Pharmacokinetics-pharmacodynamics of the helicase-primase inhibitor pritelivir following treatment of wild-type or pritelivir-resistant virus infection in a murine herpes simplex virus 1 infection model.

Herpes simplex virus (HSV) infections can cause considerable morbidity. Transmission of HSV-2 has become a major health concern, since it has been shown to promote transmission of other sexually transmitted diseases. Pritelivir (AIC316, BAY 57-1293) belongs to a new class of HSV antiviral compounds, the helicase-primase inhibitors, which have a mode of action that is distinct from that of antiviral nucleoside analogues currently in clinical use. Analysis of pharmacokinetic-pharmacodynamic parameters is a useful tool for the selection of appropriate doses in clinical trials, especially for compounds belonging to new classes for which no or only limited data on therapeutic profiles are available. For this purpose, the effective dose of pritelivir was determined in a comprehensive mouse model of HSV infection. Corresponding plasma concentrations were measured, and exposures were compared with efficacious concentrations derived from cell cultures. The administration of pritelivir at 10 mg/kg of body weight once daily for 4 days completely suppressed any signs of HSV infection in the animals. Associated plasma concentrations adjusted for protein binding stayed above the cell culture 90% effective concentration (EC90) for HSV-1 for almost the entire dosing interval. Interestingly, by increasing the dose 6-fold and prolonging the treatment duration to 8 days, it was possible to treat mice infected with an approximately 30-fold pritelivir-resistant but fully pathogenic HSV-1 virus. Corresponding plasma concentrations exceeded the EC90 of this mutant for <8 h, indicating that even suboptimal exposure to pritelivir is sufficient to achieve antiviral efficacy, possibly augmented by other factors such as the immune system.

Recent developments in anti-herpesvirus drugs.

Background Herpesviruses notably establish lifelong infections, with latency and reactivation. Many of the known human herpesviruses infect large proportions of the population worldwide. Treatment or prevention of herpes infections and recurrent disease still pose a challenge in the 21st century. Sources of data Original papers and review articles, meeting abstracts, a book (Clinical Virology; DD Richman, RJ Whitley & FG Hayden eds) and company web sites. Areas of agreement For herpes simplex types 1 and 2 and for varicella zoster, acyclovir (ACV; now increasingly replaced by its prodrug valacyclovir, VACV) and famciclovir (FCV) have greatly reduced the burden of disease and have established a remarkable safety record. Drug-resistance, in the otherwise healthy population, has remained below 0.5% after more that 20 years of antiviral use. In immunocompromised patients, drug resistance is more common and alternative drugs with good safety profiles are desirable. For human cytomegalovirus disease, which occurs in immunocompromised patients, ganciclovir and increasingly its prodrug valganciclovir are the drugs of choice. However, alternative drugs, with better safety, are much needed. Areas of controversy Various questions are highlighted. Should the new 1-day therapies for recurrent herpes labialis and genital herpes replace the current standard multi-day therapies? The marked differences between VACV and FCV (e.g. triphosphate stability, effect on latency) may not yet be fully exploited? Do current antivirals reduce post-herpetic neuralgia (PHN)? For immunocompromised patients with varicella zoster virus (VZV) disease, should the first-line treatment be FCV, not ACV or VACV? Should there be more support to explore new avenues for current antivirals, for example in possibly reducing herpes latency or Alzheimer's disease (AD)? Should primary Epstein-Barr virus (EBV) disease in adolescents be treated with antivirals? How can new compounds be progressed when the perceived market need is small but the medical need is great. FCV was reclassified from prescription-only to pharmacist-controlled for herpes labialis in New Zealand in 2010; should this be repeated more widely? This article reviews new drugs in clinical trials and highlights some of the problems hindering their progress.

The helicase-primase complex as a target for effective herpesvirus antivirals.

Herpes simplex virus and varicella-zoster virus have been treated for more that half a century using nucleoside analogues. However, there is still an unmet clinical need for improved herpes antivirals. The successful compounds, acyclovir; penciclovir and their orally bioavailable prodrugs valaciclovir and famciclovir, ultimately block virus replication by inhibiting virus-specific DNA-polymerase. The helicase-primase (HP) complex offers a distinctly different target for specific inhibition of virus DNA synthesis. This review describes the synthetic programmes that have already led to two HP-inhibitors (HPI) that have commenced clinical trials in man. One of these (known as AIC 316) continues in clinical development to date. The specificity of HPI is reflected by the ability to select drug-resistant mutants. The role of HP-antiviral resistance will be considered and how the study of cross--resistance among mutants already shows subtle differences between compounds in this respect. The impact of resistance on the drug development in the clinic will also be considered. Finally, herpesvirus latency remains as the most important barrier to a therapeutic cure. Whether or not helicase primase inhibitors alone or in combination with nucleoside analogues can impact on this elusive goal remains to be seen.

Antiviral drug resistance and helicase-primase inhibitors of herpes simplex virus.

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.

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 with the inhibitor.

OBJECTIVES: Previous studies suggested that helicase-primase inhibitor (HPI) resistance mutations can be selected at relatively high frequency from some isolates of herpes simplex virus type 1 (HSV-1). An intentional mismatch primer (IMP) PCR was developed to detect three known HPI resistance mutations well above the expected background frequency. The objective of this study was to provide proof that HPI resistance mutations pre-exist at relatively high frequency in some clinical isolates obtained from individuals naive to HPIs. METHODS: Three different IMP PCRs were standardized to detect critical HPI resistance mutations (K356N or K356T in UL5, or A899T in UL52) at 10-100 times the expected background frequency (<10(-6)). Thirty HSV-1 clinical isolates were then screened for the resistance mutations in the absence of the inhibitor using IMP PCR. RESULTS: Among 30 clinical isolates that were all susceptible to the HPI, BAY 57-1293, 5 were shown to contain UL5 mutations at 10-100 times higher than the expected frequency. No UL52 resistance mutations were encountered in this study. CONCLUSIONS: The detection of HPI-resistant mutations in some clinical isolates by means of IMP PCR proved that the mutations pre-exist and showed that they are not induced during incubation with the inhibitor.

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.

OBJECTIVES: To investigate the mechanism of action of the helicase-primase inhibitors (HPIs) BAY 57-1293 and BILS 22 BS by selection and characterization of drug-resistant herpes simplex virus (HSV)-1 mutants. METHODS: HSV-1 mutants were selected using BAY 57-1293 in Vero cells. Resistance mutations identified in the UL5 helicase or UL52 primase genes were validated by marker transfer. Cross-resistance to the structurally distinct BILS 22 BS was measured by ID(50) determinations. RESULTS: (i) A single mutation (UL52: A899T) confers 43-fold resistance to BAY 57-1293, but does not confer any resistance to BILS 22 BS. (ii) A double mutant (UL52: A899T and UL5: K356T) is 2500-fold resistant to BAY 57-1293, which is more than 17 times the sum of fold-resistance due to the individual mutations, UL52: A899T (43-fold) and UL5: K356T (100-fold). (iii) Virus containing the single helicase mutation and the double mutant with mutations in both helicase and primase showed equal resistance to BILS 22 BS (70-fold). CONCLUSIONS: By measuring the relative inhibitory concentrations required to overcome particular mutations in the helicase and primase proteins, evidence was obtained that BAY 57-1293 interacts with both components of the helicase-primase complex to achieve maximum potency, whereas for BILS 22BS, this may not be the case. Furthermore, our observations suggest that BAY 57-1293 interacts simultaneously with UL5 and UL52. Overall, the results suggest that these two potent HPIs interact differently with the helicase-primase complex.

Penciclovir is a potent inhibitor of feline herpesvirus-1 with susceptibility determined at the level of virus-encoded thymidine kinase.

Feline herpesvirus-1 (FHV-1) is the causative agent of a severe ocular disease in cats for which a safe potent antiviral chemotherapeutic agent is highly demanded. The sensitivity of FHV-1 to inhibition by three anti-herpetic nucleoside analogues [acyclovir (ACV), penciclovir (PCV) and cidofovir (CDV)] was tested by means of yield reduction assay. ACV showed very poor ability to inhibit FHV-1 replication. At low multiplicity of infection (MOI), both PCV and CDV were nearly equally effective with IC50 values ranging between 6 and 8 microg/ml. However, when the MOI was raised to 3PFU/cell, the activity of CDV was markedly reduced (IC50 25 microg/ml), while that of PCV remained relatively low (IC50 10 microg/ml). Although FHV-1 is normally insensitive to ACV, it exhibited >1000-fold increase in sensitivity when the thymidine kinase (TK) encoded by herpes simplex virus-1 (HSV-1) was supplied in trans. Furthermore, three PCV-resistant FHV-1 variants selected in vitro were shown to carry mutations in the TK gene. Taken together, these data provided direct evidence that PCV is a potent selective inhibitor of FHV-1 and that the virus-encoded TK is an important determinant of the virus susceptibility to nucleoside analogues.

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.

Herpes simplex virus (HSV) helicase-primase (HP) is the target for a novel class of antiviral compounds, the helicase-primase inhibitors (HPIs), e.g. BAY 57-1293. Although mutations in herpesviruses conferring resistance to nucleoside analogues are commonly associated with attenuation in vivo, to date, this is not necessarily true for HPIs. HPI-resistant HSV mutants selected in tissue culture are reported to be equally pathogenic compared to parental virus in animal models. Here we demonstrate that a slow-growing HSV-1 mutant, with the BAY 57-1293-resistance mutation Gly352Arg in UL5 helicase, is clearly less virulent than its wild-type parent in a murine zosteriform infection model. This contrasts with published results obtained for a mutant containing a different HPI-resistance substitution (Gly352Val) at the same location, since this mutant was reported to be fully pathogenic. We believe our report to be the first to describe an HPI-resistant HSV-1 mutant, that is markedly less virulent in vivo and slowly growing in tissue culture compared to the parental strain. Another BAY 57-1293-resistant UL5 mutant (Lys356Gln), which showed faster growth characteristics in cell culture, however, was at least equally virulent compared to the parent strain.

Development of a quantitative real-time TaqMan PCR assay for testing the susceptibility of feline herpesvirus-1 to antiviral compounds.

Feline herpesvirus-1 (FHV-1) is considered as the most common viral infection of domestic cats worldwide. It causes a disease characterized by upper respiratory and ocular clinical signs. Several attempts are currently underway to develop antiviral chemotherapy for treating FHV-1 infections. The availability of a rapid quantitative method for detecting FHV-1 would greatly facilitate prompt therapy, and hence enhance the success of any antiviral regime. In this study, a TaqMan real-time PCR assay was established for measuring FHV-1 DNA levels in culture supernatants. This assay was shown to be highly specific, reproducible and allows quantitation over a range of 2 to 2 x 10(8) copies per reaction. The assay was then applied to measure the reduction of FHV-1 DNA levels in the presence of increasing concentrations of acyclovir (ACV), penciclovir (PCV) and cidofovir (CDV). The 50% inhibitory concentrations (IC(50s)) obtained with the B927 laboratory strain of FHV-1 were 15.8 microM for ACV, 7.93 microM for CDV and 1.2 microM for PCV. The assay described here is sensitive, time-saving and does not involve prior titration of virus stocks or monitoring virus-induced cytopathic effects. Therefore, it is suitable for routine anti-FHV-1 drug susceptibility testing in veterinary clinics.

Antiviral Chemistry & Chemotherapy's current antiviral agents FactFile (2nd edition): DNA viruses.

Although most of the recent attempts to develop new antiviral agents have been focussed on RNA viruses (in particular, HIV and hepatitis C virus), a few new compounds are now awaiting their entry into the field of DNA viruses, particularly poxviruses, such as variola virus, because of the bioterrorist context, and herpesviruses, such as herpes simplex virus and cytomegalovirus, where the market scene has for many years been dominated by acyclovir, penciclovir and ganciclovir and their respective orally bioavailable prodrugs: valaciclovir, famciclovir and valganciclovir. Here, we review the current 'state of the art' with old compounds ready to rotate off and new compounds eagerly awaiting to appear on the continuously evolving scene of antiviral drug development.

Antiviral Chemistry & Chemotherapy's current antiviral agents FactFile (2nd edition): retroviruses and hepadnaviruses.

There are at present exactly 25 compounds that have been formally approved for the treatment of retrovirus (that is HIV) infections: seven nucleoside reverse transcriptase inhibitors (NRTIs), one nucleotide reverse transcriptase inhibitor (NtRTI), four non-nucleoside reverse transcriptase inhibitors (NNRTIs), 10 protease inhibitors (PIs), one coreceptor inhibitor (CRI), one fusion inhibitor (FI) and one integrase inhibitor (INI). Other compounds expected to be approved for the treatment of HIV infections in the near future are the NNRTI rilpivirine, the CRI vicriviroc and the INI elvitegravir. To obtain synergistic activity, enable lower dosage levels, thus minimizing toxic side effects, and particularly to reduce the risk of drug resistance development, common wisdom dictates that the HIV inhibitors should be used in drug combination regimens. Although, given the number of compounds available, the drug combinations that could be concocted are uncountable, only one triple-drug combination has so far been formulated as single pill to be taken orally once daily, namely Atripla containing the NtRTI tenofovir disoproxil fumarate, the NRTI emtricitabine and the NNRTI efavirenz. Here, we document these approved compounds along with other HIV-active compounds and, for the first time, compounds whose principal activity is against hepatitis B virus. The logic of this new division being the enzymatic similarity between the reverse transcriptase of HIV and hepatitis B virus; the strategies for the development of antiviral agents to combat them have much in common.

Antiviral Chemistry & Chemotherapy's current antiviral agents FactFile 2008 (2nd edition): RNA viruses.

Among the RNA viruses, other than the retroviruses (that is, HIV), which are dealt with separately in the current FactFile, the most important targets for the development of antiviral agents at the moment are the orthomyxoviruses (that is, influenza), the hepaciviruses (that is, hepatitis C virus [HCV]) and, to a lesser extent, the picornaviruses. Although the uncoating inhibitors amantadine and rimantadine were the first known inhibitors of influenza A, the neuraminidase inhibitors oseltamivir, zanamivir and peramivir have now become the prime antiviral drugs for the treatment of influenza A and B virus infections. For HCV infections, standard treatment consists of the combination of pegylated interferon-alpha with ribavirin, but several other antivirals targeted at specific viral functions such as the HCV protease and/ or polymerase may be expected to soon take an important share of this important market. Still untapped is the potential of a variety of uncoating inhibitors, as well as protease and/or polymerase inhibitors against the wide spectrum of picornaviruses. While ribavirin has been available for 35 years as a broad-spectrum anti-RNA virus agent, relatively new and unexplored is favipiravir (T-705) accredited with activity against influenza as well as flaviviruses, bunyaviruses and arenaviruses.

Herpes simplex virus helicase-primase inhibitors: recent findings from the study of drug resistance mutations.

After several decades during which nucleoside analogues (especially acyclovir and penciclovir and their prodrugs) have benefited many patients suffering from herpes simplex virus (HSV) infections, the discovery of the helicase-primase inhibitors (HPIs) represents an interesting new approach. Although antiviral resistance has not been a major problem for nucleoside analogues in immunocompetent patients, the problem of acyclovir resistance in immunocompromised patients is well documented. Several HPIs are extremely potent antiviral compounds and may, therefore, offer an important alternative therapy in these patients. The potential for synergy, not just for the inhibition of virus replication but also to delay the appearance of drug-resistant virus, needs to be thoroughly investigated. The study of resistance to HPIs has been important towards understanding the mechanism of action of these compounds and confirming the target function. However, during the course of our studies on HPI resistance, we have made a number of interesting observations that may be relevant to their clinical use. This article draws attention to the major observations on HPI resistance reported by others and to our own recently published observations that have extended this expanding area of antiviral research.

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.

BAY 57-1293 represents a new class of potent inhibitors of herpes simplex virus (HSV) that target the virus helicase primase complex. The present study was conducted using the zosteriform infection model in BALB/c mice. The helicase primase inhibitor, BAY 57-1293 was shown to be highly efficacious in this model. The beneficial effects of therapy were obtained rapidly (within 2 days) although the onset of treatment was delayed for 1 day after virus inoculation. The compound given orally, or intraperitoneally once per day at a dose of 15 mg/kg for 4 successive days was equally effective or superior to a much higher dose of famciclovir (1mg/ml, i.e. approximately 140-200mg/kg/day) given in the drinking water for 7 consecutive days, which, in our hands, is the most effective method for administering famciclovir to mice. In contrast to the vehicle-treated infected mice, all mice that received antiviral therapy looked normal and active with no mortality, no detectable loss of weight and no marked change in ear thickness. BAY 57-1293 and famciclovir reduced the virus titers in the skin to below the level of detection by days 3 and 7 post infection, respectively. In both BAY 57-1293 and famciclovir-treated mice, infectious virus titers in the ear pinna and brainstem remained below the level of detection. Consistent with these findings, BAY 57-1293 also showed a potent antiviral effect in an experiment involving a small number of severely immunocompromised athymic-nude BALB/c mice.

High frequency of spontaneous helicase-primase inhibitor (BAY 57-1293) drug-resistant variants in certain laboratory isolates of HSV-1.

Herpes simplex virus (HSV) helicase-primase is the target for a new group of potent antivirals that show great promise in vivo. A claimed advantage of this class of compounds is the low rate of drug resistance, which is reported to occur at a lesser rate than acyclovir (ACV)-resistance in cell culture. We confirmed that BAY 57-1293 is highly active against HSV-1 and superior to ACV when tested in Vero cells. Notably, drug resistance was detected in laboratory working stocks in two different strains of HSV at 10(-4) to 10(-5) and there was evidence that the resistant variants were present in the virus population before the selection was applied. Plaque-purified clones obtained from the parental viruses showed a lower level of resistance selection in the presence of drug (10-6) and this value is similar to published reports. In the case of HSV-1 SC16, no difference was observed between a working stock and a plaque-pure clone in the rate of resistance to the nucleoside analogue ACV. The working stocks were found to contain variants with resistance to BAY 57-1293 ranging from approximately 15-fold to 4,000-fold suggesting that these viruses have the potential to subvert effective therapy. Sequence analysis of HSV-1 helicase protein showed that most of the amino acid substitutions in the variants described in this study tallied with published results, with some interesting exceptions in the case of HSV-1 strain PDK. Resistant variants did not readily revert to a sensitive phenotype in the absence of the inhibitor and representative BAY 57-1293-resistant variants were cross-resistant to an alternative helicase-primase inhibitor, BILS 22 BS. Variants resistant to BAY 57-1293 retained sensitivity to the nucleoside analogue, ACV.

Antiviral prodrugs - the development of successful prodrug strategies for antiviral chemotherapy.

Following the discovery of the first effective antiviral compound (idoxuridine) in 1959, nucleoside analogues, especially acyclovir (ACV) for the treatment of herpesvirus infections, have dominated antiviral therapy for several decades. However, ACV and similar acyclic nucleosides suffer from low aqueous solubility and low bioavailability following oral administration. Derivatives of acyclic nucleosides, typically esters, were developed to overcome this problem and valaciclovir, the valine ester of ACV, was among the first of a new series of compounds that were readily metabolized upon oral administration to produce the antiviral nucleoside in vivo, thus increasing the bioavailility by several fold. Concurrently, famciclovir was developed as an oral formulation of penciclovir. These antiviral 'prodrugs' thus established a principle that has led to many successful drugs including both nucleoside and nucleotide analogues for the control of several virus infections, notably those caused by herpes-, retro- and hepatitisviruses. This review will chart the origins and development of the most important of the antiviral prodrugs to date.

Herpesvirus latency and therapy--from a veterinary perspective.

This short review considers how the human herpesviruses were among the first viruses to be effectively treated by means of antiviral therapy although the ability of alphaherpsviruses to establish neuronal latency with reactivation remains the major obstacle to achieving a cure. Laboratory animals played an essential role in the development of herpes antivirals including our understanding of the complexity of the neurological infection in relation to chemotherapy. The existence of natural herpesvirus infections in domestic species also contributes to our understanding of latency and reactivation relevant to antiviral therapy although the use of antivirals to treat or prevent virus infections in veterinary species has been minimal, to date. The review briefly focuses on herpes infections in the horse and cat where some progress has already been achieved in the veterinary antiviral field.

Antiviral Chemistry & Chemotherapy's current antiviral agents FactFile 2006 (1st edition).

The number of useful antiviral compounds is rapidly expanding. The current antiviral agents FactFile is a convenient key to the vital statistics of antiviral compounds to be used as an aide mémoire when reading or writing antiviral literature. A mini-portrait is provided for each of the antiviral compounds. The minimum criteria for inclusion of new compounds in the FactFile is the granting of an investigational new drug application with the realistic potential for medical or veterinary application. Several compounds that were subsequently withdrawn from further development are also included because of their historical importance or particular interest. The compounds are listed alphabetically according to their generic names together with systematic chemical names, common names and chemical structures. The compounds are grouped by virus targets; thus, the list is sub-divided into inhibitors of DNA viruses, RNA viruses, and retroviruses. The authors welcome comments and suggestions to be incorporated in future editions of the FactFile.

Temporal pattern of herpes simplex virus type 1 infection and cell death in the mouse brain stem: influence of guanosine nucleoside analogues.

Levels of bystander death occurring in herpes simplex virus type 1 (HSV-1)-infected mouse brain stems were studied, as well as the extent to which bystander death is influenced by guanosine nucleoside analogue treatment. Consecutive sections from brain stems of HSV-1-infected mice were stained alternately for (i) viral infection and (ii) cell death (TUNEL assay). Virus antigen was detectable in brain stems on day 3 of infection, while TUNEL staining was comparatively lower. An increase in the extent of TUNEL staining was observed on day 4 of infection. Despite this increase, however, the ratio of TUNEL-stained to infection marker-stained tissue still indicated that the amount of TUNEL staining remained lower than infection staining at this time point. On days 5 and 6 of infection, TUNEL staining continued to increase and the TUNEL/infection marker ratio switched on day 6 in favour of excess TUNEL staining, which was observed in and around the foci of infection, suggesting bystander death. The excess TUNEL staining on day 6 of infection was further increased on treatment with antivirals. The significance and implications of these results are discussed with respect to the nature and mechanism of action of the TUNEL assay, dynamics of primary HSV-1 infection, immunological influences and potential effects of antiviral treatment. The potential problems of the TUNEL assay are considered in the context of viral infection and the TUNEL assay, in combination with infection marker staining, may potentially provide a model system for quantitative analysis of true bystander death during HSV infection in vivo.

Antiviral activity of CTC-8 against herpes simplex virus (HSV-1) in cell culture: evidence for a selective antiviral effect via a cellular mechanism.

A synthetic programme produced a series of compounds related to natural prostaglandins, which are known to affect the growth of a number of viruses. Several of the compounds showed potent biological activity including antiviral effects. The compound CTC-8 [(S)-4-tert-butyldimethylsilyloxy-2-cyclopenten-1-one] contains the cyclopentenone ring of prostaglandin A1, but the extended side chains common to the prostaglandin family are truncated. The present study demonstrates that CTC-8 inhibits HSV-1 replication in cell culture at sub-toxic concentrations. The antiviral effect was evidenced by reduction in infectious virus yield, although the compound was not effective in the standard plaque-reduction assay. Time-of-addition studies and other experiments provide a possible explanation for these results by suggesting that the antiviral activity is confined to a single cycle. Under the standard conditions of high-multiplicity infection in BHK cells it was notable that CTC-8 is most effective when added for a short period 6-8 h post-infection. Furthermore, multiple passage of HSV-1 in the presence of CTC-8 did not result in the selection of resistant mutants. The results of these and other experiments are consistent with the hypothesis that the mechanism by which CTC-8 inhibits virus replication involves a cellular target. These results encourage further research into the therapeutic potential of this series of compounds.