in a mechanistic framework that provides the basis Consideration of the monomer–dimer equilibrium for design of mechanism-based inhibitors. That frame-

Antiviral Chemistry & Chemotherapy 11:1 11 L Waxman & PL Darke

work includes the nucleophilic Ser hydroxyl attack upon HCMV protease (33% at 250 µM) (DiIanni et al., 1993; a peptide carbonyl to form a tetrahedral intermediate, Burck et al.,1994). With the aim of introducing followed by loss of water to generate an enzyme-serine HCMV protease-specific binding into the reagent, a acyl intermediate for subsequent hydrolysis. Thus, gen- peptide chloromethyl ketone was synthesized based eral serine protease inhibitors exploit nucleophilicity of upon the M site cleavage sequence, GVVNA- the serine or its adjacent partner histidine as well as the chloromethyl ketone. At 50% inhibition a histidine is oxyanion hole used in stabilization of the tetrahedral modified, but only partial protection with tight-binding intermediate. Inhibitor specificity for a given serine pro- substrates is observed, and reaction with the enzyme’s tease is most readily conferred with exploitation of the cysteine residues is also found (L Waxman, unpublished substrate binding subsites. Some general serine protease results). Phenylmethane sulphonyl fluoride (PMSF) inhibitors are reactive with the nucleophilic herpesvirus reacts with the active site serine residue and, like TPCK, protease active site triad and specific inhibitors are being only partially inhibits the HCMV protease (DiIanni et developed that combine an electrophilic carbonyl with al., 1993; Burck et al.,1994). The structurally related substrate-like binding moieties. The HCMV protease serine protease inhibitors tosyl sulphonyl fluoride and 4- has a complicating property of having nucleophilic sur- (2-aminoethyl)benzenesulphonyl fluoride also react face sulphhydryls, which nonetheless might be exploit- with thiol groups on the enzyme, as seen with the ed. Crystal structure determinations for several of chloromethyl ketones (L Waxman, unpublished results). herpesvirus proteases now allow more structure-based Holwerda (1997) has proposed that the poor reactivity evolution of inhibitors, and a significant milestone in of these reagents with the active site may be due to the the process of the rational development of specific, more absence of the negative aspartic acid residue commonly potent inhibitors is the determination of an inhibitor- found in the serine protease active sites, which renders bound structure (Tong et al.,1998). His63 less reactive toward these types of inhibitors. In any event, the presence of reactive thiol groups compli- General serine protease inhibitors cates the use of these classic covalent inhibitors of serine Labelling reactions of the active site serine residues of proteases. the HSV-1 and HCMV proteases with diisopropylfluo- Proteinaceous inhibitors of serine proteases, includ- rophosphate (DFP) provide chemical evidence that the ing bovine pancreas, lima bean and soybean trypsin herpesvirus proteases are serine proteases (DiIanni et al., inhibitors, the Bowman-Birk inhibitor and ovomucoid, 1994; Stevens et al., 1994; Holwerda et al.,1994). form tight reversible complexes with serine proteases. Although the second order rate constant for inhibition Their inability to inhibit the HCMV protease is proba- of HSV-1 by DFP is only 1 M-1s-1, as compared to 37.8 bly related to the unique sequence specificity of the viral M-1s-1 for chymotrypsin (Fahrney & Gold, 1963), other enzyme. Peptide aldehydes, potent protease inhibitors serine proteases also react more slowly with DFP than believed to act as transition state analogues, including chymotrypsin, including trypsin (15-fold more slowly) chymostatin, elastatinal, leupeptin and antipain and Staphylococcus aureus V8 protease (>100-fold) (Umezawa, 1976), are also without effect (L Waxman, (Salvesen & Nagase, 1989). Other reagents routinely unpublished observations). used to covalently inhibit serine proteases include 3,4- dichloroisocoumarin, peptide chloromethyl ketones, Peptide inhibitors and sulphonyl fluorides. Inhibition by the heterocycle Substrate and product sequences are the basis of peptide 3,4-dichloroisocoumarin is mechanism-based and this inhibitors of modest potency. Thus, Holskin et al., compound is an effective inhibitor of a wide variety of (1995) describe a HCMV protease M site peptide serine proteases including trypsin, chymotrypsin, leuko- mimic containing a reduced peptide bond at the cleav- ψ cyte elastase, cathepsin G and S. aureus V8 protease age site, RGVVNA CH2NH]-SSRLA, that inhibited µ (Harper et al.,1985). However, at the high concentra- with an IC50 value greater than 500 M. A different tions (100–1000 µM) and relatively long incubation approach was taken by LaFemina et al. (1996) in a study times (up to 30 min) required to observe inhibition of on the inhibition of the HCMV protease by M site pep- the HCMV protease, this reagent was found to react tide mimetics. They noted that the potency of the unal- with one or more of the enzyme’s five thiol groups (L tered M site peptide substrate GVVNA↓SCRLA when µ Waxman, unpublished observations). Chloromethyl- used as an inhibitor (Ki=225 M) could be increased ketones inhibit serine proteases by alkylation of the threefold by substituting alanine for serine at the P1′ ↓ µ active-site histidine by the chloromethyl moiety. The position (GVVNA ACRLA; Ki=72 M). These are chymotrypsin inhibitor tosylamido-2-phenylethyl- alternative substrates and the Km is nearly equal to the chloromethyl ketone (TPCK) is a poor inhibitor of the Ki.Some M site-derived product peptides from the N-

Table 6. Tetrapeptide inhibitors of the HCMV protease active (Shah et al., 1992; Finke et al.,1995). Based upon detailed structure–activity studies as well as X-ray crys- Peptide Sequence* Origin K (µM)† i tallography of human leukocyte elastase and the related 1 Ac-Tyr-Tbg-Asn-Gly-NH2 Random library 30 serine protease, porcine pancreatic elastase, the 2 Ac-Tyr-Tbg-Asn-Gly-OH Random library 0.9 schematic diagram in Figure 3(a) depicts how the vari- 3 Ac-Val-Val-Asn-Gly-NH M site > 300 2 ous elements of the β-lactam nucleus are believed to 4 Ac-Val-Val-Asn-Gly-OH M site 190 interact with the S1′, S1 and S3 specificity pockets of Peptide inhibitors discovered through screening of a random these proteases. Because both of these elastases have a tetrapeptide library (peptides 1 and 2) are compared to analogous preference for residues with small alkyl side chains in peptides based upon the M site. There were 21 amino acids used in the tetrapeptide synthesis, defined and known in positions 1 and 2 the P1 position (Harper et al., 1984) and will hydrolyse to generate 441 samples, and randomized in positions 3 and 4, to many of the synthetic peptide substrates cleaved by the generate a theoretical 441 peptides per sample, for a total of 194,481 peptides. Synthesis was used for deconvolution of the HCMV protease (L Waxman, unpublished observa- randomized positions. tions), compounds may already be available from elas- *Abbreviations: Ac-, acetyl; Tbg, t-butylglycine. †L Waxman, DB Olsen & PL Darke, unpublished observations. A tase inhibitor programmes with inhibitory activity temperature of 0°C was used in determinations to avoid toward the HCMV protease. The inhibitors of the dissociation of the dimeric form of the CMV protease. HCMV protease based on the monocyclic β-lactam nucleus which have been described recently are peptidyl, terminal side of the scissile bond are competitive incorporating the sequence specificity of the HCMV inhibitors, such as VVNA (Ki=1.36 mM). protease, as well as non-peptidic (Borthwick et al., 1998; The screening of a totally random combinatorial Deziel & Malenfant, 1998; Yoakim et al., 1998a,b). library of tetrapeptides, made from both natural and Acylation of the active site serine takes place in minutes, unnatural amino acids reveals competitive inhibitors in and the acylated serine is more slowly hydrolysed to the micromolar range of potency (Table 6). Remarkably, regenerate the active enzyme. The more active com- µ the optimal sequence uncovered from the random mix- pounds range in IC50 from 70 nM to 20 M (Figure 3b) tures (Tyr-Tbg-Asn-Gly) has elements of substrate with good selectivity against other serine proteases. A recognition in it, with Tyr at P4, as in the R site sub- series of novel non-peptidic β-lactam inhibitors incor- strate, Tbg at P3 resembling the natural Val in that posi- porating a benzyl or heterocyclic thiomethyl side-chain tion and Asn in P2, as in the M site substrate. at C-4 and a substituted urea functionality at N-1 with

Noteworthy is the enhanced potency of Gly over Ala in IC50 values in the low micromolar range have been syn- the P1 position of that sequence (not shown). thesized and tested in cell culture in a plaque reduction Substitution of Gly into the P1 position of the afore- assay (Yoakim et al., 1998a,b) (Figure 3c). Although µ mentioned VVNA inhibitor also reduces the Ki by an several show low toxicity (TC50 values >250 M), the µ order of magnitude (Table 6). most potent have EC50 values of 50–80 M, much less The bound conformations of both M and R site- effective than in the enzymatic assay. derived peptides spanning the P9–P1 positions have Acylation of HCMV protease and the concomitant recently been investigated by NMR transferred NOE opening of the β-lactam ring allows positioning of a methods and molecular modelling (LaPlante et al., leaving group next to the released amide nitrogen. On 1998). In the region P4–P1, the peptides take on an this basis a novel fluorogenic β-lactam derivative has extended conformation and the binding of these lig- been synthesized, 4S-(4-methylumbelliferone)-3R- ands is mediated primarily by these four residues. methylazetidin-2-one-1-carboxylic acid (4-meth- Although herpesvirus proteases require occupancy of ylpyridyl) amide. Upon acylation of the enzyme, the an extensive binding site for efficient cleavage of pep- fluorescent umbelliferone moiety is released, forming tides with natural amino acid sequences, smaller pep- the basis for a very sensitive assay. This versatile tool tides interacting with only three or four subsites on enables continuous monitoring of β-lactam hydrolysis the enzyme may form the basis for further inhibitor and has been used to provide acylation and deacylation development. rates of 0.02 s-1 and 0.0004 s-1,respectively. In addition, the compound can serve as an active site titrant of Mechanism-based inhibitors HCMV protease concentration (Bonneau et al.,1999) The monocyclic β-lactam nucleus has been used as a Benzoxazinones with two substitutions are well- scaffold to design time-dependent, mechanism-based characterized as mechanism-based inhibitors of mam- inhibitors of the serine protease human leukocyte elas- malian serine proteases of the chymotrypsin tase. This approach has led to the development of high- super-family (Teshima et al., 1982). Examples are the ly potent inhibitors of elastase which are also orally highly potent and selective 2-amino-5-methyl-benzox-

Antiviral Chemistry & Chemotherapy 11:1 13 L Waxman & PL Darke

Figure 3. Representative structures of inhibitors of herpesvirus proteases, not including peptide-based inhibitors

′

β β (a) -lactam template. (b) Substrate-based -lactam inhibitor (IC50 70 nM against HCMV protease) (Deziel & Malenfant, 1998). (c) Non-peptidic β µ µ µ -lactam (IC50 10 M against CMV protease and EC50 53 M) (Yoakim et al., 1998a). (d) 2-Amino benzoxazinone (IC50 5 M against HSV-1 pro- µ µ tease). (e) Thieno[2,3-d]oxazinone (IC50 1.6 M against HSV-2 protease and 1.3 M against CMV protease) (Jarvest et al., 1997). (f) µ µ Spirocyclopropyl oxazolone (IC50 0.14 M against HSV-2 protease and 0.16 M against CMV protease) (Pinto et al., 1996). (g) Benzylidene N- µ µ sulphonyloxyimidazolone (IC50 0.4 M against CMV protease) (Pinto et al., 1996). (h) CL13933 (IC50 5 M against CMV protease) (Baum et al., µ µ 1996b). (i) PTH2 (IC50 0.3 M against CMV protease) (Baum et al., 1996c). (j) Benzimidazole sulphoxide (IC50 1.9 M against CMV protease and µ µ µ EC50 18 M) (Flynn et al., 1997). (k) Salcomine (IC50 1.4 M) ( Watanabe et al., 1998). (l) Bripiodionen (IC50 30 M) (Shu et al., 1997). (m) Sch µ µ 65676 (IC50 24 M) (Chu et al., 1996). (n) Enedione derivative of 5-methylthieno[2,3-d]oxazinone (IC50 30 nM against CMV protease, 2.7 M against HSV-2 protease and 1 µM against VZV protease) ( Pinto et al., 1999).

azinone inhibitors of human leukocyte elastase, which inhibitors in terms of structure–activity and mechanism are stable in aqueous solution (Krantz et al.,1990). are peptides directed toward the HCMV protease with Inhibition is by formation of a stable acyl-enzyme com- a C-terminal activated ketone (Bonneau et al., 1997; plex by attack of the active site serine on the carbonyl Ogilvie et al., 1997). This classical serine protease group of the inhibitor. X-ray crystallography has been inhibitor design renders competitive inhibitors, with the used to obtain the three dimensional structure of an formation of reversible hemiketal adducts with the acyl-enzyme inhibitor complex of elastase hydroxyl group of the catalytic serine residue. The basis (Radhakrishnan et al., 1987). Building upon these find- for this series of compounds is the N-terminal side of ings, Jarvest et al. (1996) synthesized benzoxazinone the M site and the observation that the minimum inhibitors of the HSV-1 protease with IC50 values in the length required for an inhibitor with low micromolar low micromolar range and half-lives ranging from hours activity is the tetrapeptide, as in acetyl-VVNA-trifluo- µ to days (Figure 3d). A 1:1 adduct is formed with the romethyl ketone (IC50 3 M). With the minimum core protease, consistent with formation of an acyl-enzyme established, these investigators systematically examined complex ( Jarvest et al.,1996). Substituted 2-amino- each amino acid moving from P1 to P4 with the aim of benzoxazinone analogues inhibit the HCMV protease optimizing the side chain at each position. With P1 µ with IC50 values of 0.2–2 M and half-lives in plasma of through P3 optimized, the amino acid at P4 can be 0.5–2 h (Abood et al.,1997). Several of these have mod- replaced with a capping group, resulting in the com- est selectivity relative to chymotrypsin and elastase and pound N-tert-butylacetyl-L-tert-butylglycyl-L-N_,N_- antiviral activity in cell culture. Based upon an earlier dimethylasparagyl-L-alanyl-trifluoromethyl ketone µ study, which recognized that the inhibitory potency of (IC50 1.1 M) (Figure 4). A shorter peptide region these compounds was inversely dependent on the size of results in a loss in potency by nearly an order of magni- the 5-substituent ( Jarvest et al., 1996), the steric bulk at tude, in accord with the observation that occupancy of this position was reduced by replacing the benzene ring the P3 position is necessary for cleavage of peptide sub- with a thiophene ring ( Jarvest et al., 1997). The result- strates (see ‘Substrate specificity and assays’). Variations of ing thieno[2,3-d]oxazinone inhibitors (Figure 3e) have the group activating the carbonyl group have been micromolar activity against HSV-1, HSV-2 and incorporated successfully into the design of pep- HCMV proteases ( Jarvest et al.,1997). The struc- tidomimetic inhibitors of other proteases (Mehdi, ture–activity relationships of these compounds for HSV 1993). Thus for HCMV, an increase in potency is µ and HCMV do not run in parallel. The finding of achieved for the pentafluoroethyl ketone (IC50 0.1 M) α µ micromolar potency for both HSV and HCMV pro- and the -ketoamide (IC50 0.2 M) (Figure 4). There is teases has led these researchers to speculate whether, in good selectivity against human leukocyte elastase, chy- spite of their sequence divergence (30% identity), it may motrypsin and the thiol protease cathepsin B. Some of be possible to design a generic inhibitor of herpesvirus these compounds are weak inhibitors of porcine pancre- proteases ( Jarvest et al.,1997). Recently, N-acyl ana- atic elastase which, like HCMV protease, shows a pref- logues of 5-methylthieno[2,3-d]oxazinone have been erence for alanine at P1. The antiviral activity in cell µ described with submicromolar potencies against the culture (EC50) is in the 100 M range, suggesting lack HCMV and HSV-2 proteases and nanomolar potency of cell permeability or degradation of the compounds. against the VZV protease ( Jarvest et al., 1999). In HSV- It is worth noting that the formation of a reversible, 2 virus-infected cells several of these compounds have covalent bond from the active site Ser to the carbonyl of good selectivity, inhibiting protease processing at the an inhibitor would be expected to add significantly to the µ 50% level at concentrations near 10 M while the TC50 binding affinity of the supporting inhibitor scaffold. Yet values were 30-fold higher. the IC50 values of the activated carbonyl compounds dis- Two unique families of serine protease inhibitors cussed above are only slightly lower than for a similar were identified by screening those compounds with the tetrapeptide carboxylate, which will not form such a link- potential to form stable acyl-enzyme adducts with the age, as shown in Table 6. It may be that the binding mode active site serine. The spirocyclopropyl oxazolones of the carboxylate is fundamentally different, wherein the (Figure 3f ) and the benzylidine N-sulphonyloxyimida- large increase in potency upon change from amide to car- zolones (Figure 3g) have been shown to be submicro- boxylate is accompanied by a shift in the binding mode of molar inhibitors of HSV-2 and HCMV proteases with the P1 residue. A shift of the carboxylate toward the some selectivity relative to the mammalian serine pro- Arg166 in the oxyanion hole is conceivable. teases elastase, trypsin and chymotrypsin (Pinto et al., The rates of association of the activated-carbonyl pep- 1996). tide inhibitors with HCMV protease have been investi- Perhaps the best characterized herpesvirus protease gated by changes in fluorescence and near-UV circular

Antiviral Chemistry & Chemotherapy 11:1 15 L Waxman & PL Darke

dichroism upon inhibitor binding (Bonneau et al., 1997). peptidyl inhibitor complexed to the enzyme (Tong et al., An identical blue shift in the enzyme fluorescence max- 1998). The existence of these ligands in solution in the imum occurs in the presence of numerous peptide deriv- bioactive conformation with respect to their backbone atives, suggesting a similar structural reorganization of geometry can account in part for the observed inhibito- the enzyme upon inhibition. In addition, the slow onset ry activity of activated ketone inhibitors with similar of inhibition of substrate hydrolysis observed with triflu- peptidyl sequences. In particular, having a similar con- oromethyl ketone and pentafluoroethyl ketone deriva- formation in the unbound and bound state effectively tives correlates with the kobs for the time-dependent reduces the loss of entropy owing to conformational change in the emission spectra. The observations are adjustment that typically occurs when a ligand binds. consistent with inhibition of the HCMV protease by Although the X-ray crystallographic comparisons peptidyl ketones involving a conformational change in and fluorescence studies summarized above support the the enzyme. However, the α-ketoamide and methyl view of the HCMV protease as an induced-fit enzyme, ketone derivatives exhibit spectral changes too rapid to perhaps the best evidence was provided by the observa- be measured. The trifluoromethyl and pentafluoroethyl tion of a conformational change during substrate derivatives are hydrated to hemiketals in aqueous solu- hydrolysis (LaPlante et al., 1999). The induced-fit tion, while the α-ketoamide and methyl ketone deriva- mechanism is consistent with the activating effect of tives are not, suggesting that slow inhibition kinetics for salts on the catalytic efficiency of herpesvirus proteases the fluorinated compounds is a result of an essential (Hall & Darke, 1995; Yamanaka et al., 1995). Thus, a dehydration prior to productive binding in the active site. blue shift in the fluorescence emission maximum upon

It is worth noting that covalent binding is not a prereq- addition of increasing concentrations of Na2SO4 was uisite for the spectrally observed conformational transi- observed in parallel with a 75-fold increase in catalytic tion to occur, as evidenced by NMR data showing that efficiency mostly owing to a 20-fold decrease in Km the 13C-labelled methyl ketone derivative is not attacked (LaPlante et al., 1999). by the active site serine (Bonneau et al., 1997). What are the implications of these observations for Bonneau et al. (1997) have proposed that the confor- drug design? In the case of the peptidyl activated car- mational change may be required to correctly position bonyl inhibitors, both the peptide moiety required to the active site residues for facile attack on the carbonyl, induce the transition to the active form of the enzyme leading to the formation of a stabilized tetrahedral and the electrophilic keto group that reacts to form the adduct, that is, an ‘induced fit’. This property sets it covalent complex mimicking the transition state are apart from classical serine proteases such as chy- important. Interactions between the enzyme and the motrypsin, which appear to be ‘lock and key’ enzymes as amino acid side chains of the inhibitor could be further determined from X-ray crystallographic studies. In fact, optimized, as evidenced by the poor binding of the it is now clear from X-ray crystallography structures that methyl ketone (Figure 4), with the support of molecular there is substantial accommodation of the inhibitor by modelling and X-ray crystallography. However, the the enzyme, with enhancement of the binding pockets >1000-fold increase in potency in going from the methyl S1 and S3 (Tong et al.,1998). Interestingly, a similar ketone to the trifluoromethyl ketone (Figure 4) suggests suggestion was made for human leukocyte elastase by that the strongest effects on potency would be achieved Stein et al. (1987a,b) that substrate binding is required upon changes in the activated carbonyl moiety. On the to engage the catalytic triad. other hand, Sali et al. (1992) have proposed that mole- These ideas have been amplified recently in a study cules that bind, but are not able to induce conforma- with important implications for the design of inhibitors tional changes associated with an induced-fit enzyme, of herpesvirus proteases (LaPlante et al., 1999). Using would have greater potential as inhibitors because the NMR techniques to study the conformation of peptidyl free energy of binding would not be channelled into ketone inhibitors related to those shown in Figure 4, bringing about structural changes in the protein. The β- these investigators concluded that these compounds lactam inhibitors, which do bind at the active site, do exist in solution primarily in a rigid, extended peptide not induce structural changes in the HCMV protease as structure and that the bulky side chains, in particular the determined by fluorescence emission (LaPlante et al., P3 tert-butyl group, are important in maintaining this 1999). Thus, in contrast to the peptidyl inhibitors, conformation. Transferred nuclear Overhauser effect intrinsic binding energy appears to be a more important studies showed that the enzyme-bound conformation of factor in determining potency. These β-lactam these inhibitors was similar to the free solution structure inhibitors are very slowly cleaved by the protease with little conformational adjustment, in agreement (Bonneau et al., 1999), but the Km is 1000-fold lower with the X-ray crystallographic structure of a related than a typical substrate, as would be expected for the

Figure 4. Peptide-based inhibitors, with a ketone formation. CL13933 (1,1′-(dithio-di-o-phenylene)-bis- positioned for reaction with active site serine (5-phenylbiguanide), Figure3(h) is a symmetrically sub- µ stituted disulphide with an IC50 of 5 M that is reversed

by the reducing reagent bismercaptoethyl sulphone (Baum et al., 1996b). At high CL13933 concentrations,

covalent adducts form with the protease on all five Cys residues. Modification of both Cys161 and Cys138 is

required for inhibition; mutants with either Cys161 or Cys138 changed to Ala are not inhibited by CL13933.

At lower concentrations where adduct formation is not observed, CL13933 promotes formation of specific intramolecular disulphides between Cys84 and Cys87, µ and between Cys138 and Cys161, whereas Cys202

remains unpaired. Only the disulphide bond between

Cys138 and Cys161 is responsible for inhibition, and

inhibition occurs via thiol-disulphide interchange. The second compound identified by Baum and col-

leagues is 1,4-dihydro-7,8-dimethyl 6H-pyrimido[1,2- b]-1,2,4,5-tetrazin-6-one (PTH2) (Figure 3i), a

compound structurally related to flavins, which has an µ IC50 of 0.3 M (Baum et al., 1996c). The actual Data from LaPlante et al. (1999). inhibitor is the oxidized form, PT, which is generated in

solution from PTH2 in the presence of dissolved oxy- tighter binding of an inhibitor that does not induce the gen. PT is rapidly reduced by the enzyme’s thiol groups protease-activating conformational change. Thus an resulting in the formation of disulphides between Cys84 ideal inhibitory molecule may be one that is able to bind and Cys87, and between Cys138 and Cys161. That a in the absence of the conformational change needed to cyclic redox-dependent mechanism is responsible is activate the enzyme, as with a β-lactam, but which is not based upon the observation that substoichiometric susceptible to turnover by the unactivated enzyme. amounts of PTH2 inhibit the protease in a time-dependent manner. As demonstrated with CL13933, loss of

Cysteine-specific reagents activity by PTH2 as well as by flavin can be ascribed to HCMV protease is sensitive to cysteine-specific the formation of the Cys138/Cys161 disulphide pair. reagents including iodoacetic acid and N-ethyl- How might generation of the Cys138/Cys161 disul- maleimide (Baum et al., 1993; Burck et al., 1994; phide result in loss of activity? One possibility suggest- Stevens et al.,1994). All five Cys residues of the native ed by the X-ray structure (Tong et al., 1998) is that the HCMV protease monomer are reduced, and four can be disulphide is near the protease active site and interferes titrated with 5,5′-dithiobis-(2-nitrobenzoic acid) sterically with substrate binding. A second is that the (DTNB). The fifth is accessible only after protein disulphide prevents the conformational change that denaturation (Flynn, et al. 1997). Only Cys161 is con- occurs upon binding substrate (Bonneau et al., 1997). served among the herpesvirus proteases and mutagene- Flynn et al. (1997) have also recognized the potential sis studies of the proteases from HSV-1, simian CMV of targeting the cysteine residues of the HCMV pro- and HCMV demonstrate that this residue is not essen- tease. Their approach takes as precedent the successful tial for catalytic activity (Cox et al., 1995; Liu & anti-ulcer agent omeprazole, a member of the benzimi- Roizman, 1993; Welch et al., 1993; Stevens et al., 1994; dazolyl-methyl sulphoxide class whose mechanism Baum et al., 1996b). Baum et al. (1996b,c) have also involves activation at low pH to the sulphenamide which shown that activity is retained when each of the five cys- then reacts with a sulphhydryl group on the H+/K+- teines of the HCMV protease is individually mutated to ATPase to form the disulphide-inhibitor complex Ala, or when multiple cysteines are replaced (Lindberg et al., 1987). Although omeprazole itself does (Cys84Ser/Cys87Ser/Cys202Ala and Cys138Ala/ not inhibit the HCMV protease, related compounds are Cys161Ala). active against HCMV protease, such as Figure 3(j), µ In a series of elegant studies, Baum et al. (1996b,c) which has an IC50 1.9 M in the enzymatic assay and identified two compounds by random screening that exhibited antiviral activity in a cell-based assay (EC50 18 inhibit by catalysing intramolecular disulphide bond µM). Its mechanism of action requires an accessible

Antiviral Chemistry & Chemotherapy 11:1 17 L Waxman & PL Darke

µ sulphhydryl group on the enzyme, so the compound does Daltons) having IC50 values near 30 M. Quanolirones not inhibit a variety of mammalian serine proteases. I and II, two new naphthacenequinone glycosides hav- Studies with the Cys138Ala mutant have implicated this ing masses of 564 and 694 Daltons, inhibit the HCMV µ residue of the protease in the mechanism of inhibition. protease with IC50 values of 14 and 35 M, respectively Since dimerization of the 16 kDa and 14 kDa pieces of (Qian-Cutrone et al., 1998). Currently no information is the two chain enzyme was observed to take place when available regarding the mechanism of inhibition of any incubation mixtures were analysed by non-reducing of these compounds or their specificity. SDS-PAGE, a second sulphhydryl, perhaps Cys161, may be involved via a disulphide exchange. Conclusions Pinto et al. (1999) have taken advantage of the properties of Cys161 and designed a class of bifunctional Significant progress has been made recently in the dis- inhibitors of the HCMV protease (Figure 3n). These covery of new leads for the development of herpesvirus compounds are enedione derivatives of the potent protease-based therapeutic agents. In particular, the thieno[2,3-d]oxazinones described by Jarvest et al. short peptide-based inhibitors for HCMV protease have (1999) which were discussed previously in ‘Mechanism- advanced in terms of understanding their mechanism of based inhibitors’. However, they not only acylate the cat- action, and structure–activity relationships are being alytic serine residue of the HCMV protease but also elaborated. The peptide nature still present may preclude alkylate Cys161 via a Michael type addition. Such a antiviral potency, but precedent in the conversion of pep- mechanism could account for the nanomolar potency of tides to cell-penetrating peptidomimetics is good. A rel- these compounds which is not achieved by the corre- evant precedent in this regard is the success with sponding saturated analogues (700-fold higher) which inhibitors of the HIV protease, for which medicinal are unable to react with Cys161. Modelling studies of chemists were forced to begin with hexapeptide-based one dual inhibitor show Cys161 lying in close proxim- inhibitors having no cell penetration, but ultimately were ity to the amide terminus of the dione double bond, able to develop orally bioavailable, non-peptide well situated for Michael addition. Interestingly, a sim- inhibitors of great therapeutic benefit. Also potentially ilar interaction with the corresponding cysteine in the significant as leads for herpesvirus therapeutics are the VZV or HSV-2 protease did not appear possible mechanism-based benzoxazinones and lactams. because of the enedione function being twisted out of The inhibitor leads at this time are of modest poten- conjugation by the A6 helix in these enzymes. cy;however, the proven active site targeting and the Accordingly, these compounds are much poorer recent crystallographic definition of the binding cleft inhibitors of the VZV and HSV-2 proteases. The [2,3- bode well for further binding optimization. In addition, d]thienooxazinone enediones may be the most potent the prospect of attaining a useful therapeutic index with inhibitors of the HCMV protease and they are also very potent inhibitors of the herpesvirus proteases is toxic (Pinto et al., 1999), presumably owing to enhanced by the lack of any known herpesvirus protease Michael-acceptor reactivity. homologues in human cells. With the strong beginnings Watanabe et al. (1998) have recently found that sal- described herein, it appears likely to us that improved comine [N,N′-bis(salicylidene)-ethylenediamino-cobalt therapy can be achieved, driven by a sustained medicinal (II)] (Figure 3k) as well as several derivatives inhibit the chemistry effort that is coordinated with the biochem- µ HCMV protease with an IC50 value of 1.4 M. Derivatives istry, structural biology and virology now available for lacking cobalt or those in which cobalt is replaced by nick- this family of viruses. el are not inhibitory. With other serine proteases, the IC50 of salcomine is in excess of 200 µM. In cell culture, plaque Acknowledgements µ formation is inhibited by an EC50 value of 2–3 M, whereas the concentration required for 50% toxicity (TC50) is 36 The authors would like to thank D Olsen, L Kuo, D µM. The mechanism of inhibition of the protease or of Hall, M Stahlhut and J Cole for their useful editorial viral replication by salcomine is not known. suggestions and substantive contributions to our understanding of herpesvirus proteases. Natural product inhibitors Three preliminary reports have described the isolation References of inhibitors of the HCMV protease from microbial fer- mentation broths. Bripiodionen (Shu et al., 1997) Abood NA, Schretzman LA, Flynn DL, Houseman KA, Wittwer AJ, Dilworth VM, Hippenmeyer PJ & Holwerda BC (1997) (Figure 3l) and Sch 65676 (Chu et al., 1996) (Figure Inhibition of human cytomegalovirus protease by benzoxazinones 3m) are both low molecular mass compounds (300–400 and evidence of antiviral activity in cell culture. Bioorganic and

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Received 20 May 1999; accepted 3 August 1999