The herpesvirus proteases as targets for antiviral chemotherapy

22 Pages·2003·0.51 MB·English

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: [phone] ; Fax: [phone]; 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 Herpesviridaefamily 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
characterization
enzymes are essential for herpesvirus replication in cell
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)
Apeler et al (1997)
protease for viral replication was implicated by the dis-
Weinheimeret 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-
Burcket al.(1994)
tion in replication have appeared,including the preven-
LaFeminaet 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 (Gaoet al.,1994;Matusick-Kumaret 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)
HHV-8 E. coli Unal et al.(1997)
pesvirus proteases, which impact inhibitor discovery,
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 (Flynnet 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 (Thomsenet 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 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 sub-
strate (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 (Welchet al.,1993;Joneset 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; Welchet 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 (Tigueet 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 (Holwerdaet 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 (Welchet 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
4 ©2000 International Medical Press
Herpesvirus proteases as antiviral targets
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
proteases while emphasizing recent progress as it relates
HSV TYLQASEKFK R site
to the development of assays useful for screening and
ALVNASSAAH M site
CMV SYVKASVSPE R site mechanistic studies.
GVVNASCRLA M site
DDVEAATSLS I site Substrate specificity and kinetic parameters
As discussed above,the herpesvirus proteases are synthe-
Well conserved amino acids are shown in bold.
The sequences shown are taken from Welch et al.(1993). sized as precursorsthat 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 pro- teases. For example, early reports for k /K values for
cat m
vide a starting point for the design of inhibitors which the HSV-1 and HCMV proteases of 38 M-1s-1and 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
Substrate Conditions* K (mM) k (min-1) kcat/Km (M-1s-1) Reference
m cat
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(NO) 25% glycerol 0.125 35 4667 Margosiak et al. (1996)
2
Abz-VVNA↓SSRLY(NO)R 0.5 M NaSO 0.76 12.0 260 Bonneau et al. (1998)
2 2 4
Abz-Tbg-Tbg-Asn(NMe)A↓SSRLY(NO)R 0.5 M NaSO 0.0032 3.06 15940 Bonneau etal. (1998)
2 2 2 4
Ac-Tbg-Tbg-Asn(NMe)A↓AMC 0.5 M NaSO 0.0132 2.1 2650 Bonneau et al. (1998)
2 2 4
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↓NH 20% glycerol 0.417 0.633 25.3 Waxman, unpublished
2
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, -SCHCH or thiobenzyl ester.
2 6 5
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 104M-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 105M-1s-1found 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 βcarbon 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 K for peptide substrates, and the additional
m
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 k /K (Sardana et al., 1994), but larger side reconcile the marked preference for P3 hydrophobic
cat m
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.
6 ©2000 International Medical Press
Herpesvirus proteases as antiviral targets
(1998) have combined a useful N-dimethyl-asparagine of detection,a rapidly cleaved substrate offers ease of use
[Asn(NMe)] substitution at P2 with the aforemen- with nanomolar concentration of enzyme that is desir-
2
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(NO )] (Handa et al,
2
(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(NMe )- able HCMV protease substrate, DABCYL-
2
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(NO) (Table 3) provides adequate sensitivity to
2
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 sub-
strates 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 sub- strates and has suggested that modified peptide sub-
strate requirements, such as in the studies cited above, strates have a lower K than their unaltered peptide
m
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(NO)R can be modified
2
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
Substrate sequence K (µM) k (min-1) k /K (M-1 s-1)
m cat cat m
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(NMe )], to give a substrate with a k Protease hydrolysis of thioesters is often more rapid
2 cat
240-fold greater and a k /K 60-fold greater than that than with the corresponding nitroanilide or AMC
cat m
of the equivalent non-optimized substrate (Bonneau et amide substrates due to the high reactivity of thioester
al.,1998) (Table 3). bonds.K values for thioester hydrolysis by serine pro-
m
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(NMe )-Ala↓AMC (Bonneau et al., are rapidly hydrolysed by the CMV protease (Table 3).
2
1998) (Table 3) (See ‘Substrate specificity and kinetic As with other serine proteases,the k is 200–500-fold
cat
parameters’). In this regard, the herpesvirus proteases greater than for amide substrates,but the K values are
m
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↓NH to RWGVVNA↓S-Abu- reagents (see ‘Inhibitors of herpesvirus proteases’).
2
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(NMe )-Ala↓AMC raises k /K 100-fold and be analysed by HPLC. In consideration of the large
2 cat m
brings K and k parameters into useful ranges (Table increases in specificity constants for optimized
m cat
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
8 ©2000 International Medical Press
Herpesvirus proteases as antiviral targets
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 (DiIanniet al.,1994).Similarly,Ser132 of HCMV pro-
& Roizman, 1993; Welch et al., 1993; McCann et al., tease is so modified (Stevenset 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 transactivity as well as of trypsin, mutation of the active site Asp102 to
for autocleavage of the precursor (Welch et al, 1993). asparagine reduces k by a factor of 104, and a base
cat
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 (Craiket 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-
low K of 3 µM (Table 3). It may be that binding of ter resemble the catalytic machinery of trypsin,still per-
m
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
Protease K (µM) Temperature pH Solution components* Method of analysis Reference
d
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 K.
d
†Application of the size exclusion chromatography method used with CMV protease to the determination of K for HSV protease was
d
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 (Lianget 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 (K ) in the
d
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 K values reported for the dimerization of the
d
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
both Arg165 and Arg166 (Qiu et al., 1996), which are HCMV protease the K is about 1 µM in the presence
d
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 K measurement using enzyme
d
10 ©2000 International Medical Press