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Novel Targets for the Development of Anti-Herpes Compounds

A. Greco*,1, J-J. Diaz1, D. Thouvenot2 and F. Morfin2,3
Universit de Lyon, Lyon, F-69003, France; Universit Lyon 1, Lyon, F-69003, France; CNRS, UMR5534, Centre de Gntique Molculaire et Cellulaire, 16 rue Dubois, Villeurbanne, F-69622, France, 2Laboratoire de Virologie Est, Hospices Civils de Lyon and UMR5537 CNRS - Universit Lyon 1, 8 avenue Rockefeller, F-69373 Lyon cedex 08, France, 3Universit Lyon 1, Institut des Sciences Pharmaceutiques et Biologiques, 8 avenue Rockefeller, F-69373 Lyon cedex 08, France
Abstract: Herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) are members of the Herpesviridae family. HSV infections have been known since ancient times and are one of the most common communicable diseases in humans. Although infections are often subclinical, HSV can cause mild to severe diseases, especially in immunocompromised patients. Herpes simplex viruses establish latency in the nuclei of neuronal cells and may reactivate, with or without symptoms, throughout the host's lifetime. Over one third of the world's population suffer from recurrent HSV infections several times a year and are thus capable of transmitting HSV by close personal contact. There are few drugs licensed for the treatment of HSV infections. Most target the viral DNA polymerase, and indeed acyclovir remains the reference treatment some thirty years after its discovery! Extensive clinical use of this drug has led to the emergence of resistant viral strains, mainly in immunocompromised patients. This highlights the crucial need for the development of new anti-herpes drugs that can inhibit infection by both wild-type viruses and drug-resistant strains. Over the last few years, significant efforts have been made to set up a range of strategies for the identification of potential new anti-viral drugs. One alternative is to develop drugs with different mechanisms of action. The present article reviews potential viral and cellular targets that are now known to be involved in HSV infection and for which specific inhibitors with anti-HSV activity, at least in cell culture, have been identified.
1

Keywords: HSV, resistance, antiviral drugs, viral target, cellular target. INTRODUCTION Herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2) are responsible for a variety of clinical manifestations in humans. They are distributed throughout the world and are transmitted from infected hosts to susceptible individuals by close personal contact. Transmission is broadly favored by the considerable number of asymptomatic infections, associated with viral shedding. Herpes infections are generally localized on the face and trunk in the case of HSV-1 and in the genital sphere in the case of HSV-2, although an increasing proportion of genital infections are associated with HSV-1 [1]. Herpes simplex viruses (HSV) possess an envelope. The central electron-dense core containing the linear double-stranded DNA genome is surrounded by an icosahedral capsid and a tegument layer. The outer envelope presents spike structures corresponding to viral glycoproteins [2]. During the entry step, viral membrane proteins bind to cellular receptors, prior to fusion of the two membranes. Nucleocapsids containing the viral genome are released into the cytoplasm and transported to nuclear pores. Viral DNA is then released into the nucleus. The viral replication cycle is relatively short (18 to 24 hours) and results in cytolysis. The viral DNA encodes at least 80 genes whose expression is sequentially and temporally regulated by complex mechanisms. Viral genes can be divided into imme-diate-early, early and late genes, according to their expression kinetics. Immediate-early proteins are involved in the regulation of the synthesis of early and late proteins. Early proteins participate in viral DNA replication, and late proteins form the structural components of the viral particles. Viral replication and encapsidation take place in the nucleus of the infected cell. Capsids containing the newly replicated viral DNA leave the nucleus and new virus particles are released into the extracellular space via a number of complex processes, including budding of nucleocapsids through the inner nuclear membrane, transport of
*Address correspondence to this author at the Universit de Lyon, Lyon, F69003, France; Universit Lyon 1, Lyon, F-69003, France; CNRS, UMR5534, Centre de Gntique Molculaire et Cellulaire, 16 rue Dubois, Villeurbanne, F-69622, France; Tel: +33 472 447926; Fax: +33 472 432685; E-mail: greco@cgmc.univ-lyon1.fr

mature virus particles through the cytoplasm by exocytic vesicles and fusion with the plasma membrane. The HSV-1 DNA polymerase (pol) has been extensively studied as a target for antiviral drugs. Indeed, some of the many nucleotide and pyrophosphate analogues that have been tested for their ability to inhibit viral replication have led to the development of antiviral drugs - notably acyclovir (ACV) and foscarnet. Acyclovir is of significant therapeutic value and is considered as the "gold standard" in HSV therapy. However, the emergence of ACV-resistant virus strains affects about 5% of immunocompromised patients receiving long-term prophylactic treatment with ACV [3]. Hence, resistance is an important clinical problem that may lead to ineffective therapy (for a review, see [4]). One alternative is to develop antiviral drugs with different mechanisms of action. In principal, all the essential viral proteins involved in HSV replication can be considered as potential targets for chemo-therapy. These include any key protein in any step in the viral cycle, from HSV attachment to the host cell to the release of newly synthesized virions. Most current investigational drugs target viral enzymes involved in key steps of the viral DNA replication, since HSV-1 or HSV-2 encode several viral proteins involved in this function. The latter include DNA-metabolizing enzymes - ribonucleotide reductase (RR), thymidine kinase and dUTPase - and seven proteins that are strictly required for HSV DNA replication, since mutations in the corresponding genes abrogate viral replication. The seven proteins correspond to the origin-binding protein UL9, the single stranded DNA-binding protein ICP8, DNA pol and its accessory protein, encoded by UL30 and UL42 respectively, and the UL5, UL9 and UL52 gene products which form a stable complex with helicaseprimase activities. Some of these viral proteins have been reported in the literature as alternative targets for the development of new kinds of anti-herpes compounds [5-10]. In addition, new antiviral molecules directed against the viral DNA pol are in development; they target other domains of the protein than those targeted by the commercially available drugs. Another alternative strategy in the development of new antiherpes drugs is to target cellular proteins, since many cellular factors are required in the different steps of HSV infection Fig.
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Fig. (1). Strategies for the development of anti-herpes therapies. In the conventional strategy, the essential viral proteins involved in HSV replication are considered as potential targets for chemotherapy. The alternative strategy is to target cellular proteins required in the different steps of HSV infection.

(1). In the last 10 years, it has been shown that cellular proteins can indeed be relevant targets for anti-HSV drugs. The first and most documented example is cyclin-dependent kinase 2 (cdk2) [5, 11, 12]. The present review will focus on the viral and cellular functions that are now known to be involved in HSV infection and for which specific inhibitors with anti-HSV activity, at least in cell culture, have been identified. HSV INFECTIONS Following the primary infection of mucosal surfaces, HSV is transported by retrograde axonal transport from the infected mucosal area and then establishes a latent infection, in the trigeminal ganglia for HSV-1 and the sacral ganglia for HSV-2. This latent infection, in which viral DNA is retained in neuronal nuclei in the absence of viral production, persists throughout the host's lifetime [13]. Despite the development of an immune response, reactivations from this source are triggered by various internal or external stimuli, such as stress, menses, fever, UV-light exposure and immunosuppression. From a clinical standpoint, HSV can cause diseases ranging from mild conditions to morbid or even lethal infections, especially in neonates and immunocompromised patients. Primary infections and reactivations are often subclinical. Asymptomatic shedding of HSV, prompted by subclinical reactivations, occurs frequently in infected individuals. HSVs cause mucocutaneous lesions presenting as vesicles or ulceration in a range of body sites. Oropharyngeal infection often results in gingivostomatitis, which is the most common form of symptomatic, primary HSV infection of the oropharynx. After a short incubation period (4 days), progression is usually rapid (15 days) and may be complicated by auto-inoculation at other sites such as the skin, fingers, nose, eyes, genital area, etc. Recurrent infections are usually described as herpes labialis and last for about 7 days. Genital HSV infection is often more severe and associated with symptoms such as pain. Each episode lasts about 3 weeks in primary infection and 2 weeks for reactivation. Genital herpes may

be complicated by meningitis [14]. The most important difference between HSV-1 and HSV-2 genital infections is that HSV-1 is rarely associated with reactivations, whereas HSV-2 may be associated with 3 to 9 reactivations per year [15]. HSV-1 is involved in ocular infections and may cause keratitis (presenting as unilateral or bilateral conjunctivitis) with preauricular lymphadenopathy, followed by an unilateral, characteristic, dendritic ulcer which usually lasts for 3 weeks [16]. In 20 to 30% of patients, these infections are associated with recurrence within 2 years. Multiple infections can result in loss of vision; indeed, in developed countries, ocular HSV infection is the most common cause of corneal blindness. Neonatal herpes is a serious infection predominantly caused by HSV-2. HSV-1 can also be responsible, as transmission may occur with a primary HSV-1 genital infection or via an oral herpes from the mother, her relatives or hospital staff. Literature reports of incidence of neonatal herpes vary considerably and range from 1 case per 2,500 live births in the USA [17] to 1.65 per 100,000 live births in the UK [18]. American studies suggest that there are three types of disease presentations: (i) localized infections with skin, eye and/or mouth lesions, (ii) infections associated with neurological symptoms and (iii) disseminated disease (the most severe form). In the absence of antiviral therapy, the mortality rate reaches 80% in cases of disseminated disease, warranting early antiviral therapy for all neonatal HSV infections. Another serious form of HSV infection is encephalitis, which is confined to the frontotemporal and parietal areas of the brain and may progress rapidly to coma and death. HSV encephalitis is a concern for all age groups, in both men and women. The incidence of HSV encephalitis ranges from about 1 case per million population per year to 1 case per 200,000 [17]. Most cases are caused by HSV-1. In untreated patients, mortality reaches 70% and only 2.5% of surviving patients have no neurological sequellae [17]. HSV infection may be particularly severe in immunocompromised patients in general and those with cell-mediated immunity deficits in particular. These patients present extensive lesions that

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may disseminate and/or progress chronically. These infections require prophylactic and/or curative antiviral chemo-therapy. HSV infection diagnosis relies on virus detection in mucocutaneous samples via antigen or nucleic acid detection and culture [19, 20]. Serology can also be of interest. HSV culture is sensitive and easy to perform; furthermore, it is required for testing HSV's sensitivity to antiviral drugs using phenotypic methods. Despite the fact that vaccination against HSV has been evaluated since the 1920s, a HSV vaccine is still not available (for a review see [21]). However, there are a few drugs licensed for the treatment of these infections. Most target the viral DNA pol, and ACV remains the reference treatment. AVAILABLE ANTI-HERPES DRUGS A number of antiviral drugs for the treatment of HSV infections have been developed since the mid 1950s. The first were idoxuridine and trifluridine, which are pyrimidine analo-gues that are phosphorylated by viral and cellular kinases. Given their toxicity, these molecules could only be used for topical treatment of HSV infections. In the 1980s, vidarabine became available as a systemic treatment for HSV encephalitis. This molecule is an adenosine analogue that is phosphorylated only by cellular kinases. There are currently three classes of drugs licensed for the treatment of HSV infections, and all target viral DNA replication: (i) the acyclic guanosine analogue ACV and related compounds, such as penciclovir and their prodrugs (valacyclovir and famciclovir, respectively), (ii) the acyclic nucleotide analogue cidofovir and (iii) the pyrophosphate analogue foscarnet. Despite numerous attempts to improve on ACV, the latter remains the gold standard for HSV treatment - some thirty years after its discovery! ACV has to be phosphorylated three times before its incorporation into viral DNA. Since the first phosphorylation is achieved by the viral TK, ACV becomes active only in infected cells. The second and third phosphorylations are carried out by cellular thymidylate kinases. ACV triphosphate is a competitive inhibitor of viral DNA pol and it is a DNA chain terminator [22]. Penciclovir has a very similar mechanism of action. ACV and penciclovir have very poor oral bioavailability and synthesis of the prodrugs, valacyclovir and famciclovir, respectively, has improved this parameter. ACV and penciclovir have very few side effects. Some nephrotoxicity has been reported but is uncommon and always reversible [23]. ACV-resistant HSV strains were first reported in 1982 [24-26], with most being recovered from immunocompromised patients previously treated with ACV [27-29]. ACV resistance may be associated with a mutation occurring spontaneously in one or both of the two viral enzymes targeted by ACV's mecha-nism of action (TK and DNA pol). Three resistance mechanisms have been reported: a loss of TK activity (TK-deficient virus), an alteration in TK substrate specificity (TK-altered virus) and/or an alteration in DNA pol activity [30]. The most frequent mutations occur in the viral TK gene, and 95% of ACV-resistant isolates present a TKdeficient phenotype [31]. Resistance associated with a mutation in the viral DNA pol gene occurs rarely [32] because a functional DNA pol is essential for viral replication (unlike viral TK). Large-scale studies have shown that resistance is mainly a concern for immunocompromised patients, and several reports have described a mean prevalence below 1% in immunocompe-tent populations [27, 29, 33]. Recently, another large survey reported a prevalence of 0.32% [3], i.e. a value that has not increased since the first studies were performed ten years previously, despite a major increase in ACV use. According to the literature, most ACVresistant HSVs isolated from immuno-competent individuals have been detected in a context of recurrent genital herpes, and the observed prevalence ranged from 3.5% to 8.6% [34, 35]. However, in most cases, the clinical course of the infection was unaffected.

The prevalence of ACV resistance in immunocompromised patients is about 5%, according to several published reports [27-29]. This incidence depends on the type of immunosuppression. In a recent survey, resistance was detected in 2.8% of solid organ transplant patients, 3.5% of human immunodeficiency virusinfected patients and 14% of bone marrow transplant patients [3]. In this latter population, the incidence of resistance was 2% for autologous transplant patients and 29% for allogeneic transplant patients. Another recent study revealed that patients receiving bone marrow of either autologous or allogeneic origin have about the same risk of developing an HSV infection (9%) but that resistance to ACV was only detected in allogeneic transplant patients, with a prevalence of up to 30% in this population [36]. These findings indicate that the prevalence of ACV-resistant HSV is steady, relative to studies published more than 10 years ago. This stability is noted for both immunocompetent and immunocompromised patients. Finally, as suggested by other studies, ACV resistance is restricted to allogeneic bone marrow transplant patients but is a major concern for these individuals. In immunocompromised patients, resistance is associated with clinical failure after 1 to 2 weeks of treatment. Lesions may present as large, ulcerated and extensive. These infections are often serious, and may become chronic [37]. After healing, recurrent infections are most often associated with an ACV-sensitive strain, although some cases of recurrences due to ACV-resistant viruses have been reported [38-41]. One way of managing ACV-resistant infection consists in improving the patient's immune status, when possible, by decreasing immunosuppressive treatments [42]. In addition, several alternative antiviral drugs may be used. Most ACV-resistant HSV isolates are also resistant to penciclovir, although ACV-resistant, penciclovir-susceptible isolates have been reported from time to time; the ACV resistance mechanism of these strains was related to either an altered TK or a mutation in the viral DNA pol [43, 44]. Foscarnet and cidofovir act directly on viral DNA pol without the need for prior activation by viral TK, and so both of these molecules are active against ACV-resistant viruses which carry a mutation in the TK gene [39, 45]. However, in clinical practice, these drugs may be associated with a significant level of toxicity. Foscarnet inhibits the pyrophos-phate binding site of DNA pol. Cidofovir is a monophosphory-lated derivative of cytidine that is phosphorylated twice by cellular kinases before being incorporated in the DNA chain by viral DNA pol. Foscarnet-resistant clinical isolates of HSV have been isolated, albeit rarely, from AIDS patients [46] and bone marrow transplanted patients [47]. Foscarnet resistance is associated with viral DNA pol mutations [46]. Cidofovir has been used for the treatment of ACV- and foscarnetresistant HSV infections [48]. To date, resistance to cidofovir has not been reported for HSV. POTENTIAL VIRAL TARGETS Numerous investigations using a variety of approaches have focused on the identification and validation of viral targets. Proteins involved in viral DNA replication are still attractive targets; however, viral proteins that are essential in other steps of the viral infection also represent potentially valuable targets as well Fig. (1), Table (1). Attachment and Penetration Penetration of HSV-1 into the cell involves at least five (gC, gB, gD, gH and gL) of the dozen or so of the viral envelope glycoproteins, together with three classes of host-cell membrane receptors; herpes virus entry mediator (HVEM, a member of the tumor necrosis factor receptor family), nectin-1 and nectin-2 (two members of the immunoglobulin superfamily) and cell surface glycosaminoglycans (preferentially heparan sulfate) [49].

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Table 1. Potential Viral and Cellular Targets for the Development of Anti Herpes Drugs. The Potential Viral and Cellular Targets and their Inhibitors Described in the Review are Presented

Potential targets Viral Glycoproteins CarraguardTM Emmelle/dextrin-2-sulfate

Drugs

polystyrene sulfonate PSS, PRO2000/5TM UshercellTM, Virend Dendrimers Ribonucleotide reductase Thiosemicarbazone family Peptidomimetic inhibitors Antisens oligomers DNA polymerase 4-hydroxyquinoline-3-carboxamide (PNU-182171 and PNU-183792); thieno[2,3-b]pyridine carboxamide Peptidomimetic inhibitors Small molecules i.e. BP5 Helicase-primase complex (UL5, UL8 and UL52) 2-amino-thiazole (T157602) (helicase domain) Aminothiazolyl-phenyl compounds (BILS 179 BS and BILS 45 BS) Thiazolylsulfonamide compounds (BAY 57-1293) siRNAs (UL5) Cellular Cyclin-dependent kinase 2 Polyamine pathway Pharmacological cdk inhibitors (olomoucine, roscovitine) Inhibitors of SAMDC Inhibitors of ornithine decarboxylase Entry receptors (HVEM, extracellular proteoglycans) Anti-HVEM antibodies; Apolipoprotein E-derived peptides

SAMDC: S-adenosyl methionine decarboxylase; HVEM: human virus entry mediator.

At present, none of the antiherpes drugs currently licensed for the treatment of HSV infections displays a well-demonstrated mode of action consisting of the inhibition of viral entry [50]. The lack of molecules with this type of mechanism of action is certainly due to the fact that virus penetration is a very complex, incompletelydeciphered process that involves numerous viral and cellular molecules that are difficult to target simultaneously with small molecule drugs. Nevertheless, many studies provide convincing evidence that certain types of molecules interfere more or less specifically with virus entry (by interacting either with the viral particle or with cellular receptors or both) and thus display antiherpes activity. Hence, these data strongly support the notion that blocking viral entry could be used to develop new kinds of antiviral compounds; targeting the process of virus penetration has the advantage of developing molecules that do not need to enter the cell to be effective, thus facilitating their development during the drug discovery process. To illustrate this kind of approach, one might mentioned the development and use of several microbicides against herpes infections, although this type of molecules are not generally classified as antiviral compounds, due to their mode of action and usage restricted to local applications. Nevertheless, several microbicides are now being tested in Phase II or III clinical trials for their ability to prevent virus shedding and its transmission by close personal contact. A few of these are sulfated polymer-based inhibitors that interact directly with viral envelope glycoproteins and prevent viral attachment to the cell (CarraguardTM, Emmelle

/dextrin-2-sulfate, polystyrene sulfonate PSS, PRO2000/5TM, UshercellTM, Virend, dendrimers) [8, 51]. In addition, various plant-extracted compounds with chemical structures of varying complexity derive their valuable antiherpes activity, at least in part, by acting on the virus attachment and penetration stages [52, 53]. It was recently shown that a phosphorothioate oligonuc-leotide displays a strong virucidal activity by inhibiting virus attachment. The mechanism of action of this oligonucleotide is not well determined although it was proposed that it is able to induce a conformational change in gB that results in inactivation of infectivity [54]. Other types of molecules have been shown to interfere with viral entry with limited specificity. The lactoferrin protein and lactoferricin, a small peptide fragment from lactoferrin's N-terminal domain, are prototypes of this family of compounds, whose mechanism of action is currently receiving much attention. Lactoferricin interacts with cell surface heparan sulfate to block viral entry. In contrast, lactoferrin's mechanism of action has not yet been elucidated in detail, although it is clear that lactoferrin must be located at the cell surface to exert its anti-HSV activity and that lactoferrin probably interferes with viral binding to heparan sulfate [55]. Modeling of the anti-HSV activity of lactoferricin analogues is currently being performed, in order to design more efficient antiHSV peptides [56]. More generally, small amphiphilic peptides that are able to form beta-sheet structures display some antiviral activity - either by binding to the virus themselves or by binding to the cell surface and

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therefore interfering with virus attachment [56, 57]. However, given that these compounds are not very specific and frequently produce a high degree of hemolysis, their potential use would be restricted to topical treatments in order to reduce viral spread and duration and to ease the symptomatology of cutaneous HSV disease. Viral DNA Replication Ribonucleotide Reductase Herpes simplex viruses encode their own RR, which converts ribonucleoside diphosphates to the corresponding deoxyribonucleotides required for viral DNA synthesis. Herpesviridae RRs are highly conserved and are composed of two homodimeric subunits, RR1 (large) and RR2 (small), whose association is essential for enzymatic activity. For the last 20 years or so, HSV RR has been considered as a potential target for antiviral chemotherapy. The first anti-RR products to be identified belonged to the thiosemicarbazone family. However, the latter inhibit cellular RR as well as viral RR. Thereafter, highly potent and specific peptidomimetic viral RR inhibitors that impair association of the two subunits have been developed. Given that the C terminus of RR2 is critical for this association, peptides corresponding to the sequence of the last few amino acids of this subunit have been synthesized. These peptides inhibit HSV RR activity in vitro by inducing disso-ciation of the two subunits [58-60] and suppress the replication of HSV-1, HSV-2 and ACV-resistant HSV strains in cell culture. They also reduced the severity of HSV-1-induced keratitis in a murine ocular model [61, 62]. Studies conducted with a viral strain selected in vitro for its resistance to one of these peptides demonstrate that mutations are located within the C terminus of the RR2 gene; however, viral RR is not required for HSV replication in exponentially growing cells and so drugs that prevent RR function have poor antiviral activity in this context [63, 64]. However, recent studies have also revealed that the large (RR1) subunit of HSV-2 RR contains a protein kinase function in addition to its well known RR function, and that HSV-2 RR1 is involved in immediate early gene transcription and virus growth [65]. Indeed, antisense oligonucleotides complementary to HSV-2 RR1 inhibit HSV-2 RR1 expression and HSV-2 replication. In addition, the protein kinase function of HSV-2 RR1 may contribute to HSV-2 reactivation, and it has been reported that antisense oligomers inhibit HSV-2 reactivation from latently infected ganglia [66-68]. Hence, the RR1 protein kinase domain, required for HSV-2 replication and reactivation from the latent state in neurons, represents a potential target in the field of anti-HSV-2 drug development. DNA Polymerase The core of the viral DNA pol from HSV-1-infected cells consists of a heterodimer of the DNA pol catalytic subunit and the accessory subunit, encoded by UL30 and UL42, respectively. HSV DNA pol is still an attractive target. ACV resistance has prompted attempts to develop anti-herpes drugs that inhibit DNA pol activity by other mechanisms. The combination of high-throughput screening of small molecules that specifically inhibit herpes DNA polymerases and structure-activity relationship studies resulted in the discovery of a novel class of non-nucleoside inhibitors, the 4-hydroxy-quinoline3-carboxamide compounds. These molecules appear to be specific for a broad range of herpes DNA pols, including those of HSV-1 and HSV-2 [69, 70]. In fact, a derivative of 8-hydroxyquinoline was shown to prevent HSV-1 infection as early as 1976 [71]. The PNU182171 and PNU-183792 derivatives of 4-hydroxyquinoline-3carboxamide and the 10c and 14 deriva-tives of a novel series of thieno[2,3-b]pyridine carboxamides have anti-HSV activity in cell culture via inhibition of viral DNA pol [72, 73]. These compounds are competitive inhibitors of deoxynucleoside triphosphate binding

to DNA pol. However, they maintain potent antiviral activity against ACV-resistant HSV strains, indicating that the mechanisms of action are different. Laboratory HSV mutants selected for their resistance to PNU-182171 are resistant to hydroxyquinoline as well as to thieno[2,3-b]pyridine derivatives. Sequence analysis of DNA pol from these mutants revealed that a single amino acid change of valine to alanine at position 823 (V823A) within the conserved domain III is responsible for the resistance. V823 is conserved in 6 of the 8 herpes DNA polymerases. Since domain III plays a critical role in the interaction between the incoming nucleotide and primer template DNA, these inhibitors could bind to the 823 region of the DNA pol and may disrupt the pol's interaction with the DNA template strand, thus interfering with the binding or incorporation of the incoming dNTP [73]. The crystal structure of the HSV-1 DNA pol catalytic subunit has been published very recently. In the proposed model based on results of co-crystal-lization and soaking experiments, the PNU-183792 non-nucleoside inhibitor competes with incoming nucleotides and dislocates the template from the active site [74]. PNU-183792 does not exhibit any cytotoxicity in a range of cell lines at relevant drug concentrations, and is orally bioavailable. These carboxamide derivatives are active against a broad range of herpes viruses and their DNA pols. Preclinical animal studies are ongoing, particularly for human cytomegalovirus infections [73]. The accessory subunit of the DNA pol complex is encoded by the UL42 gene. It binds directly to DNA and increases the processivity of polymerization by (i) increasing the affinity of the DNA pol catalytic UL30 subunit for the 3' terminus of the primer template and (ii) decreasing the probability that the DNA pol will dissociate from the template after each catalytic cycle. Specific interaction between DNA pol and the UL42 accessory protein is necessary for full DNA pol function and for viral replication [7577]. Since the UL30-UL42 interaction is critical for viral replication, its disruption represents a potential target in the design of novel antiviral agents. Three peptides corresponding to the last 38, 27 and 18 Cterminal residues of HSV-1 DNA pol catalytic subunit have been synthesized. In vitro, they inhibit the UL30-UL42 interaction and UL42-dependent long-chain DNA synthesis by HSV DNA pol but not the activity of the cellular Pol -PCNA [78-80]. When the 27mer peptide of the HSV-1 DNA pol C-terminus is fused with the enterotoxin B peptide, the resulting molecule is targeted into the nucleus of HSV-1-infected cells and inhibits viral replication by disruption of the UL30-UL42 interaction [79]. This UL30-UL42 interaction has been extensively characterized by muta-tional and structural analysis [81-83]. Biophysical studies have identified specific hydrogen bonds that are crucial for the inter-action. In particular, a hydrogen bond network connects the R1229 residue of DNA pol to the Q171 residue of UL42, which in turn bonds to the F1211 residue of DNA pol [84]. Recently, small molecules that specifically interfere with the UL30-UL42 interaction in HSV-1 have been selected using high-throughput screening. One of these, called BP5, inhibits the interaction in vitro. In addition, BP5 also prevents HSV-1 infection in Vero cells as efficiently as ACV, although it is more toxic. The mechanism of action is still unknown. However, results from a HSV mutant selected for its resistance to BP5 suggest that BP5 is virus-specific. More potent and less toxic molecules are being developed [85]. Helicase-Primase The helicaseprimase complex comprises three viral proteins encoded by the UL5, UL8 and UL52 gene products. It unwinds double-stranded viral DNA and generates primers for DNA synthesis by the viral DNA pol. UL8 is not required for enzy-matic activities but may be involved in recruiting HSV-1 DNA pol into the viral DNA replication complex [86]. Therefore, the helicaseprimase complex plays an essential role in HSV DNA replication and thus constitutes an excellent target for antiviral therapy.

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Three new types of herpes inhibitors that target the HSV helicase-primase complex have recently been discovered. High throughput screening of a molecule library with detection of the HSV1 UL5/UL8/UL52 complex's helicase activity led to identification of T157602, a 2-amino-thiazole. Genetic analysis demonstrated that T157602 targets the helicase domain of the complex. It also inhibits HSV replication in cell culture [87]. Viruses selected for their resistance to T157602 presented a mutated UL5 gene [88]. Another high throughput screening program coupled with lead optimization studies, led to aminothiazolyl-phenyl-containing compounds that act by increasing the affinity between enzyme and DNA and prevent the propagation of HSV helicase-primase catalytic cycles. Two of these, BILS 179 BS and BILS 45 BS, are much more active than ACV against wild-type laboratory and clinical HSV-1 strains and ACV-resistant HSV isolates. The antiviral activity of BILS 179 BS is HSV-specific, since it does not inhibit replication of the other members of the herpes family. BILS 45 BS displays very effective oral therapy of experimental ACVresistant HSV-1 infections in nude mice [89, 90]. Single amino acid changes in the UL5 protein have been identified in mutant viruses selected for their resistance to a BIL 45 BS analog [91]. Thiazolylsulfonamide compounds also target the viral primasehelicase complex and inhibit its ATPase activity [92, 93]. Of these, BAY 57-1293 has potent anti-herpes activity in vitro and is active against ACV-resistant mutant strains. BAY 57-1293 is more potent than ACV in vitro and in vivo, and is especially effective when treatment is delayed. It has a lower resistance rate than ACV in vitro. In animal models BAY 57-1293 has superior efficacy against HSV and also shows favorable pharmacokinetic parameters. It almost completely suppresses recurrent disease and asymptomatic virus shedding, and no detectable HSV replication takes place in the neuronal tissues of animals treated with BAY 57-1293. A very different approach has demonstrated that mice are protected from lethal infection after vaginal instillation of siRNAs designed to inhibit the HSV-2 UL5 gene expression, thus strengthening the latter's status as an important viral gene [94]. Hence, the viral helicase-primase complex represents a very promising target in the development of new anti-herpes drugs. POTENTIAL CELLULAR TARGETS Although the viral genome encodes many functions that are necessary for viral propagation in host cells, cellular proteins are also required. Much effort has been devoted to identifying the latter Fig. (1), Table (1). Within the last 10 years, attention has been focused on cellular protein kinases as antiviral targets. Recent studies have shown that replication of HSV requires specific cyclin-dependent kinases (cdks). These Ser/Thr kinases regulate the cell division cycle, apoptosis, transcription, differentiation and numerous neuronal functions. They are active when they are associated with a cyclin or another regulatory partner. Upon HSV infection, the cdk2/cyclin E complex is activated. Many pharmacological cdk inhibitors (PCIs) are available since the latter have been developed as anticancer agents. Some of these, such as olomoucine and roscovitine, have potent in vitro antiviral activity against HSV-1 and HSV-2. Roscovitine belongs to a class of specific cdk inhibitors which inhibit cdk1, 2, 5 and 7. The inhibition of HSV-1 infection by PCIs occurs by a variety of mechanisms. The PCIs inhibit activation of immediate-early and early gene expression and viral DNA synthesis. Roscovitine interferes with phosphorylation of the viral proteins ICP4 and ICP0; it inhibits ICP0's transactivating activity but not the latter's ability to disperse or degrade the cellular proteins associated with ND10 nuclear structures. Moreover, roscovitine also affects replication of

HSV-1 DNA by a mechanism which remains to be elucidated [95100]. Interestingly, cdk2 is not expressed in quiescent neurons, whereas nuclear cdk2 is induced in neurons submitted to a stressful stimulus that induces HSV reactivation. Since HSV-1 reactivation is inhibited in the presence of a cdk2-specific inhibitor, it has been speculated that cdk2 is one of the factors that determine the outcome of HSV infection in neurons [101]. Co-crystallization studies indicate that all PCIs analyzed to date target the ATP-binding pocket of the kinase's catalytic site (for a review, see [11]). PCIs inhibit the replication of wild-type and multi-drug resistant strains of HSV-1 and HSV-2. Despite repeated attempts, PCI-resistant HSV-1 strains have never been isolated. This suggests that PCIs do not act by inhibiting a single viral target. Many PCIs are being developed as antivirals and are scheduled to enter clinical trials (for a review, see [12]). A recent report has demonstrated that the polyamine biosynthetic pathway remains active in HSV-1 infected cells and that expression of the two key enzymes of this pathway, S-adenosyl methionine decarboxylase (SAMDC) and ornithine decarboxylase, is upregulated [102, 103]. This argues for a role of polyamines in the HSV-1 replication cycle. Indeed, HSV-1 particles contain two types of polyamine, spermidine in the envelope and spermine in the nucleocapsid. Furthermore, polyamines are involved in the replication of HSV-1 DNA [104, 105]. The use of methylglyoxal bis(guanylhydrazone), a specific SAMDC inhibitor, reveals that SAMDC activity is required for HSV-1 and HSV-2 replication in cell culture. Furthermore, HSV-1 infection is severely repressed when cells are pretreated with methylglyoxal bis(guanylhydrazone) and when treatment is delayed until the viral adsorption or the immediate-early and early steps of infection. Moreover, inhibition of SAMDC activity prevents the in vitro replication of HSV-1 and HSV-2 laboratory strains and of clinical mutant isolates that are resistant to conventional antiviral drugs such as ACV and foscarnet [103]. Hence, SAMDC and other key enzymes in the polyamine pathway represent potentially interesting anti-HSV targets. A few reports indicate that molecules that target cellular receptors or extracellular proteoglycans can, under some circumstances, efficiently block viral penetration into a specific tissue or organ. Infection of primary cultures of human T lymphocytes by HSV-1 can be inhibited by an HVEM-specific antiserum [106]. Moreover, it has been demonstrated that an anti-HVEM antibody inhibits the entry of HSV-1 into primary cultures of human ocular trabecular meshwork cells, suggesting that molecules inhibiting the interactions between HVEM and viral glycopro-teins could be a valid strategy to prevent the eye infections, at least with HSV-1, whose complication contributes to the development of blinding disease [107]. Recently, it was shown that the apoEdp fragment of the apolipoprotein E is able to inhibit viral infection via a mechanism that has not yet been defined but that is partially mediated by direct inhibition of viral attachment, perhaps with the blockage of extracellular proteoglycan sites or via binding to low-density lipoprotein receptor-related proteins [108]. Cellular topoisomerases are essential enzymes in the unwinding of the double-stranded DNA and have also been considered very recently as possible anti-herpes targets. Herpes virus infections are known to require topoisomerases I and II; inhibitors of topoisomerase II derived from substituted acridines are able to inhibit herpes infections, most probably by blocking topoiso-merase binding to DNA [109, 110]. CONCLUSIONS Despite intensive research, no prophylactic HSV vaccine has proven to be effective. An alternative approach to the control of HSV infections is to develop chemotherapeutic drugs with anti-

Novel Targets for the Development of Anti-Herpes Compounds

Infectious Disorders - Drug Targets 2007, Vol. 7, No. 1

17

HSV activity. The emergence of virus strains which are resistant to the available anti-herpes drugs has accelerated the antiviral research process. Until the last decade, viral functions have been the targets of choice in the anti-herpes drug discovery field, essentially because drugs directed against viral targets were expected to have high selectivity and low toxicity and because almost all the essential viral proteins have been identified. The resulting molecules have very specific antiviral activity, since they are restricted to herpes infections. In fact, these drugs inhibit infection of just one or a few members of the herpes family and rarely have broad-spectrum activity. Moreover, the occurrence of resistance to these new potential antiviral drugs, or at least those targeting the helicase primase complex, appears to be less frequent than for ACV. More recently, a range of studies have highlighted the importance of host cellular factors in regulating HSV infection. Some of these cellular proteins have been used as novel targets for antiviral therapeutics. Drugs targeting cellular functions are expected to have different advantages, including lower selectivity (i.e. inhibition of a broader range of viruses) and the diminished appearance of resistant viral strains. Indeed, cdk2 inhibitors prevent infection by HSV-1, HSV-2, other herpes viruses, drug-resistant viruses and even unrelated viruses that require active cdk2 for replication [111-113]. One can speculate that inhibitors of cellular proteins necessary for viral replication would be active against a broad range of viruses, and that targeting cellular proteins needed for viral infection may also help to prevent or slow the emergence of drug-resistant viral strains. One drawback of this strategy is that molecules acting on cellular proteins could display cytotoxic effects. This point should be taken into account for the future developments of this type of antiviral molecules. It is interesting to note that some drugs targeting HSV-2 ribonucleotide reductase or the cellular cdk2 seem also to inhibit HSV-2 and HSV-1 reactivation, respectively. Significant progress has been made in understanding the dynamics of viral infection and reactivation from latency. This will help in the identification of new, relevant drug targets and therefore in the development of new antiviral drugs with greater efficacy and safety. Management of viral infections will be facilitated by using unrelated target proteins to develop drugs with non-overlapping mechanisms of action. In theory at least, every protein whether or cellular or viral origin involved in each step of the virus cycle still represents a potential target for the development of anti-herpes drug. How-ever, not enough data are yet available to clearly demonstrate that proteins other than those described in this review have been identified as good potential targets. The alternate use of drugs in development, the combination of these drugs with conventional ones or the combination of drugs directed respectively against viral and cellular targets all constitute promising strategies for reducing the appearance and dissemination of drug-resistant viruses and preventing reacti-vation from latency. ACKNOWLEDGEMENTS We are thankful to Dr Henri Agut for his comments and suggestions about this review. A. Greco and J-J Diaz are members of the Institut National de la Sant et de la Recherche Mdicale (INSERM). ABBREVIATIONS ACV = Acyclovir cdk = Cyclin-dependent kinase HSV = Herpes simplex virus HSV-1 = Herpes simplex virus type 1 HSV-2 = Herpes simplex virus type 2

HVEM PCI Pol RR SAMDC TK

= = = = = =

Herpes virus entry mediator Pharmacological cdk inhibitor Polymerase Ribonucleotide reductase S-adenosyl methionine decarboxylase Thymidine kinase

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Received: July 11, 2006

Accepted: October 13, 2006