Antiviral drug resistance and helicase–primase inhibitors of herpes simplex virus

Drug Resistance Updates 14 (2011) 45–51

Contents lists available at ScienceDirect

Drug Resistance Updates journal homepage: www.elsevier.com/locate/drup

Antiviral drug resistance and helicase–primase inhibitors of herpes simplex virus Hugh J. Field a,∗ , Subhajit Biswas b a b

Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB30ES, United Kingdom University of Cambridge, National Health Services Blood & Transplant Centre, Long Road, Box-96, Cambridge CB2 0PT, United Kingdom

a r t i c l e

i n f o

Article history: Received 27 July 2010 Received in revised form 22 November 2010 Accepted 23 November 2010

a b s t r a c t A new class of chemical inhibitors has been discovered that interferes with the process of herpesvirus DNA replication. To date, the majority of useful herpesvirus antivirals are nucleoside analogues that block herpesvirus DNA replication by targeting the DNA polymerase. The new helicase–primase inhibitors (HPI) target a different enzyme complex that is also essential for herpesvirus DNA replication. This review will place the HPI in the context of previous work on the nucleoside analogues. Several promising highly potent HPI will be described with a particular focus on the identification of drug-resistance mutations. Several HPI have good pharmacological profiles and are now at the outset of phase II clinical trials. Provided there are no safety issues to stop their progress, this new class of compound will be a major advance in the herpesvirus antiviral field. Furthermore, HPI are likely to have a major impact on the therapy and prevention of herpes simplex virus and varicella zoster in both immunocompetent and immunocompromised patients alone or in combination with current nucleoside analogues. The possibility of acquired drug-resistance to HPI will then become an issue of great practical importance. © 2010 Elsevier Ltd. All rights reserved.

1. Effective HSV antiviral therapy in its third decade

2.1. Thymidine kinase

It is well known that nucleoside analogues have dominated the therapeutic armory for treating herpes simplex virus (HSV) since the introduction of 5-iododeoxyuridine, adenine arabinoside and triflurothymidine in the early 1960s. The principal compounds that are currently used, and have been over the past three decades, are acyclovir (ACV), penciclovir (PCV) and their respective orally bioavailable prodrugs valaciclovir (VACV) and famciclovir (FCV) (Field and De Clercq, 2004).

It soon became apparent that mutations that result in defective TK activity exist in plaque-pure isolates of HSV at relatively high frequency (e.g. >10−4 PFU) (Parris and Harrington, 1982). TK activity is not an essential function in tissue culture and TK-defective strains grow with equal rate and to equal titres compared with wild-type (Field and Wildy, 1978), furthermore, there is no requirement for compensating mutations to allow such strains to multiply in cell culture. Numerous different mutations give rise to the TK-defective viruses including insertions or deletions in homopolymeric G–C nucleotide stretches resulting in premature termination yielding no product or a truncated polypeptide. It has been shown that many such mutations result in the resistant phenotype (Andrei et al., 2001; Frobert et al., 2008). Single base substitutions in, or close to, the active site of the TK gene were also shown to confer nucleoside analogue resistance (Darby et al., 1981) by means of altered substrate specificity although conditions must be arranged to select for functional TK in tissue culture (Larder et al., 1983) and such mutations occur with relatively low frequency (≤10−6 ).

2. Drug resistance and the nucleoside analogues HSV readily multiplies in tissue culture and plaque-formation is a convenient assay. It was quickly shown after their discovery that virus cultured in the presence of ACV or PCV would yield resistant plaques within one or two passages. Furthermore, resistance mutations exclusively mapped to the genes coding for thymidine kinase (product of UL23 gene) and DNA polymerase (product of UL30) (Coen and Schaffer, 1980; Schnipper and Crumpacker, 1980).

2.2. DNA polymerase ∗ Corresponding author. E-mail addresses: [email protected] (H.J. Field), [email protected] (S. Biswas). 1368-7646/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.drup.2010.11.002

Nucleoside analogue triphosphates as well as the phosphonyl derivatives (e.g. adefovir), aphidicolin, and pyrophosphate ana-

46

H.J. Field, S. Biswas / Drug Resistance Updates 14 (2011) 45–51

logues (e.g. phosphononformate), all ultimately interact with HSV DNA polymerase. The compound or metabolite thus either inhibits the DNA polymerase directly or is incorporated into HSV-DNA leading to chain termination or other defect. HSV DNA polymerase is an essential enzyme for virus replication therefore mutations leading to loss of this enzyme activity render the virus non-viable. Consequently, drug-resistance mutations in DNA polymerase occur following amino acid substitutions that allow the enzyme to retain function (Larder and Darby, 1985). In tissue culture, in our experience, such mutations occur with relatively low frequency (≤10−6 PFU). Others have claimed a higher frequency of DNA polymerase mutations in clinical isolates based on the detection of ACV-resistant virus with co-resistance to phosphonoacetic acid (e.g. Parris and Harrington, 1982). However, although those early studies were carried out very carefully, the location of mutations to the DNA polymerase gene was inferred indirectly from plating efficiency in the presence of an alternative inhibitor and without the benefit of modern sequencing technology to confirm the sites of resistance mutations. Higher frequencies of HPMPC-resistance have been reportedly obtained from patients who were possibly immunocompromised and had received antiviral therapy (Sarisky et al., 2000). 2.3. Difference between HSV-1 and HSV-2 The majority of HSV isolated from the most-common mucocutaneous lesions of the face and mouth are HSV-1. HSV-1 is also a cause of the severe (albeit rare) condition of herpes encephalitis and, more commonly, recurrent ocular disease – herpes keratitis. By contrast, genital herpes is usually caused by the related virus, HSV-2, although this anatomical differentiation is not universal. As mentioned above, mutations leading to nucleoside analogue resistance occur with relatively high frequency. It has been noted in several studies, however, that the frequency of resistance mutations is higher in HSV-2 than HSV1 in both laboratory and clinical isolates (Sarisky et al., 2000). The biological reasons that underlie these observations remain unclear. 3. Mutation frequency/rate in tissue culture Although DNA viruses are generally held to have a lower “mutation rate” than RNA viruses this is not necessarily easy to measure. There is also difficulty in distinguishing mutation “frequency” from “rate” (Smith and Inglis, 1987; Drake and Holland, 1999) that needs to be defined in terms of the number of substitutions with time or genome replications (Vere Hodge and Field, 2010). For HSV, frequency can be determined by culturing a known titre of plaqueforming units (PFU) in the presence of an inhibitor, e.g. 10 ␮M ACV (10-times the IC50 of approximately 1 ␮M in cell culture). As mentioned above, for the TK-defective phenotype this is approximately 10−4 PFU. Since a single plaque of HSV produces of the order 105 PFU this means that resistance to ACV can appear within a single passage in tissue culture. In order to detect substrate-specificity mutants in either DNA polymerase or TK, however, a greater number of virions must be screened (>106 PFU). The number of host cells in the culture dish then becomes a practical limitation and it may be necessary to passage virus several times in the presence of a sub-inhibitory concentration of the inhibitor in order to detect resistant plaques. Furthermore, for the TK-dependent nucleoside analogues, culture conditions must be arranged to select against TK-defective strains which occur relatively frequently. Alternatively, HSV-TK was provided in trans in order to maintain high levels of the nucleoside triphosphate in the infected cells (Larder et al., 1983).

4. Mutation frequency/rate in vivo 4.1. HSV nucleoside analogue-resistance in the immunocompromised host HSV antiviral drug resistance is a well-recognized problem in immunocompromised patients and frequencies of the order 5% among clinical isolates have been commonly reported (Field, 2001). Even higher rates (14–30%) have been claimed in bone-marrow recipients (Frobert et al., 2008). Virus isolates from immunocompromised patients may be mixtures of various resistant and sensitive viruses (Christophers and Sutton, 1987). In these patients the selection of resistant variants appears to be similar to that encountered in tissue culture with mutations in TK and DNA polymerase. Furthermore, infections may become resistant to multiple agents, e.g. both nucleoside analogues and foscarnet. 4.2. HSV nucleoside analogue resistance in immunocompetent individuals In contrast to the immunocompromised population where HSV is prone to acquire resistance to antivirals, this is extremely rare in either genital or oral infections in otherwise normal patients and large surveys of isolates (>2000) have shown no obvious trends to decreasing sensitivity during a period of more than 30 years (Christophers et al., 1998). It is true that ACV-resistant viruses are occasionally encountered (Kost et al., 1993; Swetter et al., 1997), however, this remains extremely unusual. 4.3. Special cases – nucleoside analogue-resistance in ocular infection (keratitis) and neonatal disease (generalised herpes) While nucleoside-resistant HSV remains a very rare phenomenon in the majority of patients, there are two exceptions to this rule. The first is herpes keratitis where corneal isolates may be mixtures of ACV-sensitive and ACV-resistant viruses leading to a relatively high frequency of recurrent lesions that are refractory to therapy (Duan et al., 2008). Another situation where resistance has been encountered is in the severe disease encountered in neonatal herpes (Whitley, personal communication). In both these situations, it may be argued that immune control is compromised in respect of the anatomical site of virus infection although the patients’ immune systems may be otherwise intact. 5. The discovery of helicase–primase (H–P) as an antiviral target After nearly half a century of nucleoside analogue domination, the Twenty-first Century heralded several new and potentially important targets for HSV antivirals. Among the first compounds directed at one of these novel targets to reach the clinic are the helicase–primase inhibitors (HPI) (Crute et al., 2002; Kleymann et al., 2002). Prior to the action of herpesvirus DNA polymerase, which incorporates nucleotides into the growing DNA strand to execute DNA replication, the double-stranded herpesvirus DNA genome must first be unwound and synthesis primed. These functions are carried out by a complex of three proteins (Matthews et al., 1993) that in HSV are the products of UL5 (helicase), UL52 (primase) together with an accessory protein (the product of UL8) (Fig. 1). The heterotrimeric complex comprising the gene products of UL5, UL8 and UL52 provides several of the essential enzymatic functions required for HSV double-stranded DNA replication (reviewed by Weller, 2006). The sub-complex formed by the products of UL5 and UL52 alone were shown to be sufficient for the helicase and primase functions respectively although the presence of the UL8 protein stimulated these activities (Dodson and Lehman, 1991). It

H.J. Field, S. Biswas / Drug Resistance Updates 14 (2011) 45–51

47

(a) CH3 N

O

O

N S N

S

O

NH2

CH3

(b)

S O O

N

NH2

N NH

Fig. 1. Schematic diagram showing the site of action of nucleoside analogue triphosphates (e.g. acyclovir or penciclovir) and helicase–primase inhibitors (e.g. BAY 57-1293, BILS 22 BS, or amenamevir).

was shown subsequently that the UL52 protein contains the entire primase active site required for phosphodiester bond formation while the UL5 protein makes a minimal contribution to primer synthesis. However, the presence of the UL52 protein in the complex appeared to be an essential requirement for the helicase activity of the UL5 protein (Cavanaugh et al., 2009). Thus, this heterotrimeric protein complex (products of UL5/8/52) Exhibits 5 –3 helicase, a single-stranded DNAdependent NTPase, and primase activities (Dodson and Lehman, 1991). The precise function of UL8 is unknown but, as stated above, it has been shown to increase the rate of primer synthesis by UL5–UL52 although it has no detectable role in the DNA-unwinding activity (Cavanaugh et al., 2009). At the start of the new Millenium, drug discovery groups associated with several pharmaceutical companies identified potent inhibitors of helicase–primase (H–P) including a number of aminothiazole derivatives (reviewed by Kleymann et al., 2002; Field and Vere Hodge, 2008). From the initial lead compounds, several series of HPI emerged as potent selective inhibitors of HSV or varicella-zoster virus. 6. Development of inhibitors of helicase–primase as antiherpesvirus antivirals The thiazolylphenyl derivatives BILS 179BS and BAY 57-1293 (Fig. 2a and b), showed remarkable in vivo efficacy in animal models of HSV-1 and HSV-2 infection (Crute et al., 2002; Kleymann et al., 2002). It was shown using the murine HSV skin infection model that the compound was equally effective or superior to oral famciclovir (Biswas et al., 2007a). A dose of 15 mg/kg per day per os for 4 days starting one day after inoculation prevented mortality, markedly lessened clinical signs and reduced infectious virus in the tissues to below the level of detection within a few days. BAY 57-1293 was also very effective in an immunocompromised (athymic) HSV murine infection model (Biswas et al., 2007a). These compounds appear to act by enhancing the affinity of helicase–primase complex for the HSV DNA. The compound, BILS 22 BS (Liuzzi et al., 2004) (Fig. 2b) was among the first to be reported to have antiviral potential in vitro and in vivo, however its development was discontinued. A different series of HPI was reported by Kleymann et al. (2002) and BAY 57-1293, the lead compound (Fig. 2a) is currently under clinical development. In a recent press

(c)

O

N

CH3

H3C

O S

O N

O N

NH

O Fig. 2. The chemical structures of three potent HPI active against HSV or VZV. (a) BAY 57-1293: N-methyl-N-(4-methyl-5-sulfamoyl-1, 3-thiazol-2-yl)2-[4-(pyridin-2-yl)phenyl]acetamide. (b) BILS 22 BS: N-(2-{[4-(2-amino-1,3thiazol-4-yl)phenyl]amino}-2-oxoethyl)-N-benzylbenzamide (c) ASP 2151 – amenamevir: N-(2,6-dimethylphenyl)-N-(2-{[4-(1,2,4-oxadiazol-3-yl)phenyl]amino}2-oxoethyl)etrahydro-2-H-thiopyran-4-carboxamide 1,1-dioxide.

release, it was reported that three phase I studies have been completed with BAY 57-1293 and the drug was generally well-tolerated and showed high and long-lasting exposures in the human subjects (Aicuris, 2009). A separate development programme conducted by the Yamagouchi Corporation and later Astellus, led to the discovery of the HPI known as ASP2151 (Fig. 2c). The first reports of the preclinical efficacy of ASP2151 have recently been published (Chono et al., 2010). ASP2151 was shown to be orally effective and more potent compared to VACV in reducing cutaneous lesions in an HSV-1-infected, hairless mouse model (Katsumata et al., 2009). The various HPI, currently under development are more or less equally active against HSV-1 and HSV-2. ASP2151 is the only reported HPI, to date, to show activity against VZV (Suzuki et al., 2009) and ASP2151 was shown to be 30–76 times more potent than ACV against VZV determined by plaque reduction assays in HEF cells (Chono et al., 2010). ASP2151 is currently undergoing phase II clinical trials in man for the treatment of HSV-2 and VZV. 7. Selection of drug resistance confirms the mechanism of action of HPI As for all of the nucleoside analogues described above, the hall-mark of selective antiviral activity is the ability to select inhibitor-resistance mutations. This was found to be true for HPI and in the development of these compounds the selection of resis-

48

H.J. Field, S. Biswas / Drug Resistance Updates 14 (2011) 45–51

Fig. 3. The helicase protein of HSV showing the sites at which amino acid substitutions confer resistance to BAY 57-1293 or BILS 22BS. a Recent observation, not yet confirmed by marker transfer.

tance confirmed that the mechanism of action is directed to the H–P complex. Mutant strains of HSV that have acquired resistance to HPI are readily selected in tissue culture. This may be shown by inoculating ≤106 PFU of sensitive virus into ≥106 susceptible cells in the presence of an inhibitory concentration of the HPI. The resultant yield is likely to contain HPI-resistant virus. Plaques can be picked and their resistance, measured by means of a plaque-reduction assay, may range from moderate resistance (barely detectable) up to ≥5000-fold, implying that a variety of different mutations are responsible for loss of sensitivity to the HPI (Biswas and Field, in press). 8. Location of HPI drug-resistance mutations DNA sequencing shows that, all but one resistance mutation, to date, map to the helicase (UL5) gene (Biswas and Field, 2008), (Fig. 3). In addition a single resistance mutation has been located in the HSV-1 primase (UL52) gene (Biswas et al., 2008a) (Fig. 4). The mutations were checked by marker transfer which confirmed that the substitution accounted for the acquired resistance. In some cases resistant virus selected using the above method are shown to have more than one mutation, e.g. in both UL5 and UL52 (Biswas et al., 2008a). Based on sequence comparison between helicase genes from multiple organisms, together with site-directed mutagenesis stud-

ies, Zhu and Weller (1992) defined six conserved functional motifs in UL5 that, in HSV-1, codes for a protein comprising 882 residues. All HPI-resistance mutations described so far are close to the 4th functional motif that spans HSV-1 amino acid residues N342 NKRCVEHE350 (Fig. 3). The majority of HSV-1 HPI-resistance mutations are located just down-stream from this motif and result from substitutions in positions G352, M355 and K356 (Biswas and Field, 2008). A substitution (N to K) at position 342 (the first residue of the 4th functional motif) has also been shown to confer resistance to BAY 57-1293 (Biswas et al., 2009) and the analogous mutation (N336K) has been shown to confer resistance to VZV (Chono et al., 2010). The single UL52 (primase) resistance mutation reported to date lies close to the terminus of the HSV-1 primase at amino acid A899 of a protein that comprises 1058 residues (Biswas and Field, 2008) (Fig. 4). 9. HPI-resistance mutations in HSV-1 and HSV-2 Although more work on HPI-resistance has been reported on HSV-1, the pattern appears to be the similar for HSV-2 (Kleymann et al., 2002). However, the HSV-2 helicase gene codes for one residue (Leu20) less compared to HSV-1, thus for example, the HSV-2 resistance mutations N341K, G351R (Field, unpublished), and K355N (Spector et al., 1998), are equivalent to N342K, G352R, and K356N in HSV-1. To the best of our knowledge, no primase mutation that confers HPI-resistance has yet been reported in an

Fig. 4. The primase protein of HSV showing the site in HSV-1 where a single amino acid substitution confers resistance to BAY 57-1293.

H.J. Field, S. Biswas / Drug Resistance Updates 14 (2011) 45–51

HSV-2 strain but the mutation analogous to the A899T substitution in UL52 has yet to be investigated. 10. HPI resistance mutations – cross-resistance to other inhibitors

49

showed no evidence of reduced virulence in the murine infection model (Liuzzi et al., 2004; Sukla et al., 2010b). Some examples of the effects of HPI resistance mutations on growth rate and pathogenicity are summarized in Table 1.

10.1. Nucleoside analogues

12. The frequency of HPI drug-resistance mutations in HSV

It is to be expected that all viruses with acquired resistance to HPI should be sensitive to current nucleoside analogues, all of which target the herpesvirus DNA polymerase. To date, no exceptions to this rule have been reported and all HPI-resistant viruses tested show no change in sensitivity to alternative compounds, e.g. ACV or PCV. Furthermore, when a series of well-characterized ACVresistant mutants with mutations in either DNA polymerase or TK were tested against BAY 57-1293 all were found to be sensitive (Field, unpublished observations).

A proposed advantage of the HPI has been claimed to be the relatively low rate of selection of resistant mutants (≤10−6 PFU) (Betz et al., 2002; Kleymann et al., 2002; Spector et al., 1998) compared with resistance to nucleoside analogues that arises in tissue culture at ≤10−4 (Sarisky et al., 2000). Our own observations confirmed this for recently plaque-purified stocks of HSV-1. However, it was shown that several laboratory strains of HSV-1 contain BAY 57-1293 resistance mutations at 10–100 times higher frequency (Biswas et al., 2007d). Furthermore, similar to the results with laboratory working stocks, some clinical isolates of HSV-1 obtained from patients thought never to have been treated with HPI were also shown to contain HPI drug-resistant mutants at relatively high frequency (Biswas et al., 2007c). Recently a similar pattern (i.e. individual viruses with a relatively high frequency of resistance mutations) has been confirmed for recent clinical isolates of HSV-2 (Field, unpublished observations). These results raised the question as to whether the resistance mutations were pre-existent in the virus population or could be induced in the presence of the inhibitor. The latter explanation appeared unlikely since the selection was carried out in high concentration of compound using cells that had been pre-incubated in the inhibitor that was replenished during the selection procedure. Thus virus replication during the selection procedure should have been suppressed. Final proof of the pre-existence of the resistance mutations at frequencies of ≥10−5 comes from recent experiments described by Sukla et al. (2010a). An intentional mis-match primer PCR was used to amplify the K356N or K356T resistance mutations. The conditions were designed to detect these mutations in the presence of wild-type at 10–100 times the back-ground frequency. Using this methodology in a recent survey 5/30 recent HSV-1 clinical isolates were shown to have pre-existing HPI resistance mutations thus explaining the relatively rapid selection of the resistance mutations using the classic tissue culture method.

10.2. Alternative HPI For the majority of HPI strains selected for resistance to BAY 571293 it was shown that their fold-resistance to BILS 22 BS which is an alternative HPI was similar suggesting an identical mode of action and molecular target. However one mutant was resistant to BAY 57-1293 but was fully sensitive to BILS 22 BS. In this case, resistance was conferred by the amino acid substitution A899T in the HSV-1 UL52 (primase) protein (Biswas et al., 2008a). This implies that the two HPI differ with respect to their interaction with the primase function and suggests that there is scope for further research in relation to potential drug combinations (see below) in order to exploit both targets. 11. HPI-resistance mutations and virus fitness For all forms of antiviral drug resistance, a matter of major concern is whether or not resistance mutations compromise the virus’ ability to replicate efficiently in the host. Viruses in which this is clearly the case are described as “less fit”. It is interesting to note, therefore, that several HPI resistance mutations are clearly associated with a reduction in growth rate in tissue culture. This was shown for BAY 57-1257-selected mutations N342R, G352R and M355T. When inoculated into a laboratory mouse HSV skin infection model it was not surprising, therefore, that the first two viruses showed evidence of reduced virulence (Biswas et al., 2008b, 2009). Not all HPI resistance mutations, however, had a markedly deleterious effect on virus growth. For instance, the mutation K356Q actually increased the rate of virus replication in tissue culture compared to wild type at both high and low multiplicity of infection (Biswas et al., 2007b). Similar results were obtained for a recombinant virus constructed to contain the same mutation (Biswas et al., 2007b). This virus showed no evidence of reduced virulence in the murine infection model. Furthermore, the K356N mutation which confers approximately 5000-fold resistance and the G352V (400fold resistant) mutants both grew normally in tissue culture and

13. The implications of HPI drug resistance mutations for the patient 13.1. Immunocompetent patients It remains to be seen whether or not patients infected with strains that contain such relatively high frequencies of mutants will be more likely to develop resistance to therapy. Our opinion is that this will not be so in typical patients, based on previous experience with nucleoside analogues such as ACV and FCV (Sarisky et al., 2003; Christophers et al., 1998; Field, 2001).

Table 1 The effect of single HPI resistance mutations on the growth of HSV-1 in tissue culture and pathogenicity in a murine skin infection model. Mutation in UL5 (helicase) gene

Fold resistance to BAY 57-1293

Growth in Vero cellsa

Pathogenicity in Balb/C micea

N342K G352V G352R M355T K356N K356Q

40 400 3000 5 >5000 150

Slow Normal Slow Slow Normal Fast

Less fit Virulent Less fit n/d Virulent Virulent

a

Original data may be found in Biswas et al. (2007b, 2008b, 2009) and Sukla et al. (2010b).

50

H.J. Field, S. Biswas / Drug Resistance Updates 14 (2011) 45–51

13.2. Immunocompromised patients and atypical herpes

15. Drug combinations with HPI

It is more likely, however, that HPI resistance will be more significant in immunocompromised patients. In this context, this group should include special cases such as generalized herpes in the newborne and probably those suffering from recurrent herpes keratitis. In all these cases it is likely that less fit viruses are able to replicate without exposure to the full battery of the host’s innate and adaptive immune responses. Furthermore, prolonged virus replication increases the likelihood of the selection of resistant variants. In a recent study it was shown that mice infected with deliberate mixtures of wild-type HSV-1 and the K356N mutant (in ratio of 1/50 or 1/500) could be effectively treated with optimum dose of BAY 57-1293; however HPI-resistance could be detected in a few mice infected with the 1/50 mixture following therapy (Sukla et al., 2010b). The pre-existence of the highly resistant K356N mutant that is not associated with any obvious loss of fitness at relatively high frequency in some isolates suggests that this mutant, at least, has a fair chance of being encountered in the clinic in the future. This is particularly likely in these patient groups where frequent resistance to nucleoside analogues has already been encountered.

Clearly, there is much potential for exploring combinations of HPI with other compounds, the most obvious being the nucleoside analogues. It is expected that additive effects or mild synergy will be obtained and research is proceeding on these lines although, to our knowledge, little has yet been published.

14. HPI and the problem of herpesvirus latency Herpesviruses generally have an extraordinary propensity to establish life-long infections with the host. For HSV and VZV this is accomplished by establishing neuronal latency in peripheral ganglionic neurons. Reactivation of the quiescent virus from time to time results in virus shedding with the possibility of transmission to new susceptible hosts and recurrent disease in the patient. Nucleoside analogues such as ACV have been unable to remove latent HSV or VZV during periods of latency and animal models demonstrate that although therapy before, during or shortly after initial infection may reduce the number of latent foci (Field and Thackray, 2000) no therapy, to date has been able to prevent latency from being established or block the subsequent pattern of latency and recurrence (Field and Thackray, 1997). The site of action of HPI differs from that of nucleoside analogues since they act at a different step in DNA replication. However, since it is unlikely that any of the target proteins are expressed during latency it may be inferred that, as for nucleoside analogues, latent HSV or VZV will not be susceptible to therapy. When VACV or FCV are administered once or twice daily during latency, both have been successful, in suppressing recurrent disease for many months or even years. No human trials have yet been conducted with HPI in this mode, however, given the pharmacological profile and promising animal data (Baumeister et al., 2007; Kleymann et al., 2002) it should be possible to maintain drug concentrations in tissue above that required to inhibit virus replication. Suppression therapy would, therefore, be feasible provided there are no adverse safety issues. Notwithstanding these pessimistic predictions regarding the unlikely efficacy of HPI in relation to established latency, BAY 57-1293 has been claimed to suppress reactivation from latency and reduce virus load in guinea pig sacral dorsal root ganglia (Baumeister et al., 2007) after genital infection, or mice or rabbit trigeminal ganglia following ocular inoculation (Kaufman et al., 2008). These early preclinical findings are encouraging, however, those who have worked in the field for a long time will remain skeptical until further studies have been carried to confirm and extend these claims in a variety of models. It remains to be seen whether HPI alone, or in combination with other classes of inhibitor, will have a practical bearing on the establishment or maintenance of herpesvirus latency, or on the frequency of recurrences, or severity of recurrent lesions.

16. Conclusions and future prospects The emergence of HPI is an exciting development in the herpes antiviral field that has seen little major advance since the introduction of the nucleoside analogue prodrugs, VACV and FCV. The key issue now for HPI is that of safety. The nucleoside analogues, ACV and PCV were considered with great caution when they were first introduced into the clinic because of the theoretical possibilities for genetic or other forms of toxicity. In the event, these nucleoside analogues are now widely regarded as remarkably safe drugs that have been taken by individuals for many months or years for suppressive therapy with no obvious adverse effects. It remains to be seen whether or not any HPI can match this enviable safety record. There have been toxicity issues during the development of several therapeutic HPI candidates and only time will tell whether or not the current leading compounds will emerge with a clean record. If confidence in safety can be achieved, then we are sure that HPI will make a major impact and we will enter an interesting era where the pros and cons of nucleoside analogues versus HPI will be tested in clinical trials. In this chapter, we have discussed the possible development of HPI drug-resistant mutants. The previous experience with resistance to ACV and similar drugs leads us to be cautiously optimistic about this. Ultimately, it is likely that HPI will be used in combination with other herpes antivirals. Particularly in those who suffer from frequent recurrences or severe or chronic herpes lesions including the immunocompromised patients, it is likely that the availability of HPI will have a major impact on herpes antiviral chemotherapy in the near future. Acknowledgements During the period of research on helicase–primase inhibitors described in the review, HJF and SB received a small grant-in-aid from Arrow Therapeutics, London. HJF has an on-going research collaboration with AiCuris GmbH and Co. KG, Wuppertal, Germany from whom we have also received financial support. References Aicuris. www.aicuris.com, 09 November 2009. Andrei, G., Fiten, P., De Clercq, E., Snoeck, R., Opdenakker, G., 2001. Evaluating phenotype and genotype of drug-resistant strains in herpesvirus. Mol. Biotechnol. 18, 155–167. Baumeister, J., Fischer, R., Eckenberg, P., Henninger, K., Ruebsamen-Waigmann, H., Kleymann, G., 2007. Superior efficacy of helicase–primase inhibitor BAY 57-1293 for herpes infection and latency in the guinea pig model of human genital herpes disease. Antivir. Chem. Chemother. 18, 35–48. Betz, U.A.K., Fischer, R., Kleymann, G., Hendrix, M., Rübsamen-Waigmann, H., 2002. Potent in vivo antiviral activity of the herpes simplex virus primase–helicase inhibitor BAY57-1293. Antimicrob. Agents Chemother. 46, 1766– 1772. Biswas, S., Field, H.J., 2008. Herpes simplex virus helicase–primase inhibitors: recent findings from the study of drug resistance mutations. Antivir. Chem. Chemother. 19, 1–6. Biswas, S., Field, H.J. (in press). Helicase–primase inhibitors—a new approach to combating herpes simplex and varicella-zoster viruses. In: E. De Clercq (Ed.), Antiviral Drug Strategies, Whiley. Biswas, S., Jennens, L., Field, H.J., 2007a. The helicase primase inhibitor, BAY 57-1293 shows potent therapeutic antiviral activity superior to famciclovir in BALB/c mice infected with herpes simplex virus type 1. Antivir. Res. 75, 30–35. Biswas, S., Jennens, L., Field, H.J., 2007b. Single amino acid substitutions in the HSV1 helicase protein that confer resistance to the helicase–primase inhibitor BAY

H.J. Field, S. Biswas / Drug Resistance Updates 14 (2011) 45–51 57-1293 are associated with increased or decreased virus growth characteristics in tissue culture. Arch. Virol. 152, 1489–1500. Biswas, S., Kleymann, G., Swift, M., Tiley, L., Lyall, J., Aguirre-Hernández, J., Field, H.J., 2008a. A single drug-resistance mutation in HSV-1 UL52 primase points to a difference between two helicase–primase inhibitors in their mode of interaction with the antiviral target. J. Antimicrob. Chemother. 61, 1044–1047. Biswas, S., Muguel, R.N., Sukla, S., Field, H.J., 2009. A mutation in helicase motif IV of herpes simplex virus type 1 UL5 that results in reduced growth in vitro and lower virulence in a murine infection model is related to the predicted helicase structure. J. Gen. Virol. 90, 1937–1942. Biswas, S., Smith, C., Field, H.J., 2007c. Detection of HSV-1 variants highly resistant to the helicase–primase inhibitor BAY 57-1293 at high frequency in two of ten recent clinical isolates of HSV-1. J. Antimicrob. Chemother. 60, 274–279. Biswas, S., Swift, M., Field, H.J., 2007d. High frequency of spontaneous helicase primase inhibitor (BAY 57-1293) drug-resistant variants in certain laboratory isolates of HSV-1. Antivir. Chem. Chemother. 18, 13–23. Biswas, S., Tiley, L.S., Zimmermann, H., Birkmann, A., Field, H.J., 2008b. Mutations close to functional motif IV in HSV-1 UL5 helicase that confer resistance to HSV helicase–primase inhibitors, variously affect virus growth rate and pathogenicity. Antivir. Res. 80, 81–85. Cavanaugh, N.A., Ramirez-Aguilar, K.A., Urban, M., Kuchta, R.D., 2009. Herpes simplex virus-1 helicase–primase: roles of each subunit in DNA binding and phosphodiester bond formation. Biochemistry 48, 10199–10207. Chono, K., Katsumata, K., Kontani, T., Kobayashi, M., Sudo, K., Yokota, T., Konno, K., Shimizu, Y., Suzuki, H., 2010. ASP2151 a novel helicase–primase inhibitor, possesses antiviral 1 activity against varicella-zoster virus and herpes simplex virus types 1 and 2. J. Antimicrob. Chemother. 65, 1733–1741. Christophers, J., Clayton, J., Craske, J., Ward, R., Collins, P., Trowbridge, M., Darby, G., 1998. Survey of resistance of herpes simplex virus to acyclovir in northwest England. Antimicrob. Agents Chemother. 42, 868–872. Christophers, J., Sutton, R.N., 1987. Characterization of acyclovir-resistant and sensitive clinical isolates of herpes simplex virus isolates from an immunocompromised patient. J. Antimicrob. Chemother. 20, 389–398. Coen, D., Schaffer, P.A., 1980. Two distinct loci confer resistance to acycloguanosine in herpes simplex virus type 1. Proc. Natl. Acad. Sci. U.S.A. 77, 2265–2269. Crute, J.J., Grygon, C.A., Hargrave, K.D., Simoneau, B., Faucher, A-M., Bolger, G., Kibler, P., Liuzzi, M., Cordingley, M.G., 2002. Herpes simplex virus helicase–primase inhibitors are active in animal models of human disease. Nat. Med. 8, 386–391. Darby, G., Field, H.J., Salisbury, S., 1981. Altered substrate specificity of herpes simplex thymidine kinase confers acyclovir-resistance. Nature 289, 81–83. Dodson, M.S., Lehman, I.R., 1991. Association of DNA helicase and primase activities with a subassembly of the herpes simplex virus 1 helicase–primase composed of the UL5 and UL52 gene products. Proc. Natl. Acad. Sci. U.S.A. 88, 1105–1109. Drake, J.W., Holland, J.J., 1999. Mutation rates among RNA viruses. Proc. Natl. Acad. Sci. U.S.A. 96, 13910–13913. Duan, R., de Vries, R.D., Osterhaus, A.D., Remeijer, L., Verjans, G.M., 2008. Acyclovirresistant corneal HSV-1 isolates from patients with herpetic keratitis. J. Inf. Dis. 198, 659–663. Field, H.J., 2001. Herpes simplex virus antiviral drug resistance—current trends and future prospects. J. Clin. Virol. 21, 261–269. Field, H.J., De Clercq, E., 2004. Antiviral drugs: a short history of their discovery and development. Microbiol. Today 31, 58–61. Field, H.J., Thackray, A.M., 1997. Can herpes simplex virus latency be prevented using conventional nucleoside analogue chemotherapy? Antivir. Chem. Chemother. 8 (Suppl. 1), 59–66. Field, H.J., Thackray, A.M., 2000. Early therapy with valaciclovir or famciclovir reduces but does not abrogate herpes simplex virus neuronal latency. Nucleos. Nucleot. Nucl. Acids 19, 461–470. Field, H.J., Wildy, P., 1978. The pathogenicity of thymidine kinase-deficient mutants of herpes simplex virus in mice. Journal of Hygiene, Cambridge 81, 267–277. Field, H.J., Vere Hodge, R.A., 2008. Antiviral agents. In: Mahy, B.W., Van Regenmortel, M.H.V. (Eds.), Encyclopedia of Virology, I, third ed. Elsevier, Oxford, pp. 142–154. Frobert, E., Cortay, J.C., Ooka, T., Najioullah, F., Thouvenot, D., Lina, B., Morfin, F., 2008. Genotypic detection of acyclovir-resistant HSV-1: characterization of 67 ACV-sensitive and 14 ACV-resistant viruses. Antivir. Res. 79, 28–36. Katsumata, K., Chono, K., Kontani, T., Sudo, K., Shimizu, Y., Suzuki, H., 2009. Efficacy of acute oral treatment with ASP2151 or valacyclovir (VACV) against cutaneous infection with herpes simplex virus (HSV) type 1 in mice. In: Poster V-1742, Interscience Conference on Antimicrobial Agents and Chemotherapy, September 12–15, San Francisco, USA.

51

Kaufman, H.E., Varnell, E.D., Gebhardt, B.M., Thompson, H.W., Atwal, E., RübsamenWaigmann, H., Kleymann, G., 2008. Efficacy of a helicase–primase inhibitor in animal models of ocular herpes simplex virus type 1 infection. J. Ocul. Pharmacol. Ther. 24, 34–42. Kleymann, G., Fischer, R., Betz, U.A., Hendrix, M., Bender, W., Schneider, U., Handke, G., Eckenberg, P., Hewlett, G., Pevzner, V., Baumeister, J., Weber, O., Henninger, K., Keldenich, J., Jensen, A., Kolb, J., Bach, U., Popp, A., Maben, J., Frappa, I., Haebich, D., Lockhoff, O., Rubsamen-Waigmann, H., 2002. New helicase–primase inhibitors as drug candidates for the treatment of herpes simplex disease. Nat. Med. 8, 392–398. Kost, R.G., Hill, E.L., Tigges, M., Straus, S., 1993. Recurrent acyclovir-resistant genital herpes in an immunocompetent patient. N. Engl. J. Med. 329, 1777–1782. Larder, B.A., Darby, G., 1985. Selection and characterization of acyclovir-resistant herpes simplex virus type 1 mutants inducing altered DNA polymerase activities. Virology 146, 262–271. Larder, B.A., Cheng, Y.C., Darby, G., 1983. Characterization of abnormal thymidine kinases induced by drug-resistant strains of herpes simplex virus type 1. J. Gen. Virol. 64, 523–532. Liuzzi, M., Kibler, P., Bousquet, C., Harji, F., Bolger, G., Garneau, M., Lapeyre, N., McCollum, R.S., Faucher, A.-M., Simoneau, B., Cordingley, M.G., 2004. Isolation and characterization of herpes simplex virus type 1 resistant to aminothiazolylphenyl-based inhibitors of the viral helicase–primase. Antivir. Res. 64, 161–170. Matthews, J.T., Terry, B.J., Field, A.K., 1993. The structure and function of the HSV DNA replication proteins: defining novel antiviral targets. Antivir. Res. 20, 89–114. Parris, D.S., Harrington, J.E., 1982. Herpes simplex virus variants resistant to high concentrations of acyclovir exist in clinical isolates. Antimicrob. Agents Chemother. 22, 71–77. Sarisky, R.T., Bacon, T.H., Boon, R.J., Duffy, K.E., Esser, K.M., Leary, J., Locke, L.A., Nguyen, T.T., Quail, M.R., Saltzman, R., 2003. Profiling penciclovir susceptibility and prevalence of resistance of herpes simplex virus isolates across eleven clinical trials. Arch. Virol. 148, 1757–1769. Sarisky, R.T., Nguyen, T.T., Duffy, K.E., Wittrock, R.J., Leary, J.J., 2000. Difference in incidence of spontaneous mutations between herpes simplex virus types 1 and 2. Antimicrob. Agents Chemother. 44, 1524–1529. Schnipper, L.E., Crumpacker, C.S., 1980. Resistance of herpes simplex virus to acycloguanosine: role of the viral thymidine kinase and polymerase loci. Proc. Natl. Acad. Sci. U.S.A. 77, 2270–2273. Smith, D.B., Inglis, S.C., 1987. The mutation rate and variability of eukaryotic viruses: an analytical review. J. Gen. Virol. 68, 2729–2740. Spector, F.C., Liang, L., Giordano, H., Sivaraja, M., Peterson, M.G., 1998. Inhibition of herpes simplex virus replication by a 2-amino thiazole via interactions with the helicase component of the UL5–UL8–UL52 complex. J. Virol. 72, 6979–6987. Sukla, S., Biswas, S., Birkmann, A., Lischka, P., Zimmermann, H., Field, H.J., 2010a. Mismatch primer-based PCR reveals that helicase–primase inhibitor resistance mutations pre-exist in herpes simplex virus type 1 clinical isolates and are not induced during incubation wth the inhibitor. J. Antimicrob. Chemother. 65, 1347–1352. Sukla, S., Biswas, S., Birkmann, A., Lischka, P., Ruebsamen-Schaeff, H., Zimmermann, H., Field, H.J., 2010b. Effects of therapy using a helicase–primase inhibitor (HPI) in mice infected with deliberate mixtures of wild-type HSV-1 and HPI-resistant UL5 mutant. Antivir. Res. 87, 67–73. Suzuki, H., Chono, K., Katsumata, K., Kobayashi, M., Kontani, T., Sudo, K., Shimizu, Y., Takakura, S., ASP2151, a novel helicase–primase inhibitor, possesses antiviral activity against varicella-zoster virus as well as herpes simplex virus type 1 and 2 2009. Poster V-1741, Interscience Conference on Antimicrobial Agents and Chemotherapy, September 12–15, San Francisco, USA. Swetter, S.M., Hill, E.L., Kern, E.R., Koelle, D.M., Posaved, C.M., Laurence, W., Safrin, S., 1997. Chronic vulvar ulceration in an immunocompetent woman due to acyclovir-resistant thymidine kinase deficient herpes simplex virus. J. Inf. Dis. 177, 543–550. Vere Hodge, A., Field, H.J., 2010. General mechanisms of antiviral resistance. In: De Clercq, B.W. (Ed.), Genetics and Evolution of Infectious Diseases. Elsevier, pp. 339–362, Chapter 1. Weller, S.K., 2006. In: Sandri-Glowin, R.M. (Ed.), HSV-1 DNA Replication in Alphaherpesviruses: Molecular and Cellular Biology Edition. Caister Academic Press. Zhu, L.A., Weller, S.K., 1992. The six conserved helicase motifs of the UL5 gene product, a component of the herpes simplex virus type 1 helicase primase, are essential for its function. J. Virol. 66, 469–479.