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Arvin A, Campadelli-Fiume G, Mocarski E, et al., editors. Human Herpesviruses: Biology, Therapy, and Immunoprophylaxis. Cambridge: Cambridge University Press; 2007.

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Human Herpesviruses: Biology, Therapy, and Immunoprophylaxis.

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Chapter 68Candidate anti-herpesviral drugs; mechanisms of action and resistance


Clinical Virology, GlaxoSmithKline, Research Triangle Park, NC, USA

Research into the molecular biology of herpes replication in recent years has revealed novel targets for drug development (Fig. 68.1). The characterization and functional assay of these targets have been facilitated by advancements in gene expression, protein purification, proteonomics, bioinformatics, and efficient robotic screening technologies. The pipeline for new herpes drugs has been expanding as drug candidates have evolved more rapidly due to improvements in chemical synthesis (i.e., combinatorial and parallel synthesis methods), and with aids for drug design (X-ray crystallography, in silico computer modeling tools, as well as chemoinformatics). Many new herpes inhibitors have been reported, and most of these possess novel modes of actions. Several have entered clinical evaluation, with some later discontinued because of safety issues. This chapter will describe promising drug candidates in early development that appear to act at individual steps of the viral replication cycle, and focus on those that have the most potential for success (Table 68.1).

Fig. 68.1. Herpesvirus replication cycle illustrating precedented and novel drug targets (numbered) and their general stage of function in the viral replication cycle.

Fig. 68.1

Herpesvirus replication cycle illustrating precedented and novel drug targets (numbered) and their general stage of function in the viral replication cycle. (Adapted from Roizman et al., 1993.)

Table 68.1. New anti-herpes inhibitors in the discovery and development pipeline.

Table 68.1

New anti-herpes inhibitors in the discovery and development pipeline.

The chemotherapy of herpes infections was markedly advanced by the discovery of the first, highly selective antiherpetic agent, acyclovir (ACV, Zovirax®; [9-(2-hydroxyethoxymethyl)guanine]) (Elion et al., 1977). Since the introduction of this agent, there has been only incremental progress in new drug approvals for the myriad of diseases caused by this family of diverse pathogens. The drugs approved since the introduction of ACV include valacyclovir (VACV, Valtrex®, the L-valine ester prodrug of ACV, penciclovir (PCV; [9-(4-hydroxy-3-hydroxymethylbutyl-1-yl) guanine]), a related nucleoside analogue with a similar basis for drug action against HSV and VZV, and its prodrug, famciclovir (FCV, Famvir®). More efficacious treatment of CMV disease was achieved with yet another guanosine analogue ganciclovir (GCV, Cymevene®, Cytovene®; 9-(1,3-dihydroxy- 2-propoxymethyl)guanine), and its recently approved prodrug, valganciclovir (Valcyte®; L-Valine, 2-[(2-amino-1,6-dihydro-6- oxo-9H-purin-9-yl)methoxy]-3-hydroxypropyl ester, monohydrochloride),. The treatment of ocular CMV infections was advanced by the antisense agent ISIS 2922 (formivirsen, Vitravene®). Broad-spectrum antiherpetics include the pyrophosphate analogue foscarnet (PFA, Foscavir®; trisodium phosphonoformate, phosphonoformic acid) and the nucleotide analogue cidofovir (CDV, HPMPC, Vistide®; (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine).

Although these agents have proven efficacious in the prophylaxis and treatment of herpes infections, there remains a need for drugs with higher potency, more rapid and durable antiviral action, more convenient dosing regimens, and importantly, fewer and less severe side effects. Because these systemic drugs ultimately target the viral DNA polymerase, and the nucleoside analogs are viral TK-dependent, cross-resistance can occur (Erice, 1999). New drugs with novel mechanisms of actions would provide valuable alternatives. Ideally, the new drug would eliminate the latent reservoir; a challenging goal since the herpesviruses have evolved complex strategies to persist under the reach of host defense mechanisms.

The drug development process: an overview

The scientific literature abounds with reports of the in vitro anti-herpetic activities of diverse organic molecules and biological products, and in many cases, the selectivity index (SI; the ratio of cellular toxicity to antiviral potency in vitro) appears promising (Snoeck et al., 2002). However, the demonstration of in vitro activity is only the first step in the long and arduous journey from the laboratory bench into the clinic (Scolnick et al., 2001). Other considerations, particularly absorption, distribution, metabolism and excretion (ADME) parameters are key to success. Lipinski’s rule-of-five analysis (Lipinski et al., 2001) which established guidelines regarding structural properties most often associated with viable drug candidates has been embraced by the pharmaceutical industry.

The discovery and development process is slow and costly. The industry average to bring a new drug to market was estimated at over $500 million dollars for drugs introduced in 1990 and is undoubtedly higher today (Boston Consulting; Pharmaceutical R&D Costs, 1993). The time from synthesis of a new drug to regulatory approval has grown to over 14 years from an average of 8 years in the 1960s, according to analyses by the Tufts Center for the Study of Drug Development. Figure 68.2 provides a schematic review of some of the major preclinical development activities generally required to advance a drug to approval.

Fig. 68.2. Schematic overview of drug development process.

Fig. 68.2

Schematic overview of drug development process.

Historically, anti-infectives have been discovered through the screening of compound libraries directly against the replicating organism. This classic approach was used in the discovery of all the currently approved systemic antiherpetics. ACV, which was originally synthesized to potentiate the anticancer activity of the nucleoside analogue, cytosine arabinoside (Ara C), by inhibition of adenosine deaminase (the enzyme responsible for its metabolic breakdown), emerged as a potent antiherpetic during random screening in herpes simplex virus (HSV)-infected cells (Elion et al., 1977).

Screening of diverse chemical libraries in virally-infected cell cultures, although less efficient than individual enzyme or target-based screens (discussed below), provides the opportunity to identify new viral targets. Active inhibitors can then serve as laboratory tools to probe the biology of viral replicative events. The mechanisms of novel action are defined by identifying changes in the phenotype of infected, treated cells, often aided by time of inhibitor addition and withdrawal experiments. Antiviral selectivity is indicated by the ability of the virus to develop resistance to escape drug inhibition, and genetic mapping of resistance mutations identifies the viral target(s).

A second approach to the discovery of new inhibitors consists of direct screening of compounds against a catalytic enzyme or other biological function. Often screening and inhibitor optimization may be directed by structural modeling. This approach is highly efficient, and has been particularly successful in the human immunodeficiency virus (HIV) arena, producing candidates or approved drugs targeting HIV entry, fusion, genetic integration, protease, and the HIV reverse transcriptase. Significant efforts to identify herpes protease inhibitors have not been successful to date, but direct screening of herpes DNA polymerases and the helicase-primases have produced exciting drug candidates with strong development potential.

A few principles emerge from the collective experiences in the field of antiviral drug discovery. The ideal target will be unique to the virus, or sufficiently distinct from the host cell counterparts to allow preferential inhibition, and will be essential for viral replication (in vitro for assay purposes) and for disease pathogenesis in the host. The genetic barrier to resistance will be high, and drug-resistant variants will pay a penalty in replication competence or tissue tropism. In the case of the alpha herpesviruses, drug-resistant variants will lack the ability to reactivate from latency.

An active antiviral compound must have other attributes in order to progress successfully through the drug development process. The chemical and pharmaceutical development criteria of cost-effective manufacturing, product stability, good solubility, oral bioavailability, and acceptable protein binding preclude development of high molecular weight compounds, or complex biological products, except as topical or injectable formulations. For the well-defined catalytic sites of many target enzymes, small molecule inhibitors (≤600 Da MW) can provide the ideal “fit.” However, for the less well-defined catalytic sites of some enzymes, or the broad surfaces of protein–protein interactions (Tsai et al., 1997), larger MW inhibitors, such as protein mimetics, may be required. Protein-protein interactions comprise many opportunities for antiviral intervention, but from a pharmaceutical perspective, these remain challenging targets (Arkim and Wells, 2004).

Host cell targets as an approach to virus inhibition

Drugs that target host cellular functions have been considered as potential antiviral agents (Shugar, 1999) and the herpes virus literature documents the antiherpes activity of a variety of host cell kinase inhibitors, such as roscovitin and inhibitors of p38 and cdk-E (Schang, 2002; Schang et al., 2002; Chang, 2003). Investigators have looked at the effects of various antimetabolites on the ability of host cells to support viral replication. Although these are useful probes for discerning the contributions of host functions to viral growth, none have yet been shown to have clinical development potential as systemic therapies for the herpes diseases.

Reveratrol, a natural plant phytoalexin, was recently shown to inhibit HSV 1 and 2 replication early in the infection cycle by inhibition of ICP-4 (Docherty et al., 1999). The mechanism may not directly involve a virus target, but could have application as a topical agent similar to the anti-inflammatory effect of n-docosanol 10% cream (Abreva®). One category of host targets which may be fruitful for the development of novel therapeutics that deserves mention here are immune response modulators. The herpes viruses have developed multiple ways to evade the host’s innate immunity and to block antigen presentation required for the adaptive immune responses. Thus, agents which protect or augment host defense mechanisms by interfering with viral immune evasion functions may provide additional tools in disease management. Such agents could complement antiviral drugs and may potentiate vaccine efficacy (Miller et al., 2002). For example, Imiquimod, an immune response modifier approved for the topical treatment of external genital and perianal warts, has recently been shown to be an agonist to the toll-like receptor 7 (TLR-7) (Hemmi et al., 2002). The related analog, resiquimod, has also been shown to have immunomodulatory properties and efficacy as a topical therapeutic agent in genital HSV-2 infections models (Bernstein et al., 2001; Miller et al., 2002). TRL-7 may be involved in the induction of cytokines, especially alpha interferon, and other antiviral effector molecules which may enhance cell mediated immunity (Wang et al., 2005). Moreover, Imiquimod has also been shown to have potent adjuvant/priming properties in several vaccine models (Rechtsteiner et al., 2005; Thomsen et al., 2004). Several candidate TLR agonists, plus other immunomodulatory agents are in development for various viral diseases, but will not be discussed further.

Antiviral targets in early replication events

In principle, an inhibitor that blocks the very earliest steps in the invasion of a cell by a virus should effectively restrict the spread of infection and could also serve as a prophylactic agent. However, inhibition of herpes viral attachment and uncoating may not be feasible, since there are no unique, restrictive mechanisms for herpes entry that could be exhaustively (and presumably safely) disabled. This is in contrast to the prospects for the antagonist of the CCR5 host receptor element required for HIV infection (Baba et al., 1999), the recently approved HIV fusion inhibitor (LaBranchea et al., 2001) in HIV infection, and the rhinovirus uncoating blocker (Diana et al., 1987).

Immediate early gene (IE) expression or the transactivation functions of their products could after the earliest replication events for intervention, and their inhibition should effectively restrict productive viral infections and potentially decrease reactivation from latency. However, since the transcription of viral genes and the viral-mediated transactivation intimately involve host transcriptional machinery and factors, selectively would be difficult to attain.

Three or four series of compounds with designated early modes of action have emerged from cell-based screening programs. Time-of-addition studies or quantitation of viral transcription and translation indicated that these classes of compounds acted after the adsorption phase of HSV or CMV infection but before viral DNA synthesis. Two striking features of their mechanism of action (MOA) were common to these agents: their in vitro potency was compromised by increased multiplicity of infection (MOI), and the investigators were unable to select resistant viruses, despite repeated attempts to passage virus in the presence of the inhibitors. These features, particularly the latter, are consistent with inhibition of a cellular target. In the case of CMV423 (see below), the inhibition of a host protein kinase is demonstrated. Although the in vitro SIs for these agents indicate at least a preferential inhibition of viral growth, drug development potential of these compounds is doubtful. They will be briefly described.


The activity of the benzothiophene class was identified in a random compound screening program. The lead compound, PD146626 (9-(methyloxy)-3,4-dihydro[1]benzothieno[2,3-f][1,4]thiazepin-5(2H)-one), was shown to inhibit HSV type 1 (HSV-1) replication by blocking immediate early viral gene expression, specifically VP16 and ICPO expression. However, viruses deleted for the VP16 and ICPO loci (“knock-out” viruses) lacked resistance to PD146626, and the compound showed apparent anti-viral activity against CMV, which lacks VP16 and ICP0 homologues. Moreover, PD146626 could exert this inhibitory effect in cells pretreated before viral infection, or in cells treated with only short exposures (up to 2 hours) during viral infection. Thus, PD146626 apparently targets a cellular function critical for the expression of HSV-1 immediate early genes (Boulware et al., 2001). Extensive SAR studies that focused on stereospecific substitution on the diazepine ring and optimal nitrogen substitution achieved striking improvements as evidenced by a 2-log enhancement in potency and a 3-log improvement in therapeutic index. However, in vivo efficacy could not be determined due to metabolic issues, and thus the safety consequences of this inhibitory mechanism remains to be determined (Hamilton et al., 2002).

Non-nucleoside pyrrolopyrimidines 828, 951, and 1028

Three structurally distinct analogues in a series of non-nucleoside pyrrolo[2,3-d]pyrimidines emerged from a cell-based screening program (Jacobson et al., 1999). At low MOIs compounds 828, 951, and 1028 all showed potency comparable to that of GCV. One of these compounds, 828, was tested for toxicity and shown to be less toxic against human bone marrow progenitor cells than GCV, a key improvement.


Perhaps the best-studied agent with activity early in the herpes life cycle is CMV423, 2-chloro-3-pyridin-3-yl-5,6,7,8- tetrahydroindolizine-1-carboxamide. This tetrahydroindolizine derivative is active against CMV, HHV-6, and HHV-7 at low concentrations, but shows only modest activity against HSV-1 and -2 and none against varicella-zoster virus (VZV) (Snoeck et al., 2002; De Bolle, 2004). The synergistic activity against CMV observed when CMV423 was combined with GCV, PFA, or CDV suggested that CMV423 was inhibiting a different step in viral replication, most likely an earlier one, than these DNA polymerase inhibitors. A series of studies aimed at defining the point of inhibition, using the low multiplicity of infection (MOI) multi-cycle format and probing for the expression of the IE (immediate early) and E (early) antigens showed transient reductions in the levels of IE antigen detectable on days 1 and again on days 4 and 5, in concert with first- and second- round of viral replication. Interestingly, CMV423 was able to block substantial expression of IE antigen at the viral input of 0.1 PFU/cell, an MOI at which antiviral activity is lost, indicating that low IE expression is sufficient to overcome the block to replication. This result suggests that any drug directed against the CMV IE gene would have to be almost 100% effective to produce the desired virus suppression.

Further work on the mechanism of inhibition of human herpesvirus (HHV)-6 replication again pointed to a cell target, as inhibition occurred in a cell-line dependent fashion (De Bolle et al., 2004). The molecular target in HHV-6 is most likely a different early event, occurring before viral DNA synthesis but after IE antigen production. Based on the similarity of the action of herbimycin, which is known to inhibit cellular tyrosine kinase activity through binding to heat shock protein (Cirone et al., 1996) and to block infection of human T lymphoid cells by HHV-6, the antiviral action of CMV423 is likely to be mediated through inhibition of a host cell tyrosine kinase. Preclinical safety and pharmacokinetic studies on this interesting inhibitor continue (Aventis, data on file; Bournique et al., 2001). The clinical relevance of the MOI-dependence, and the safety margin with host inhibitory mechanisms, remain to be determined.

ISIS 13312

One very specific and clinically validated inhibitor of CMV IE gene expression is the approved anti-sense agent formivirsen (ISIS 2922, Vitravene®). Its utility is greatly limited due to the need for monthly intravitreal injections, and the occurrence of adverse ocular reactions. ISIS 13312, an analogue of ISIS 2922, has been shown to have a longer half-life than ISIS 2922 (approximately 2 months in monkey retina) and could provide the advantage of less frequent dosing (Henry et al., 2001). However, ISIS 13312 is not currently in clinical development.

Antiviral targets in the herpesvirus DNA replication complex

The six or seven essential proteins that comprise the herpes viral DNA replication machinery offer several attractive enzyme targets for drug development, as well as some of the more challenging protein-protein interactions (Matthews et al., 1993; Anders and McCue, 1996; Loregian and Coen, 2006). These components of the HSV replication machinery include the single-stranded DNA binding protein (ICP8, pUL29), the polymerase accessory factor (pUL42), the helicase-primase complex (pUL5, pUL8, pUL 52) and the viral DNA polymerase (pUL30). HSV requires an origin-binding protein specifically (pUL9). These proteins work in concert, are co-localized within specific intranuclear structures, and are found in association with other viral and host proteins involved in the replication cycle events. In principle, points of antiviral intervention could include those that affect protein recruitment and transport (post-translational modification), the sequentially–ordered protein–protein binding events in replisome assembly, and the individual catalytic functions of the enzymes involved in the DNA synthetic process.

The direct inhibition of the DNA polymerase function through nucleoside/nucleotide substrate analogues or pyrophosphate mimics, such as seen with the ACV, GCV, and PFA, is a clinically-validated approach. Discovery efforts continue to exploit this well-validated target, usually in concert with the viral encoded nucleoside kinase for added selectivity in the monophosphorylation step (or also including thymidylate kinase activity). Successful inhibition of the HIV RT through non-catalytic mechanisms (non-nucleotide reverse transcriptase inhibitors such as nevarapine and efaverenz) have prompted the search for similar agents in herpes discovery screening programs. Representatives in both these categories are in advanced preclinical or early clinical testing. Also in the pipeline are agents that interfere with the function of the helicase primase complex (Fig. 68.3).

Fig. 68.3. HSV DNA replication targets.

Fig. 68.3

HSV DNA replication targets. (Adapted from Crumpacker and Schaffer, 2002.)

Herpesvirus DNA polymerase inhibitors

The herpes virus DNA polymerase is a multifunctional enzyme that possesses both a deoxynucleotide polymerizing activity and a 3′–5′ exonuclease proof-reading function, and the structure of the herpes virus replicating complex has been modeled (Franklin et al., 2001). The polymerase polypeptide shares regions of sequence similarity with the catalytic subunits of other alpha – like DNA polymerases of eukaryotes (Braithwaite and Ito, 1991). The conserved regions involved in substrate recognition within the polymerase have been determined by comparative modeling with the Klenow polymerase, and by genetic analysis of mutants resistant to nucleoside/nucleotide analogs (Gibbs et al., 1998; Larder et al., 1987). These regions (Ⅰ, Ⅱ, Ⅲ, Ⅴ, Ⅶ and the delta ∗region C) are non-contiguous, indicating the broad areas of contact across the polymerase polypeptide during the catalytic polymerization process. The ability of an inhibitor to interfere with correct folding through binding outside of the catalytic sites could impair enzyme function, although mutational escape may be more feasible, based on precedence in the HIV NNRTI series (Spence et al., 1995).

Nucleoside/nucleotide analogue inhibitors

The apparent tolerance of the herpes DNA polymerases for modified acyclic and carbocyclic sugar moieties, exemplified by ACV, GCV, and PCV, drove additional exploration in the purine nucleoside series during the late 1980s and the early 1990s. The discovery of oxetanocins with the structural characteristic of two hydroxymethyl groups located on a rigid 4-membered ring led to the synthesis and antiviral evaluation of a number of related compounds and investigation of their antiviral properties (Sakuma et al., 1991; Sekiyama et al., 1998). Compounds in the oxytanocin series of base analogues, characterized by a carbocyclic sugar moiety, were investigated and showed broad-spectrum activity against the herpesviruses.


Lobucavir, (R)-9-[4-hydroxy-2-(hydroxymethyl)butyl] guanine], (LBV, cygalovir, BMS 180194), a cyclobutyl analogue of guanine arose from the oxytanocin series and was advanced through early clinical evaluations (Yang et al., 1996a, b). The ultimate outcome serves to illustrate the risks associated with the discovery and development process.

LBV has antiviral activity against HIV, hepatitis B virus, and most herpesviruses, and the triphosphate of LBV is a potent inhibitor of hCMV DNA polymerase in vitro. However, Tenney et al. (1997) showed that this nucleoside analogue is phosphorylated intracellularly to its triphosphate form in both infected and uninfected cells, with phosphorylated metabolite levels only two- to three-fold higher in CMV-infected cells compared to uninfected cells. The lack of selective anabolism in virally infected cells (a factor contributing to the broad antiviral activity) provides the potential for substrate utilization by host cell DNA polymerases with corresponding safety risks.

LBV was advanced to the clinic. Preliminary human data showed a dose-related anti-CMV effect (Dunkle, 1996). A clinical study in HIV- and CMV-co-infected patients demonstrated a 50% reduction in CMV viruria and a greater than 1 log reduction in HIV viral load from semen at the highest dose. Side effects were dose-related, and included mild to moderate diarrhea and nausea in 10%–20%, and 7%–12% of recipients respectively (Lalezari et al., 1997).

Despite promising early clinical results, an international Phase Ⅲ study of LBV as therapy for hepatitis B was suspended in February 1999 due to safety concerns. Toxicologic studies in rodents had suggested increased incidence of stomach, vaginal and cervical cancers with long-term exposure.

Omaciclovir H2G and its pro-drug

Another carbocyclic guanosine analogue, H2G, (R)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine (omaciclovir), was shown to be a potent broad-spectrum antiherpes agent especially active against VZV (Abele et al., 1991). The MOA is similar to that of ACV, with less selectivity as a substrate for TK. Resistance mechanisms at the TK locus overlap with those of ACV (Ng et al., 2001). H2G is not an obligate chain terminator, although once incorporated, H2G-MP will only support limited chain elongation (Lowe et al., 1995). The triphosphate of H2G has a considerably longer intracellular half-life in infected cells than does ACV-triphosphate (Lowe et al., 1995), a feature that could provide dosing advantages over VACV if clinically validated. Preclinical efficacy studies in the simian varicella model indicated superior potency over ACV (Soike et al., 1993). However, species-specific differences in metabolism of ACV make such comparisons misleading. ACV oral bioavailability is lower in monkeys than in humans, and the higher aldehyde oxidase levels in monkeys result in faster metabolic clearance in monkeys than in humans, dogs, and rodents (de Miranda and Burnette 1994; de Miranda and Good, 1992).


(ABT-606; [L-valine-(2-hydroxy-4-hydroxymethyl-butylyl) guanine]) is a prodrug of H2G that significantly enhances its oral bioavailability. MIV-606 is quickly converted to H2G, with undetectable concentration of parent prodrug MIV-606 (Medivir AB, Huddinge Sweden, unpublished data). – Three phase I studies with a total of more than 100 volunteers, including subjects 65 years of age and older, demonstrated that MIV-606 was safe and well tolerated after multiple dosing up to total daily doses of 1500 mg.

A phase Ⅱ study comparing 250, 500, and 750 mg twice daily of MIV-606 with 800 mg five times a day of ACV in zoster patients has also been completed. Trial results suggested equivalent or superior efficacy of MIV-606, compared to ACV, at significantly lower doses. If this claim can translate into an improved therapeutic effect of MIV-606 at a more convenient dose, it could provide enough improvement over current therapies to justify further development. Availability of MIV-606 could potentially lead to much wider treatment of zoster, and the broad spectrum of action could benefit patients with compromised immune function, such as transplant recipients, cancer patients, and AIDS patients (Medivir AB, Huddinge Sweden, unpublished data).

Alkoxyalkyl esters of cidofovir (CDV)

CDV is a nucleotide (monophosphate) analog with broad spectrum anti-herpes activity that is licensed for the intravenous treatment of CMV retinitis in HIV-infected patients. CDV is phosphorylated by cellular enzymes, and the CDV-diphosphate (DP) is a competitive inhibitor of viral DNA polymerase (Safrin et al., 1997). Mechanistically, CDV-DP inhibits many viral DNA polymerases, and recent studies document activity against pox viral infections (Neyts et al., 2004; De Clercq, 2002). In CMV, resistance to CDV can arise from single point mutations in the polymerase locus, usually mapping to the exonuclease functional domains (Chou et al., 2003).

CDV exhibits a number of drawbacks that greatly limit its utility as an anti-herpetic agent. Oral bioavailability is low (<5%) requiring Ⅳ administration, usually on a weekly or semi-weekly basis, and dose-dependent nephrology may require pre-hydration, dose reduction and/or co-treatment with probenecid. Other safety liabilities were documented in preclinical toxicology studies Vistide® [package insert], Gilead Sciences, 1999.

Prodrug strategies, accelerated by the threats of bioterrorism, have been employed to increase oral bioavailability and improve the safety profile of CDV (Huggins et al., 2002). The basic prodrug design exploited a natural fatty acid (lysophosphatidylcholine) molecule as carrier to facilitate drug absorption in the gastrointestinal tract. The lipid ester conjugates were much more active in vitro (EC50 values at least 100-fold lower) than CDV or cyclic CDV against a range of herpes viruses, including strains of HSV, VZV, CMV, EBV, HHV-6, and HHV-8. SAR of the ether lipid ester analogs defined a 20 atom optimum for alkyl chain length, and explored the nature of the linker group (Williams-Aziz et al., 2005). Consistent with the observed increase in antiviral potency of the 1-O-hexadecyloxypropyl conjugate of CDV in cell culture, studies with radiolabelled compound confirmed increased cell penetration (10–20 fold) and higher intracellular levels (100-fold) of the active antiviral form CDV-DP than those measured in cells treated with CDV parent drug (Aldern et al., 2003).

These lipid carrier prodrugs showed significant advantages over CDV in several in vivo models of murine and human CMV (Bidanset et al., 2004; Kern et al., 2004a; Wan et al., 2005; Kern et al., 2004b). Higher levels of protection were also achieved with the CDV oral prodrugs in a lethal cowpox challenge model (Huggins, 2002). Studies evaluating the oral bioavailability and tissue distribution of 14C-labeled hexadecyloxypropyl-cidofovir (HDP-CDV), octadecyloxyethyl-cidofovir (ODP-CDV), and oleyloxypropyl-cidofovir (OLP-CDV) in female NIH Swiss mice demonstrated that these alkoxyalkyl esters are highly orally bioavailable (88–97%) and do not concentrate in the kidney (Ciesla et al., 2003). Thus these compounds may avoid the dose-limiting toxicity of CDV, if these results translate into the clinic.

The lead compound, CMX001, will be progressed through phase I safety and pharmacokinetic studies in humans for potential use as a smallpox treatment or vaccine rescue (Painter and Hostetler, 2004). Such a therapeutic could also provide a safer salvage therapy for ACV or GCV resistant viruses in immunocompromised patients with life-threatening herpes infections.

This prodrug strategy was successfully applied to another nucleotide phosphonate 9-(S)-(3-Hydroxy-2-phosphonomethoxypropyl)adenine [(S)-HPMPA] with a similar enhancement of in vitro antiviral potency (Beadle et al., 2006).


Armed with structure activity relationship (SAR) clues from the crystal structure of the HSV-1 TK complexed with GCV (Brown et al., 1995), and substrate potency comparisons with ACV, PCV, H2G and the oxytanocins, the scientists at Ajinomoto set out to design a novel series of nucleoside analogues. Extensive exploration of the side chain conformation and enantiomeric specificity in the oxytanocin series lead to the identification of potent activity in a compound with a cyclopropyl sugar (Sekiyama et al., 1998). The lead molecule in this series, A-5021, (1′S,2′R)-9-{[1′,2′-bis(hydroxymethyl)cycloprop-1′-yl]methyl}guanine, showed superior in vitro potency over the gold standards ACV or PCV against HSV-1 and VZV in vitro; however, the difference for HSV-2 was only marginal (Iwayama et al., 1998). The compound was also active against EBV and HHV-6, but not HHV-8 (Neyts et al., 2001). Since HSV-2 infection remains the most prevalent disease worldwide, A-5021 must show other development advantages over ACV, VACV and FCV to warrant the time and development investment for the genital herpes indications.

The mechanism of action of A-5021 was investigated (Ono et al., 1998) and found to be qualitatively similar to that of ACV and PCV. A-5021 is anabolized to the monophosphate by the herpes TK enzymes and to the diphosphate by GMP kinase, as is ACV-MP. Levels of A-5021 triphosphate accumulating in HSV-1 or VZV-infected cells were higher than those for ACV-TP, but were roughly comparable to PCV-TP levels. The intracellular half life of A-5021-TP was longer than that of ACV-TP, but somewhat shorter than that of PCV-TP. However, ACV-TP had the most potency at the level of HSV DNA polymerase inhibition, with A-5021-TP intermediate in potency. Incorporation studies showed A-5201-MP could be incorporated into a growing DNA chain, although subsequent chain elongation was inefficient. The anti-HSV-1 and -2 activities were shown to be potentiated in vitro by the immunosuppressive agent mycophenolate mofetil, a finding consistent with the mechanism of competitive inhibition of GTP incorporation, since this agent is known to cause a reduction in cell dGTP pools (Neyts and De Clercq, 2001). A disadvantage of A-5021 is the likely cross-resistance with the most prevalent phenotype of ACV-resistant HSV, the TK-deficient phenotype.

A series of in vitro and in vivo studies suggested potential advantages of A-5021 over ACV for infections mediated by HSV-1. A-5021 exhibited more prolonged antiviral action than did ACV after short exposure of infected cells in vitro. This superior potency and durability carried over into several animal models of HSV-1 infection (Iwayama et al., 1999). In a comparison using once-a-day oral administration with equivalent 25 mg doses, A-5021 demonstrated advantages over ACV in reducing the severity in HSV-1 cutaneous lesions. While the oral bioavailability and AUC of A-5021 is approximately half that of ACV, the superior in vitro potency and the prolonged effect contributed to better efficacy in this cutaneous HSV-1 murine infection model. When initiation of therapy by the intravenous route (100 mg/kg) was delayed to day 4 postinfection, A-5021 again was more effective in diminishing disease spread than ACV or PCV.

A-5021 treatment also resulted in a complete survival of mice infected intracerebrally with HSV-1 after Ⅳ dosing with 25 mg/kg A-5021 TID, compared to only 50% survival at 100 mg/kg Ⅳ ACV TID dosing (Iwayama et al., 1999). The levels of A-5021 in the brain were not presented. Higher uptake of antiviral agent into the infected organ could be a major factor in the superior efficacy of A-5021 in this model, and a clear advantage of A-5021 over ACV, which has limited ability to penetrate the blood-brain barrier (de Miranda and Good, 1982).

In another variation of time of addition and withdrawal treatments in the animal model, high dose intrapertioneal (ip) infection of SCID mice was followed by once daily subcutaneous treatment (50 mg/kg). After 4 days of treatment, initiated at 1 hour, or 1 or 2 days post-infection, the delay in mortality and ultimate number of survivors was far greater in the A-5021 treated groups than in the ACV-treated groups (Neyts et al., 2001). No pharmacokinetic information was provided to allow comparisons of actual systemic exposures.

In contrast to the superior performance of A-5021 against the gold standard of ACV in all the HSV-1 infection models, the efficacy of A-5021 was not distinguished from that of PCV in a model of systemic infection with HSV-2 (Iwayama et al., 1999).

A-5021 has entered clinical development. It remains to be seen if the advantages in potency and duration of antiviral effects seen in cell culture and animal models will translate into superior efficacy in the various HSV and VZV disease indications. The ophthalmic use for herpetic keratitis is under development. Another proposed clinical application would be its use in gene therapy approaches to cancer, utilizing the HSV-1 TK vectors, since A-5021 is less cytotoxic than GCV, which is currently used (Hasegawa et al., 2000).

BCNA compounds

A new structural class of bicyclic furo pyrimidines (BCNAs) have recently been discovered that demonstrate both highly specific and selective anti-VZV in vitro activity (Balzarini and McGuigan, 2002; McGuigan et al., 2003; De Clercq, 2003a,b). The starting point for these compounds was BVDU, ((E)-5-(2-bromovinyl)-2′-deoxyuridine), which was established in the early 1980s as having good antiviral activity but low selectivity. The BCNAs are characterized by a long alkyl or alkylaryl side-chain at the base moiety that may be responsible for both their antiviral properties and their lipophilic properties. The compounds are highly potent at sub-nanomolar concentrations, and cytotoxicity has not been observed at high micromolar concentrations.

The MOA has not been fully elucidated, but the compounds lose their antiviral activity against TK-deficient VZV strains, demonstrating that phosphorylation by the VZV-encoded TK is essential. Kinetic studies with purified enzymes revealed that the compounds were indeed a substrate for VZV TK, which is able to phosphorylate the BCNA compounds to both their corresponding 5′-mono and -diphosphate derivatives; a factor in their anti-VZV selectivity. Another indication of the unusual selectivity of this class of nucleoside analogs was the lack of substrate recognition by cellular kinases which contribute to the anabolism of other pyrimidine analogs; the cytosolic or mitochondrial TKs, cyrosolic dTMP kinase, and most striking, nucleoside diphosphate kinase, the host enzyme which converts BVDU-DP to the active triphosphate form. Consistent with this observation, no 5-triphosphate of BCNA could be detected in VZV-infected cells (Sienaert et al., 2002). Information on the inhibitory effects of BCNA anabolites on the VZV DNA polymerase is not yet available.

There is no clear cut correlation between their affinity for VZV TK and the antiviral potency of the compounds, indicating that an additional SAR is likely (Balzarini and McGuigan, 2002). The closely related Simian varicella virus (SVV) is not sensitive to BCNA although in vitro studies indicate that SVV TK is able to phosphorylate BCNAs. Unfortunately this precludes the utility of the SVV animal model in the therapeutic development of BCNAs (Sienaert et al., 2004).

The BCNAs are highly stable and not liable to breakdown by nucleoside-nucleobase catabolic enzymes (Balzarini et al., 2002). The fact that they are not susceptible to degradation by thymidine phosphorylase and that they do not inhibit dihydropyrimidine dehydrogenase are key improvements over BVDU. Further clinical development is anticipated.

Non-nucleotide inhibitors


In an effort to identify non-substrate inhibitors of the herpes polymerase with broad-spectrum activity, Pharmacia researchers set up herpes polymerase-based screens and tested 80 000 representatives of different compound diversity. Selectivity was achieved by secondary evaluation of hits against the mammalian DNA polymerases alpha, gamma and delta. The systematic discovery program identified the activity of the naphthalene carboxamide series, exemplified by the initial active compound, PNU-26730 (Vaillancourt et al., 2000). Further optimization in this series led to a quinolone ring substitution and the 4-hydroxyquinoline-3-carboxamides series. Increased potency against the CMV, HSV-1 and VZV DNA polymerases was achieved by adding substitutions at the 6 position on the quinolone ring yielding the 4-oxo-dihydroquinolines (4-oxo-DHQs) series of compounds. These compounds, represented by PNU-182171 and PNU-183792, were also more active than the initial lead and the gold standard ACV against VZV and CMV, but showed no enhanced activity against HSV-1 or 2. These 4-oxo-DHQs were not active against other RNA and DNA virus tested: vaccinia, SV-40, adenovirus, HBV, influenza A, coxsackie B or VSV. (Brideau et al., 2002; Knechtel et al., 2002; Wathen, 2002).


One of the 4-oxo-DHQ compounds, PNU-183792 (N-(4-cholorobenzyl)-1-methyl-6-(4-morpholinylmethyl)-4-oxo-1,4 dihydro-3-quinolinecarboxamide) was selected for additional investigations. Efficacy studies in a murine model of lethal MCMV infection showed antiviral activity similar to GCV when treatment was initiated up to 24 hours post infection, but was less efficacious at comparable doses given 48 hours postinfection (Brideau et al., 2002). Other properties required for a drug candidate were demonstrated: for example, PNU-183792 was orally bioavailable in dogs and rodents, achieving concentrations above the IC50, with reasonable rates of clearance and a half life of 3 hours in dogs. The important measure of available drug levels is at the intracellular site of action, and although this information was not published for PNU-183792, it is likely to be similar to the actual plasma concentrations. Nucleoside and nucleotide analogues may have an advantage over non-nucleoside inhibitors of DNA polymerase in this regard, as the t½ of the triphosphate active form anabolized within the cell compartment can exceed plasma levels of unchanged drug.

The strong correlation between polymerase inhibition and viral replication inhibition in the analogue series supports inhibition of viral DNA polymerase as the MOA (Fig. 68.3). Further mechanistic studies into the nature of the polymerase inhibition showed competition with the binding of natural substrate (dTTP) to the polymerase enzyme, with a low-level affinity for the enzyme substrate complex that was not defined. Points of drug interaction with target protein were characterized by resistant virus bearing point mutation(s) in the DNA polymerase gene (Oien et al., 2002; Thomsen et al., 2003).

This promising class of compounds will be active against the drug-resistant TK variants of HSV and VZV, and the current in vitro profile shows activity against clinically relevant HSV and CMV polymerase mutants (Thomsen et al., 2003). This profile is consistent with this class of compounds interacting at a different molecular site in the polymerase polypeptide.

Information on the current development status is limited since Pharmacia was purchased by Pfizer. The selectivity screens have reduced the likelihood of mechanism-based toxicity within the series leads; and the safety profile will be defined by full in vivo toxiologic studies. Other key questions with this type of molecule and the nature of the MOA will be potency compared to valacyclovir or famciclovir, durability of action (related to frequency of dosing), and the ease of viral escape (resistance).

Herpes helicase-primase inhibitors

Efforts to identify drugs targeting other components of the DNA replication complex have focused on another enzyme, the helicase-primase (Hall and Matson, 1999). In the herpesviruses, the helicase-primase complex consists of 3 proteins that associate as a trimeric complex to carry out the essential tasks of unwinding the dsDNA in the 5′ to 3′ direction, RNA polymerase activity and ssDNA-stimulated ATPase activities (Crute and Lehman, 1991; Parry et al., 1993). In HSV, these are the gene products of UL5, UL8, and UL52 ORFs. The drug candidates recently identified from screens targeting helicase-primase represent the next generation, and an exciting new class of antiviral agents. The major unknowns with this MOA include the ease of emergence of resistance in the clinic, and the pathogenicity (including reactivation) and transmission of potential of resistant virus.


The scientists at Tularik Inc. screened a library of >190,000 samples consisting of small organic molecules and natural products, using a novel filtration assay for the detection of the helicase DNA unwinding activity (Sivaraja et al., 1998). The most active selective compound was a 2-aminothiazole compound, T157602 (Spector et al., 1998). Preliminary MOA studies suggested that T157602 stabilized the helicase-primase complex, effectively trapping the enzyme on the DNA substrate and blocking all 3 activities of the complex (Fig. 68.3). The compound was a reversible inhibitor (IC50 = 5 µM), of the helicase activity of the HSV UL5/8/52 complex, but was less active against other helicases, and could also interfere with primase activity at higher concentrations (IC50 = 20 µM).

Strains of HSV-1 and HSV-2 resistant to T157602 that were selected in the laboratory carried individual point mutations in the UL5 viral gene that resulted in amino acid substitutions in the corresponding UL5 protein. Marker transfer studies confirmed the role of these genetic changes in the resistance phenotype, both at the level of the UL5 enzyme subunit and of mutant virus. Animal-model efficacy studies were not reported for this series.

In vitro cytotoxic studies revealed no apparent cellular toxicities at concentrations exceeding 100 µM, indicating a therapeutic window greater than 30-fold. However, as of 2003, the 2-aminothiazole compounds are no longer included in the Tularik development pipeline, suggesting safety deficiencies arose during the phase I/Ⅱ clinical studies.


Related compounds with more potent helicase-primase activity emerged from enzyme-based screens at Boehringer Ingelheim (Crute et al., 2002) and cell-based viral replication screens at Bayer AG (Kleymann et al., 2002). The thiazolylphenyl-containing compounds represented by BILS 179 BS inhibited all three enzyme activities of the HSV helicase-primase complex at 100 nM or lower concentrations. The antiviral activity was specific for HSV, with no activity against VZV, and human or murine CMV. Preliminary data suggest that the mechanism of inhibition involves a stabilization of the interaction between the enzyme complex and the DNA substrate, most likely by imposing a physical constraint both to enzyme progression through the DNA-unwinding reaction and to primase catalytic activity (Fig. 68.3).

Resistant viruses were selected by serial passage in BILS 179 BS for more definitive MOA studies. Helicase-primase purified from cells infected with these resistant viruses demonstrated decreased inhibition in an in vitro DNA-dependent ATPase assay that corresponded with antiviral activity. Single base pair mutations clustered in the N-terminus of the UL5 gene that resulted in single amino acid changes in the UL5 protein were identified by marker transfer and DNA sequence analysis. These results were consistent with helicase-primase inhibitor activity mediated through specific interaction with the UL5 protein (Liuzzi et al., 2004).

BILS 179 BS was 10–15 times more active than ACV in vitro (EC50s 27 nM–100 nM). Cytotoxicity effects were somewhat dependent on cell type, and additional experiments are needed to better clarify the cytotoxicity profile of these compounds. Efficacy studies were conducted in murine models of primary cutaneous and genital disease, using ACV as the treatment comparator (Crute et al., 2002). In the cutaneous model of progressive zosterform disease (hairless SKH-1 mouse infected with HSV-1 strain KOS), BILS 179 BS demonstrated comparable efficacy to ACV when treatment was initiated 3 hours post infection (1 × or 4 × daily). However, BILS 179 BS was superior to ACV when treatment frequency was reduced, or when initial treatment was delayed by 65 hours.

Comparable results were evident in the genital disease model (Swiss Webster mice vaginally infected with HSV-2 strain HG-52). Efficacy was based on a composite disease severity scoring system that included mortality. Again, BILS 179 BS had similar efficacy to ACV when treatment was initiated at 3 hours (1 × or 4 × daily), and superior efficacy when treatment frequency was reduced or when initial treatment was delayed by 65 hours. Actual drug exposures were not reported for these studies; however, BILS 179 BS was reportedly bioavailable, and on a weight-dosing basis, BILS 179 BS showed superior activity to ACV. Disappointing findings from a drug development perspective included the identification of pre-existing resistant variants within the wild type virus population, and rapid selection of highly resistant growth-competent viruses in vitro that maintained a stable drug-resistant phenotype in the absence of drug. Of more clinical relevance was the demonstration that two resistant strains studied were fully competent for disease pathogenesis (by cutaneous or intracerebral routes in mice), and for reactivation from latency in an ocular infection model (Liuzzi et al., 2004). By contrast, ACV-resistant strains of HSV were generally less virulent in various infection models. This safety feature is based on the biology of HSV, and the mechanism of action of the drug ((Elion et al., 1977). ACV resistant clinical isolates are generally TK-deficient, and as a consequence less pathogenic in the immune competent population and less competent for reactivation from latency (recurrences are ACV-susceptible). The potential for transmission of drug-resistant strains into the general population is a public health consideration and requires careful monitoring (Shin et al., 2001; Bacon et al., 2003). The clinical development timetable for this compound is unknown at this time (mid 2006).

BAY 57-1293

The Bayer discovery program used a fluorometric high-throughput screening assay that identified inhibition in any target essential for viral replication in cell culture. Over 400,000 compounds were tested at 10 µM, and several compound classes with activity were identified (Kleymann, 2003a,b). The triazole urea class of analogues was selected for additional study. Profiling and modeling techniques were employed, and optimization to improve the solubility led to the 2-pyridyl substituent in the para position of the phenyl ring in the lead compound to create BAY 57-1293 (N-[5-(aminosulfonyl)-4-methyl-1,3-thiazol-2-yl]-N-methyl-2-[4-(2-pyridinyl)phenyl]acetamide). BAY 57-1293 inhibited the replication of HSV type 1 and 2 in Vero cells (IC50 of 20nM) with a selectivity index of 2,500; ACV had an IC50 of 1 µM and a selectivity index of 250 under comparable conditions. There was no appreciable activity against VZV or CMV. BAY 57-1293 was equally active against ACV-resistant TK or polymerase mutants, and the activity was irrespective of cell type (Kleymann et al., 2002). The in vitro replication block was reversible upon drug removal, and significant activity was still demonstrated when added late post infection.

Researchers selected virus resistant to each of the three analogues in the series. Resistance resulted from point mutations in UL5 (present in all six resistant strains), or in one case, a UL5 mutation together with a point mutation in UL52. These mutations in UL5 were clustered between nucleotides 1045 to 1077, a region that corresponds to the amino acids from 349 to 359 involved in the alpha helicase region that is most homologous across the herpesviridae (Kleymann et al., 2002). BAY 57-1293 inhibits the ATPase activity of the viral helicase-primase complex in a dose-dependent manner (IC50 of 30 nM).

The treatment potential of BAY57-1293 was investigated in several cutaneous and systemic animal models of disease (Betz et al., 2002). The activities of orally administered BAY 57-1293 for the treatment of acute HSV-1 and HSV-2 infections were assessed in a widely-used murine lethal challenge model of disseminated herpes. Mice were infected intranasally and treated, starting 6 hours later for 5 consecutive days, three times a day. BAY 57-1293 and comparator drugs (ACV, GCV, VACV, FCV, Brivudin) were tested in escalating doses, and ED50 (dose at which 50% of the infected animals survive) values calculated. With an ED50 of 0.5 mg/kg of body weight TID against HSV-1 and HSV-2, BAY 57-1293 was the most potent compound tested. Comparable values for ACV were 22 and 16 mg/kg of body weight. No toxic side effects of BAY 57-1293 treatment were apparent in the mice upon gross inspection, and the highest dose tested (60 mg/kg TID) appeared to be well tolerated. Comparable results were shown in a rat lethal challenge model.

In the cutaneous zosterform model, oral treatment of HSV-2 established by dermal scarification was delayed until establishment of disease (day 3) and then animals were treated for 5 days TID with 15 and 60 mg of BAY 57-1293 per kg and 60 and 240 mg of VACV per kg. The lower dose of BAY 57-1293 was statistically more efficacious than the highest dose of VACV used (P < 0.011), based on a compiled disease severity score.

In the guinea pig vaginal model of HSV-2 genital herpes, delayed treatment with BAY 57-1293 (20 mg/kg 2 × daily orally; days 4–14 post infection) rapidly shut down disease progression. VACV at 7.5 × higher dose levels produced only a weak response. Benefit in terms of time to healing was clearly superior in the BAY 57-1293 treatment group. Perhaps the most promising results observed in the animal studies were the observation that acute treatment in this model could reduce the number of subsequent recurrences. This latter outcome may reflect the more potent and rapid shut down of the virus feeding the latency reservoir, and illustrates the importance of rapid diagnosis and treatment of the primary infection.

In these studies, the actual drug exposures (PK parameters) for BAY 57-1293 and comparators were not reported (Betz et al., 2002) making it difficult to compare potencies. However, the PK for single 1 mg/kg dose in female BALB/C mice indicated high oral bioavailiability Cmax 4.4 µM after 1 hour, and relatively slow elimination from the plasma (Tmax = 1 hr; t½ 6 hours). Oral bioavailability >60% and an elimination half-life of >6 hours has also been observed in rats and dogs. Plasma concentrations with these properties would exceed 0.025 µM at 24 hours post dose. Under the conditions used in the cutaneous efficacy model studies with 15 and 60 mg doses administered 3 times daily, one would expect significant accumulations of drug levels above IC90 over the 5-day course of treatment; providing extended antiviral cover in the post-dosing period (Kleymann et al., 2002). BAY 57-1293 shows the potential for once daily dosing, a convenience important for chronic suppression. From a pharmaceutical manufacturing perspective, such potency has advantages in smaller pill size and burden (number of pills per dose), resulting in economic savings in the amount of drug substance.

The long duration of drug exposure can offer advantages in terms of efficiency in reduced frequency of dosing, but must be balanced by an excellent safety profile to avoid undesired consequences of toxic build up. Early toxicology studies indicated that once-daily dosing of dogs with 30, 100, and 300 mg/kg of BAY57-1293 for 28 days was well tolerated. The identical dosing protocol in rats, however, resulted in a dose-dependent transitional hyperplasia of the urinary bladder epithelium (Kleymann et al., 2002). Based on a structural similarity to the diuretic drug, Diamox® (acetylzolamide), the Bayer toxicologists hypothesized that inhibition of the carbonic anhydrase enzymes led to bladder hyperplasia. Sulfonamides with broad inhibitory activity against the carbonic anhydrases of rats, dogs, and humans, only cause this bladder hyperplasia in the rodents. The in vitro inhibition of a carbonic anhydrase standard assay by BAY57-1293 occurs at 2 µM; 100-fold above the viral inhibitory concentration. Extended toxicologic evaluations will be required to further clarify this observation. The overall preclinical profile of this compound would support clinical development, and the evidence of superior potency and more rapid onset of action, and durability compared to the gold standard therapy, make it one of the most promising agents in the development pipeline (2004).

A novel series of inhibitors of the CMV helicase primase function was identified in a cell-based viral replication (single cycle) assay (Cushing et al., 2006). The imidazolyl-pyrimidine core scaffold was substituted extensively at the 2-, 4-, 5-, and 6 positions to produce an active series of analogs with in vitro potencies ranging from 0.04–0.30 µM. The SAR revealed the importance of the imidazole-nitro group dyad, and the nature of the substituents at the 4 position. The chemical features were consistent with a mechanism of action involving a covalent binding to a target protein. Irreversible binding to the UL70 component of the CMV helicase primase complex (UL102, UL105, UL70) was demonstrated by co-immunoprecipitation of UL70 bound to radiolabeled inhibitor (Cushing et al., 2006). Resistance selection is not yet reported for these new inhibitors. Two compounds provide excellent starting points for the further optimization for the necessary properties of a viable drug candidate.

Inhibitors of DNA processing and packaging

After herpesvirus DNA replication, the concatemeric product is packaged into preformed capsids and cut into unit-length genomes by site-specific cleavage. At least seven HSV proteins have been identified as participants in this process; pUL6, pUL15, pUL17, pUL25, pUL28, pUL32 and pUL33 (Beard et al., 2002) and many of these have been confirmed as essential for viral replication. By analogy with DNA bacteriophage packaging and processing, a terminase complex binds to the capsid portal, trims the concatemeric DNA at a specific sequence with unique structural features (Adelman et al., 2004), translocates the DNA into the capsid and finally cleaves the DNA at a repeat of the specific sequence. The HSV pUL6 protein has been shown to form the capsid portal (Newcomb et al., 2001, 2003). A variety of evidence indicates that the pUL15 and pUL28 proteins form the terminase complex that cleaves the HSV DNA at the sequence before and after packaging (Beard et al., 2002; White et al., 2003; Przech et al., 2003). The packaging genes are well-conserved among the herpesviruses; for example at least six of the seven HSV genes have close homologues in CMV. Of the terminase components CMV pUL89, pUL56 and pUL104 are the homologues of HSV pUL15, pUL28 and UL6 (Fig. 68.4). Biochemical and structural studies of the hCMV pUL89 and pUL56 suggest that the pUL56 binds to the viral DNA, while pUL89 mediates DNA cleavage via an ATP-dependent-nuclease activity (Bogner et al., 1998; Scheffczik et al., 2002; Scholg et al., 2003). Since the processing and packaging of concatemeric DNA has no exact counterpart in the human cell this target presents the possibility of discovering very selective antiviral agents.

Fig. 68.4. Inhibitors of CMV DNA replisome, packaging, and nucleocapsid egress.

Fig. 68.4

Inhibitors of CMV DNA replisome, packaging, and nucleocapsid egress.

Inhibitors of the portal protein of HSV

WAY 150138

Activity of the thiourea class of compounds emerged from cell-based replication screens, revealing a striking degree of specificity within the alpha herpesviruses, but no activity across other human herpesviruses (Visalli and van Zeijl, 2003). Minor structural changes in the main scaffold resulted in >10-fold shifts in activity between HSV and VZV, and the lead HSV compound, WAY 150138, (benzamide, N-[3-chloro-4-[[[(5-chloro-2,4-dimethoxyphenyl)amino]thioxomethyl] amino]phenyl]-2-fluoro-). Identification of the portal protein as the molecular target was made by the generation and mapping of laboratory-derived resistant mutants (van Zeijl et al., 2000; Visalli and van Zeijl, 2003). The portal proteins of HSV-1 and HSV-2 share 86% amino acid identity or similarity, while VZV (pUL54) and human CMV (pUL104) portal proteins share only 44% and 27% identity/similarity, respectively. Although these homologues share a high degree of functional homology, and strong overall amino acid identity in conserved domains, no broad spectrum inhibitors have yet been identified.

The individual mutations identified in the HSV-1 strains resistant to WAY-150138 suggested points of interaction with the compound resulted from the folding of the UL6 protein in its active 3- dimensional conformation. A crystal structural model for the portal protein to aid in understanding the mechanism of UL6 binding and in further drug design was not available. Research on this class of portal protein inhibitors continues. No information is currently published on the other preclinical properties of these compounds that would indicate their potential as herpes simplex therapeutics.

Comp I

Subsequent screening of related compounds in the thiourea series revealed that a small chemical modification (addition of a spacer, -HC(CH3)-, between the aryl ring and the thiourea nitrogen) yielded compounds active against VZV, but with loss of activity against HSV. Three N-methylbenzyl-N′-arylthiourea analogues (Comp Ⅰ, Comp Ⅱ, and Comp Ⅲ) were selected for further study.

A number of MOA studies were conducted confirming that VZV DNA cleavage and packaging is inhibited by these thiourea compounds via inhibition of the ORF54 gene (Visalli et al., 2003). Resistant viral isolates were found to possess mutations in the VZV ORF54 gene, the homologue of HSV UL6, similar to the mechanism of WAY-150138 (van Zeijl et al., 2000; Newcomb and Brown, 2002). As expected, treatment of wild-type virus with the inhibitor resulted in the absence of DNA-containing capsids, and restriction in the spread of infectious VZV to adjacent uninfected cells.

Current marketed VZV antivirals all target the DNA polymerase, and virus resistant to these new thiourea compounds are not cross-resistant to the approved drugs. Thus, targeted inhibition of the VZV ORF54 protein may prove to be a productive approach in identifying new agents to complement existing antivirals in the treatment of VZV infections.

Dihydroxyacridone series

The dihydroxyacridone series was investigated in order to target a MOA other than viral DNA polymerase (Akanitapichat et al., 2000). Initial attempts focused on analogues with functional groups at the 5, 6, or 8 positions. The 5-Cl congener (5-chloro-1,3-dihydroxyacridone) was determined to be the most selective inhibitor of HSV and was selected for additional study. Mechanistic studies suggested that HSV replication was blocked after DNA and late protein synthesis. Further studies (Akanitapichat and Bastow, 2002) indicated that maturation of replicating DNA and late virion production were inhibited in the same dose-dependent manner, resulting in a two- to three-fold reduction in the production of B capsids. Of interest was the inability to isolate resistant virus, although these attempts were limited, attempts to isolate resistant virus were unsuccessful.

Additional chemical elaboration and parallel synthesis of compounds in this series (Lowden and Bastow, 2003) identified compounds that were active against CMV (ED50 value of 1.4 µM at low MOI) and some that were active against both CMV and HSV. At least one compound in this series inhibited cell replication (mean CC50s = 33 µM), but did not have antiviral activity. Preliminary mechanistic studies indicated the likelihood of diverse MOAs. While this has some appeal, more extensive work may be needed to determine the real selectivity and safety of this series of compounds.

Inhibitors of the CMV terminase complex


The first selective inhibitors of the hCMV terminase complex, TCRB (2,5,6-trichloro-1-ß-d-ribofuranosyl benzimidazole) and its analogue BDCRB (1-(ß-d-ribofuranosyl)-2-bromo-5,6-dichlorobenzimidazole) arose serendipitously from a chemistry effort to modify the broad spectrum transcriptional inhibitor DRB into an anti-tumor agent (Townsend and Revenkar, 1970). The antiviral activity was uncovered during subsequent screening for antiviral activity in the series (Townsend et al., 1995). In contrast to the action of GCV, viral DNA synthesis was unaffected in a single round of replication. The phenotype of infected, treated cells was consistent with a block in the viral DNA cleavage and packaging (Fig. 68.4).

Genetic mapping studies of the BDCRB and TCRB-resistant mutants of Towne and AD169 strains confirmed the interaction of these inhibitors with two subunits of the terminase complex (Krosky et al., 1998; Underwood et al., 1998). Specific point mutations were identified in the UL89 gene, which encodes the small subunit required for the nuclease cleavage of the precursor viral DNA into unit genome lengths (Bogner, 2002). A mutation was also selected in the UL56 gene, which encodes the large subunit responsible for sequence-specific DNA binding to the pac motifs, and ATP-dependent translocation of the viral DNA into the preformed capsids for final cleavage and packaging.

Precisely how two unrelated compound series, the β-D-ribosyl benzimidazoles and the sulfonamides, block DNA processing and packaging is not currently understood. The exact binding sites for BDCRB and BAY 38-4766 on the terminase subunits are apparently different, based on lack of cross-resistance (Evers et al., 2004). Their binding could disrupt protein-protein interaction via allosteric mechanisms, or could directly interfere with enzymatic activity. The genome maturation process is complex, and occurs at the viral replication center sites, where viral and cellular transcriptional and DNA replication machinery also assemble (Dittmer et al., 2005; McVoy and Nixon, 2005; Thoma et al., 2006). BDCRB treatment of CMV-infected cells resulted in a major block to correct unit genome clevage, and also allowed a minor level of monomer plus larger than unit product genomes resulting from skipped cleavages (McVoy and Nixon, 2005). This inhibitor also may directly interfere with the interaction of the pUL56 and the portal protein pUL104, as evidenced by the ability of BDCRB to block their co-immunoprecipitation from CMV-infected cells (Dittmer et al., 2005). Consistent with a direct interaction of the pUL56 terminase subunit with the portal protein, resistance modifications in UL89 and UL56 to BDCRB, TCRB and BAY 38-4766 were accompanied by a compensatory change in UL104, which alone did not confer resistance (Buerger et al., 2001; Reefschlaeger et al., 2001; Komazin et al., 2004). These interesting inhibitors continue to help elucidate the genome maturation events.

These two lead benzimidazole ribosides showed potent, selective activity for human CMV. However, pharmacokinetic studies indicated metabolic lability of the glycosidic bond between the base and the sugar moieties (Good et al., 1994). Recently, the identification of two cellular enzymes that catalyze the cleavage of the glycosidic bonds of BDCRB and TCRB were reported (Lorenzi et al., 2006). An active chemical program was undertaken to stabilize this linkage (Townsend et al., 1999; Chulay et al., 1999). The results of this effort were fruitful; yielding two clinical candidates, GW275175X, an inhibitor of the hCMV DNA terminase process packaging, and BW1263W94 (maribavir), an inhibitor of the pUL97 protein kinase (information as of 2003). The application of amino acid esters as pro drugs of BDCRB has also been successful, identifying L-Asp-BDCRB as a potential candidate for further development (Lorenzi et al., 2005).


A benzimidazole with a pyranosyl sugar moiety selected for further development was GW275175X (2-bromo-5,6-dichloro-1-ß-d-ribopyranosyl-1-H-benzimidazole), a compound whose antiviral activity is specific for hCMV; there was no in vitro inhibition of HSV-types 1 or 2, VZV, or other DNA and RNA viruses tested (Williams et al., 2003; Underwood et al., 2004). MOA studies (Underwood et al., 2004) supported inhibition of viral cleavage and packaging, consistent with the mechanism of BRCRB. As expected, virus with the BDCRB- and TCRB-resistant mutations in UL89 were cross resistant to GW275175X. One notable aspect of the MOA is the rapid reversibility of the BDCRB or GW275175X block in infected cell culture, with resumption of viral DNA concatamer processing and restoration of the rate of viral yield production occurring within 8–10 hours following drug washout (Underwood et al., 2004).

A battery of preclinical toxicology testing sufficient to support initial Phase I studies in humans (GSK, data on file) was completed for GW275175X. No significant adverse activity in in vivo assays designed to predict effects on metabolic parameters were exhibited. Other in vitro assessments of GW275175X were encouraging; pharmacology screens, and in vitro and in vivo mutagenecity tests were clean, and liver enzyme studies were clean. GW275175X was evaluated for toxicity in rodents (28-day acute, 6-month chronic) and primates (28-day acute). Systemic exposures at the various doses ranged from 60- to100-fold the in vitro IC50 for CMV inhibition. Importantly, penetration into the CNS and vitreous humor in primates was good, ranging from approximately 2 × the IC50 at 50 mg/kg/day to 8X the IC50 at 200 mg/kg per day. These characteristics would be important for treatment of congenital and ocular CMV disease.

Based on the overall preclinical properties and safety profile, GW275175X was progressed into Phase I single-escalating dose (100–1600 mg) studies. No drug-related or clinically significant changes from baseline were seen in vital signs, ECG, or clinical laboratory values, and all adverse events were considered mild. The pharmacokinetic profile indicated several advantages of this benzimidazole ribose over the parent BDCRB and the analogue, maribavir, including better CNS penetration, longer plasma half-life, and reduced serum protein binding.

While the overall preclinical and initial phase I data encouraged further clinical development, this compound was not advanced due to the sponsor’s decision to progress the other anti-CMV agent from the benzimidazole riboside series (1263W94, maribavir) instead.

BAY 38–4766

The non-nucleosidic BAY 38–4766, 3-hydroxy-2,2- dimethyl-N-[4([[5-(dimethylamino)-1-naphthyl]sulfonyl] amino)-phenyl]propanamide compound emerged from a cell-based discovery program as the lead highly selective inhibitor of human, monkey and rodent CMV. Resistance to this drug was genetically mapped to the UL89 and the UL56 ORFs, indicating that this class of compound also targeted the hCMV terminase complex (Reefschlaeger et al., 2001; Buerger et al., 2001). The drug susceptibilities of 36 hCMV clinical isolates to the BAY 38–4766 and GCV were evaluated in two different phenotypic assays. All isolates including those resistant to GCV were inhibited by at least 50% at a concentration of approximately 1 µM of BAY-38–4776 (McSharry et al., 2001a,b). Antiviral activity comparable to GCV was demonstrated in a hollow-fiber model where hCMV-infected human cells entrapped in hollow fibers were transplanted into immunodeficient mice (Weber et al., 2001). Viruses resistant to BAY 38–4766 are not cross resistant to current marketed CMV drugs GCV, CDV or PFA.

A favorable pharmacokinetic profile has been demonstrated in humans for the initial lead compound in this series. A phase I study in healthy male subjects after single oral doses of BAY 38–4776 (100 to 2000 mg) indicated that the drug was well tolerated with no adverse events or changes in vital signs or lab parameters. The Cmaxs ranged from 0.33 mg/l (100 mg dose) to 4.2 mg/l (2000 mg/l dose) and occurred within 0.5 to 5 hours. After Cmax concentrations were reached, drug was eliminated from plasma, with a t1/2 of 3 to 5 hours reaching a terminal half life of 12–16 hours. The increases in AUC and Cmax were dose dependent (Nagelschmitz et al., 1999).

As of 2004, work continued to advance a compound from this interesting series into full clinical development.

Protease inhibitors

Impressive success in the development of protease inhibitors (PIs) in the treatment of HIV infection has not been paralleled in the herpesviruses. The PIs for HIV infection were introduced in 1996 and quickly set a new standard of care, dramatically extending the life of patients with HIV infection. Success in the design of therapeutic agents targeting the HIV protease is partly attributed to the well-defined structural features of the catalytic site in this aspartyl protease (Supuran et al., 2003). In contrast, the herpes proteases belong to the serine protease family, and are characterized by a distinctive catalytic triad of his, his, ser within the less tractable active site. The cleavage sites are unique and highly conserved across the herpesviruses. The herpes proteases comprise the N-terminal sequence of the capsid scaffold protein. After completion of capsid assembly, auto cleavage by the protease releases the scaffold, permitting DNA packaging (Gibson et al., 1994).

The herpes protease is essential for the production of infectious virus, and therefore represents a valid target. The quest for inhibitors of these proteases was facilitated by the development of efficient enzyme-based screens and wide-ranging X-ray crystallographic structure and catalytic site features that became available in the mid-1990s (Borthwick et al., 1998; Holwerda, 1997; Qiu and Abdelmeguid, 1999; Waxman and Darke, 2000). Figure 68.5 illustrates the application of X-ray crystallographic structural information to the design and optimization of enzyme inhibitors (Hoog et al., 1997), a tool used successfully in the HIV aspartyl PI discovery programs (Erickson et al., 1990).

Fig. 68.5. The HSV-2 protease monomer.

Fig. 68.5

The HSV-2 protease monomer. Two disordered surface loops are shown as dashed lines (N-terminal residues 1–16 are disordered). The two histidines in the active site are shown in red, one on the β6 and the other on the hairpin turn between (more...)

Several major pharmaceutical companies took up this quest, and several mechanism-based peptide and heterocyclic inhibitors of either the reversible or irreversible type were identified (Borthwick, 2005). However, these were not always active in virally-infected cells, and none have been progressed into clinical development to date. Any inhibitor would have to be capable of uptake not only into cells, but also into the capsid structure within the nucleus of the infected cells. This nucleocapsid barrier may contribute to the poor antiviral activity reported for compounds that are potent inhibitors of the enzyme assay in vitro (Borthwick, 2005). The protease as a target remains to be successfully exploited.

Inhibitors of the CMV UL97 encoded protein kinase

The CMV-encoded UL97 protein kinase has several features that make it highly attractive as a chemotherapeutic target. It belongs to a family of serine-threonine protein kinases highly conserved across all mammalian herpesviruses, suggesting its potential as a broad spectrum target (Chee et al., 1989; Smith and Smith, 1989). The pUL97 shares structural features with aminoglycoside phosphotransferases; bacterial enzymes known to phosphorylate sugar-containing moieties, which may account for its fortuitous ability to monophosphorylate GCV and ACV (Littler et al., 1992; Sullivan et al., 1992; Talarico et al., 1999; Zimmerman et al., 1997; Michel and Mertens, 2004). This nucleoside phosphotransferase (ACV, GCV) activity has also been reported for the EBV homologue BGLF4 (Zacny et al., 1999), but not for the alphaherpes virus kinase homologues. Importantly, the pUL97 differs from a prototypic serine-threonine protein kinase biochemically (high pH and NaCl optima) and substrate motif specificity (He et al., 1997; Baek et al., 2002).

The study of the replication functions of these herpes protein kinases is complicated by the fact that they are not absolutely essential for growth in cultured cells. Null mutants of the protein kinases of HSV-1, VZV, and CMV have shown variable phenotypes in different cell types, under different culture conditions, complicating MOA studies and predictably, quantitative drug inhibition studies (Moffat et al., 1998; Chou et al., 2006). Studies into the function of the HSV-1 pUL13 and the VZV gene 47 homologues indicate multiple activities throughout the virus replication cycle. These include regulatory roles in early gene expression, indirect effects on host gene expression, late protein post-translational modifications associated with virion maturation, and contributions to tissue-specific pathogenesis. (Purves et al., 1993; Kato et al., 2001; Kenyon et al., 2001; Coulter et al., 1993; Overton et al., 1994; Ng et al., 1994; Michel et al., 1999; Kawaguchi et al., 1999; Moffat et al., 1998; Pritchard et al., 1999; Wolf et al., 2001; Krosky et al., 2003b; Marschall et al., 2005; Hu and Cohen, 2005).

Therefore, inhibition of such a target could cumulatively penalize viral replication. These proteins undoubtedly play essential roles in disease pathogenesis (Moffat et al., 1998). In the case of the human CMV pUL97, the high degree of interstrain sequence conservation (Lurain et al., 2001) coupled with the observations that there are no null mutants of the CMV pUL97 in clinical strains would argue that this is an ideal target. Two series of unrelated compounds are potent inhibitors of the CMV pUL97, and their MOA studies have extended our current knowledge of pUL97 function. Proof of concept for one of these has been achieved in the clinic.


Indolocarbazoles have been investigated as potential antiviral agents based on the fact that they are competitive inhibitors of the ATP binding sites of kinases in the protein kinase C family (Zimmerman et al., 2000; Marschall et al., 2001). A series of indolocarbazoles was recently analyzed and three (Go6976, K252a, K352c) were established to be highly effective inhibitors (IC50s ranging from 0.009 to 0.4 µM) of both GCV-sensitive and -resistant hCMV, with little effect against HSV. Cytotoxicity assays in proliferating cells reportedly indicated that the effective antiviral concentration of these compounds was significantly lower than those affecting cellular functioning. However, attempts to select resistant virus under the selective pressure of increasing concentrations of drug indicated that resistance was lost or restricted to low-level replication at higher drug concentrations, hinting that cellular functions may be involved (Kawaguchi and Kato, 2003). Alternative explanations include the possibility that resistance to Go6976 results in severe growth impairment, which could be established by the replication competence of a virus strain with deleted target. Growth impairment (1–2 logs titer reduction) has been reported for the deleted UL97 hCMV (Prichard et al., 1999; Wolf et al., 2001). Efforts to elucidate the exact MOA of the indolcarbazoles have focused on the hCMV pUL97 protein kinase. Indolcarbazoles with anti-CMV activity inhibited pUL97 protein kinase autophosphorylation in vitro, and mutant virus encoding a non-functional pUL97 (catalytic site mutant) deletion was completely insensitive to the indocarbazoles (Marschall et al., 2001).

A series of symmetrical indolocarbazoles were independently synthesized to investigate SARs against a range of herpesviruses (Slater et al., 1999). Several novel and potent inhibitors of hCMV were identified, although none were progressed to clinical development. Many of these had reasonable SIs in the normal diploid fibroblasts used for CMV activity, yet they were extremely toxic to human marrow stem cell differentiation. The development potential of this indolocarbazole class of compounds is unknown based on currently available information, which does not address pharmacokinetics and safety properties.

Recently a series of quinazolines with anti-CMV activity was shown to act through inhibition of the pUL97 (Herget et al., 2004). Limited testing against cell protein Kinases indicated viral PK selectivity, although they also block host EGFR. The series appears promising, based on available data.


BW1263W94 1-(β-l-ribofuranosyl)-2-isopropylamino-5,6-dichlorobenzimidazole (maribavir), was derivatized from the original benzimidazole BDCRB and TCRB (Townsend et al., 1995) as part of efforts to stabilize their metabolically labile glycosidic linkage and improve oral bioavailiability (Chulay et al., 1999; Townsend et al., 1995, 1999). The precedence of flipping the sugar conformation to the L or unnatural biologic form had been established in the nucleoside analog inhibitors of HIV RT as a way of reducing mechanism-based toxicity and rapid metabolic clearance. The potency of the β-L-BDCRB was amplified by various substitutions for the halogen in the 2-position of the benzimidazole base. The resulting development candidate BW1263W94 showed potent activity for CMV and EBV, but no other viruses, including the various animal CMV strains tested (Kern et al., 2004). An unexpected finding was the change in the MOA: maribavir no longer exerted significant inhibition of viral DNA processing and packaging. Instead, maribavir strongly reduced viral DNA synthesis in the quiescent MRC-5 lung fibroblasts used in this study. The mechanism was not mediated through a substrate analog inhibition of the CMV DNA polymerase. Maribavir was not phosphorylated in infected cells, nor did the compound itself or any of its synthetic phosphorylated derivatives inhibit the CMV DNA polymerase (Biron et al., 2002).

The discovery that maribavir was a selective inhibitor of the pUL97 came simultaneously from 2 independent approaches: selection and genetic mapping of a resistant virus strain, and fortuitously, from a broad protein kinase-inhibitor screening effort (Biron et al., 2002). Resistant virus selected with a related analog in the series encoded a Leu397Arg amino acid substitution in pUL97, which conferred a 20- to 200-fold less sensitive phenotype to maribavir. This resistant virus remained susceptible to BDCRB and other approved anti-CMV drugs, including GCV, and was fully competent for in vitro growth. Supporting evidence for the pUL97 as the target was provided by studies with the pUL97 enzyme: the wild type pUL97-catalyzed phosphorylation of histone 2b was inhibited by maribavir (IC50 = 2 nM) while pUL97 with the Leu397Arg mutation was not (IC50 > 1000 nM). A second resistance mutation in the UL97 ORF was selected in the laboratory in the context of a clinical strain following serial passage in increasing concentrations of drug. The resulting point mutation encoded a Thr409Met change, which is also located close to the ATP-binding domain, and far upstream of the GCV resistance mutations which map to the substrate binding domain (Chou, 2006; Chou et al., 2002). The resulting resistance phenotype was intermediate relative to that of the Leu397Arg mutation in AD169, conferring 80-fold vs. 200-fold increase (Biron et al., 2002; Chou, 2006). Maribavir maintains activity against all GCV resistant UL97 mutants identified to date, indicating that the interactions of these two drugs with the protein kinase are distinct (McSharry et al., 2001; Biron et al., 2002).

Reduction in viral DNA synthesis by maribavir may be a consequence of inadequate or improper phosphorylation of pUL44 by the pUL97 kinase. The pUL97 carries a NLS, and locates to the nucleus during the replication cycle (Michel et al., 1996). A direct interaction with, and phosphorylation by, pUL97 of the pUL44, the DNA processivity factor, has been reported (Krosky et al., 2003; Marshall et al., 2003). This interaction was linked to the co-localization of pUL44 and pUL97 in the replication complexes (Marshall et al., 2003).

The pUL97 has been shown to exert its major effects late in CMV replication (Wolf et al., 2001; Krosky et al., 2003a) based on studies with the RCΔ97 (Prichard et al., 1999). Consistent with studies of UL97 null virus, maribavir treatment of CMV-infected foreskin fibroblasts (HFF) also resulted in an increase of type A empty capsids (Wolf et al., 2001) or type C DNA filled capsids (Krosky et al., 2003a). These empty and precursor capsids accumulated in the nucleus of infected HFF cells at late times in the replication cycle, after infection with RCΔ97, or in wild-type infected cells treated with maribavir. “Studies with HSV (Kato et al., 2006; Simpson-Holley et al., 2004) and with the CMV UL97 deficient virus (Wolf et al., 2001; Krosky et al., 2003a) have suggested a role for these viral protein kinases in nucleocapsid egress. The large nucleocapsids cannot exit through the nuclear membrane, which is composed of a tight structural matrix of proteins called lamins, without disassembly of lamin subunits in order to relax and open the junctions. Cellular p32 protein is reported to recruit pUL97 to the lamin B receptor, where it is hypothesized that pUL97 phosphorylates specific lamin components, resulting in their redistribution (Marschall et al., 2005). Thus the viral protein kinase regulates a host protein substrate during virion maturation. The block of the CMV UL97 kinase activity by maribavir results in nuclear retention and accumulation of nucleocapsids (Wolf et al., 2001; Krosky et al., 2003a).

A second genotype has been associated with maribavir resistance following laboratory passage of both laboratory and clinical strains of CMV (Chou et al., 2004; Komazin et al., 2003; Chou, 2006). Mutations in the UL27 ORF, encoding changes of Arg233Ser, Ala269Thr, Leu335Phe, Leu335Pro, Trp362Arg, or Ala406Val-415stop, are reported to confer only a modest level of maribavir resistance (two-fold–5-fold increase in IC50s). Little is known about the function of pUL27 at this time. However, the UL27 homolog of mCMV, known as M27, was shown by mutagenesis studies to be non-essential for growth in culture, but was required for virulence and mortality in vivo (Abenes et al., 2001). The CMV UL27 (1824 bp ORF, 608 amino acids) is transcribed as an early-late gene (Stamminger et al., May 2002; Chou et al., 2004), and has been shown to be nonessential for growth in vitro. The pUL27 encodes a nuclear localization signal (NLS); deletional mutagenesis resulted in cytoplasmic retention and a one-half log reduction in viral titers (Chou et al., 2004).

Viral encapsidation and nuclear egress involve the action of a number of viral gene products (Mettenleiter, 2002), and the pUL97 clearly plays a role in the process. Data are accumulating which point to a role of the UL97 protein kinase action in CMV virion morphogenesis, perhaps by directing the normal intranuclear and intracytoplasmic distribution of viral proteins required for virion assembly and intracellular movement during the sequential steps of primary envelopment, tegumentation, translocation and final particle maturation (Azzeh et al., 2006; Chou et al., 2004; Prichard et al., 2005). The pp65 antigen, product of the UL83 ORF, is phosphorylated by the pUL97 in transiently co-transfected cells, and its phosphorylation in cells infected with wild-type AD169 is blocked by maribavir, but pp65 phosphorylation is not blocked in cell infected with the maribavir-resistant strain Leu397Arg (Sethna, GSK data on file). Consistent with these findings, the intranuclear distribution of pp65 is altered in cells infected with the UL97 deficient strain, or in cells infected with wild type virus and treated with maribavir (Pritchard et al., 2005). The pUL27 contains several proposed pUL97 substrate motifs (Baek et al., 2002; Chou et al., 2004). The elucidation of the role of pUL27 in the maribavir-resistant phenotype awaits further investigation on its function.

As maribavir progresses in clinical development, it will be important to understand the basis for drug resistance and its correlation with clinical outcome. The UL27 genetic changes that conferred resistance to maribavir resulted in only a low-grade resistance (three- to four-fold elevated IC50s) compared to the highly resistant phenotype of the L397RpUL97 strain. The gene sequence in 16 clinical isolates was 96% conserved, with no changes at the locations of the four UL27 maribavir resistance mutations noted. From a clinical perspective, the UL27 gene appears more tolerant of mutations than the UL97 gene (98% gene conservation; Lurain et al., 2001), and the resulting levels of maribavir resistance may not preclude drug efficacy. It remains to be seen whether the prolonged drug selection pressure of prophylactic or suppressive regimens results in the accumulation of additional and multiple mutations in the UL27 and UL97 target genes, and potentially in as yet unidentified maribavir targets or UL97 substrates.

Preclinical toxicology testing of maribavir has been comprehensive, with overall results establishing a good safety profile (Koszalka et al., 2002). These results contrast favorably with results of preclinical testing of GCV and CDV, both of which have a litany of toxicologic and tolerability concerns.

The pharmacokinetic profile of maribavir in rodents and primates demonstrated excellent oral bioavailability, although drug levels in the brain, cerebrospinal fluid, and vitreous humor of cynomologus monkeys were low. Maribavir is highly bound to human plasma proteins, principally the albumin fraction; however, the binding is reversible in vitro. The impact of this property on efficacious dosing regimens remains to be determined.

Maribavir has successfully completed a series of Phase I–Ⅱ clinical trials (Wang et al., 2003; Lu and Thomas, 2004). In Phase I studies in healthy volunteers and HIV-infected subjects, single oral doses of 50 to 1600 mg produced similar dose-proportional pharmacokinetics. Maribavir was rapidly absorbed following oral administration with Cmaxs occurring within 1 to 3 hours. Absorption was at least 30% to 40%, and drug was eliminated from plasma with a t1/2 of 3 to 5 hours. The plasma and urinary excretion profiles indicated that the drug was extensively metabolized, with the major metabolite identified as the N-dealkylated analogue, which did not show anti-viral activity in vitro.

A pilot study was conducted in HIV patients (n = 8) with CMV retinitis, in order to measure the steady state pharmacokinetics in ocular tissues following 8 days of oral dosing. Antiviral drug levels were achieved in ocular tissues, although substantially lower than those found in plasma. This result indicates a potential for drug efficacy in CNS infections in congenital disease. As expected, blood CMV DNA levels in those viremic retinitis patients responded with a viral load drop. Additional Phase 1 studies included the important drug–drug interaction study using the drug cocktail approach to identify liver cytochrome P-450 enzymes capable of metabolizing maribavir, or being inhibited by maribavir (Ma et al., 2006)

No safety concerns of note were observed in these studies; however, mild to moderate taste disturbance and headache were reported in 80% and 53% of the subjects, respectively (Wang et al., 2003; Lu and Thomas, 2004). The taste disturbance was presumed to be due to secretion of drug or its principal metabolite metabolite into the salivary glands after systematic absorption and while not a safety concern, could have implications for adherence.

A subsequent phase Ⅰ-Ⅱ clinical trial was conducted to further determine the pharmacokinetics of maribavir and to monitor asymptomatic CMV shedding in semen in HIV-infected men (Lalezari et al., 2002). Six dosage regimens (100, 200, or 400 mg three times a day, or 600, 900, or 1200 mg twice a day) or a placebo were evaluated for 28 days. In that proof of concept study, potent anti-CMV activity (2.9 to 3.7 log10 reductions in PFU-ml in semen) was established at all doses. The reductions in CMV titers for all regimens compared well with results reported for the approved doses of CDV (5 mg-kg) in a comparable trial (Lalezari et al., 1995). Maribavir was reasonably well tolerated and safe with taste disturbance again the most frequently reported adverse event. Other adverse events reported by a higher percentage of subjects receiving maribavir than placebo included diarrhea, nausea, rash, pruritus, and fever.

Maribavir has recently demonstrated prophylactic efficacy in a Phase Ⅱ randomized, double-blind, placebo-controlled trial in allogenic stem cell transplant patients (N = 111). Doses of 100 mg BID, 400 mg QD, and 400 mg BID for 12 weeks all reduced the rate of CMV reactivation; preemptive therapy was required for 15%, 30%, and 15% of the respective maribavir doses, relative to 57% for placebo recipients (ViroPharma Inc. press release March 29, 2006). The overall safety profile of maribavir in the 12 week study recapitulates earlier clinical results. The durability of the anti-viral effects and incidence of resistance is under study.

Maribavir is clearly the most advanced new anti herpes drug in the clinical development pipeline. Based on urgent medical need and the favorable development characteristics of maribavir, the FDA has granted Fast Track Status for the prevention of CMV infections in allogenic bone marrow and solid organ transplant patients (ViroPharma Inc. press release Feb. 7, 2005).

Antivirals with activity against EBV, HHV-6, HHV-7, and HHV-8

While anti-viral drug development efforts continue for HSV1 VZV, and CMV utilizing both old and new targets, lack of progress against EBV virus is notable. There are several reasons for this. Primary EBV infection is generally subclinical in immunocompetent individuals. However, it may cause infectious mononucleosis (generally a benign and self-limiting disease) but the window of opportunity for antiviral treatment during the clinical course is short. The lytic EBV manifestation of oral hairy leukoplakia in immunocompromised patients responds to therapy with ACV. EBV-associated lymphoproliferative diseases, Burkitt’s lymphoma and nasopharyngeal carcinoma, all of which may develop without obvious preceding immunodeficiency, remains a major unmet medical need. Latent EBV infection is considered either the etiologic agent for these conditions or a major contributing factor. (Thorley-Lawson and Gross, 2004).

From a drug development perspective; it is not yet clear what EBV or (host) functions to target; and the value of a viral replication inhibitor is unknown (Okano, 2003). The L-benzimidazole riboside, maribavir, which is in clinical developments for CMV disease, also shows activity against EBV in vitro, apparently by blocking both the appearance of linear forms of newly synthesized EBV DNA and the accumulation of the early antigen EA-D (Zacny et al., 1999). While the exact mechanism is unclear, it may be involve the EBV protein kinase, BGLF4, a close homolog of the hCMV UL97. This viral protein autophosphorylates, and has also been reported to phosphorylate the analogous DNA polymerase processivity factor, EA-D (Chen et al., 2000). In lytically infected Akata cells, the level of hyperphosphorylated EA-D was reduced by maribavir treatment, similar to the impact of maribavir treatment on the hCMV pUL44. However, direct inhibition of the phosphorylation of EA-D has not been demonstrated, and maribavir did not block the phosphorylation of EA-D by EBV protein kinase in transient co-expression assays with these two viral genes (Gershburg and Pagano, 2002). Therefore the phenotype of maribavir-treated Alcata cells is consistent with inhibition of BGLF4 function; thus mechanism studies must be addressed within the context of the infected cell. While much remains to be determined regarding maribavir’s mechanism of action against EBV, it should be considered a viable candidate for further study.

Information on the incidence of EBV-associated lymphomas in transplant patients treated prophylatically for CMV infections with maribavir may yield insights into the relationship of lytic replication to the post transplant lymphoproliferative diseases (Razonable et al., 2005).

Research targeted at developing antivirals against human herpesvirus type 6 (HHV-6), type 7 (HHV-7) and type 8 (HHV-8) has been limited. A new series of arylsulfone derivatives have been shown to have in vitro activity against HHV-6 and HHV-7, as well as CMV. While work with this series of compounds is preliminary, a new MOA of indirect inhibition of viral DNA synthesis may be involved (Naesens et al., 2006; Razonable et al., 2005). Both HHV-6 and HHV-7 have an extremely high prevalence rate of about 95% in the US, but in the vast majority of cases their presence is associated with mild self-limiting symptoms, usually fever and rash, where treatment isnt warranted. Infection with HHV-8 is more serious since reactivation is in the form of Kaposi’s sarcoma (Ablashi, 2002). Immune preservation or reconstitution in AIDS patients by highly active anti-retroviral therapy (HAART) has reduced the incidence of Kaposis, indicting the protective effects of the functional immune system in this infection.

A systematic approach to identifying antivirals against HHV-6, HHV-7 and HHV-8 has been completed (de Clercq et al., 2001b). Approved antivirals (ACV, VACV, PCV, FCV, GCV, PFA, CDV, and brivudin) and investigational compounds (Lobucavir, H2G, A-5021, D/L-cyclohexenyl G and S2242) with demonstrated activity against herpesviruses were evaluated in appropriate in vitro systems. The most potent compounds with the highest antiviral selectivity index were: (ⅰ) for HHV-6; PFA, S2242, A-5021 and CDV;(ⅱ) for HHV-7; S2242, CDV and PFA; and (ⅲ) for HHV-8; S2242, CDV and GCV.


The pipeline is indeed rich with new antiherpes agents. Many of these act by novel, and as yet unvalidated mechanisms of action from a therapeutic viewpoint. Their successful performance in the clinic will increase our understanding of the role of these new targets in viral disease.

For the two HSV helicase-primase inhibitors, a critical development milestone will be the human safety data for the thiazole class of compounds, the ease of viral escape (resistance), and the pathogenecity and transmissibility of mutant virus. The hCMV terminase inhibitor candidates start with the advantage of a highly selective and clearly essential target. However, it remains to be seen whether there are unanticipated (non-mechanism-based) toxicities in human studies with the two chemical series of inhibitors. Positive efficacy results in the Phase Ⅲ clinical studies of the candidate CMV drug, maribavir, would provide target validation for the first inhibitor of a viral protein kinase.

As these drug candidates achieve regulatory approval, and with their expanded clinical use, the impact of drug potency and MOA on the emergence of resistance in the population will become the focus of studies. Resistance to the currently licensed antivirals (ACV, PCV, and their prodrugs) is low at < based on HSV isolates, a figure that has remained relatively constant since these drugs have been on the market, in spite of their widespread use (Bacon et al., 2003). The vast majority of ACV-resistant HSV (TK-deficient) viruses are not capable of reactivating after latency, losing the transmission opportunity, and thus the presence of ACV-resistant virus in the overall population is acceptable. In an immunocompromised patient, the risk of developing resistance is much greater than in an immunocompetent individual, and clinical outcome becomes the critical issue. The availability of rescue therapies with novel MOAs will help fill this medical need.


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