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 dimer- ization 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 ©2000 International Medical Press 0956-3202/00/$17.00 1 L Waxman & PL Darke 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 cap- sids 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-dimen- sional 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. 2 ©2000 International Medical Press Herpesvirus proteases as antiviral targets 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