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The seminal discovery of acyclovir 40 years ago heralded the modern era of truly selective antiviral therapies and this drug remains the therapy of choice for herpes simplex virus infections. Yet by modern standards, its antiviral activity is modest and new drugs against novel molecular targets such as the helicase-primase have the potential to improve clinical outcome, particularly in high-risk patients. A brief synopsis of current therapies for these infections and clinical need is provided to help provide an initial perspective. The function of the helicase-primase complex is then summarized and the development of new inhibitors of the helicase-primase complex, such as pritelivir and amenamevir, is discussed. We review their mechanism of action, propensity for drug resistance, and pharmacokinetic characteristics and discuss their potential to advance current therapeutic options. Strategies that include combinations of these inhibitors with acyclovir are also considered, as they will likely maximize clinical efficacy. [ABSTRACT FROM AUTHOR]

Although neonates are commonly exposed to vaginal herpes simplex virus (HSV)-2, neonatal herpes is rare. Therefore, we analyzed paired infant and maternal HSV-2 isolates from two cases of mother-to-infant transmission to identify viral factors contributing to vertical transmission. Sixteen infant isolates with neonatal herpes and 27 genital isolates in their third trimester were included. The infant isolates were significantly more temperature-independent than the maternal isolates. Sequence comparison revealed viral UL13 protein kinase (UL13-PK) mutation in the infant isolates in both cases. In the expanded cohort, infant isolates (5/18) had significantly more UL13-PK mutations than genital isolates (1/29). Isolates within 8 days post-birth (3/4) had a significantly higher frequency of UL13-PK mutation than those after 9 days (2/14), suggesting a close association between UL13-PK mutations and vertical transmission. Elongation factor 1-delta was identified as a target of UL13-PK by proteomic analysis of UL13-PK-positive and -negative HepG2 cells. The mixed infant isolates with the intact and mutated UL13-PK conferred altered cell tropism, temperature independence adapting to fetal temperature, and better growth properties in Vero and hepatoblastoma HepG2 cells than in HSV-2 with intact and mutated UL13-PK alone, indicating that viral UL13-PK mutation is essential for vertical HSV-2 transmission., (© 2024 Wiley Periodicals LLC.)

DNA viruses are responsible for many diseases in humans. Current treatments are often limited by toxicity, as in the case of cidofovir (CDV, Vistide), a compound used against cytomegalovirus (CMV) and adenovirus (AdV) infections. CDV is a polar molecule with poor bioavailability, and its overall clinical utility is limited by the high occurrence of acute nephrotoxicity. To circumvent these disadvantages, we designed nine CDV prodrug analogues. The prodrugs modulate the polarity of CDV with a long sulfonyl alkyl chain attached to one of the phosphono oxygens. We added capping groups to the end of the alkyl chain to minimize β-oxidation and focus the metabolism on the phosphoester hydrolysis, thereby tuning the rate of this reaction by altering the alkyl chain length. With these modifications, the prodrugs have excellent aqueous solubility, optimized metabolic stability, increased cellular permeability, and rapid intracellular conversion to the pharmacologically active diphosphate form (CDV-PP). The prodrugs exhibited significantly enhanced antiviral potency against a wide range of DNA viruses in infected human foreskin fibroblasts. Single-dose intravenous and oral pharmacokinetic experiments showed that the compounds maintained plasma and target tissue levels of CDV well above the EC 50 for 24 h. These experiments identified a novel lead candidate, NPP-669. NPP-669 demonstrated efficacy against CMV infections in mice and AdV infections in hamsters following oral (p.o.) dosing at a dose of 1 mg/kg BID and 0.1 mg/kg QD, respectively. We further showed that NPP-669 at 30 mg/kg QD did not exhibit histological signs of toxicity in mice or hamsters. These data suggest that NPP-669 is a promising lead candidate for a broad-spectrum antiviral compound.

Human cytomegalovirus (CMV) is a ubiquitous β-herpesvirus that establishes latent asymptomatic infections in healthy individuals but can cause serious infections in immunocompromised people, resulting in increased risk of morbidity and mortality. The current FDA-approved CMV drugs target late stages of the CMV life-cycle. While these drugs are effective in most cases, they have serious drawbacks, including poor oral bioavailability, dose-limiting toxicity, and a low barrier to resistance. Given the clinical relevance of CMV-associated diseases, novel therapies are needed. Thus, a novel class of compounds that inhibits the early stages of the CMV life-cycle was identified and found to block infection of different strains in physiologically relevant cell types. This class of compounds, N-arylpyrimidinamine (NAPA), demonstrated potent anti-CMV activity against ganciclovir-sensitive and -resistant strains in in vitro replication assays, a selectivity index >30, and favorable in vitro ADME properties. Mechanism of action studies demonstrated that NAPA compounds inhibit an early step of virus infection. NAPA compounds are specific inhibitors of cytomegaloviruses and exhibited limited anti-viral activity against other herpesviruses. Collectively, we have identified a novel class of CMV inhibitor that effectively limits viral infection and proliferation., Competing Interests: Declaration of competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Varicella zoster virus (VZV) causes chicken pox and shingles and is prevalent worldwide. Acyclovir and penciclovir (and its prodrugs) are first-line treatments for VZV infections, but they are not highly potent against VZV and resistance may arise in immunocompromised people on long-term therapy. HPMPC (cidofovir) is active against VZV, but cidofovir is not approved for treating VZV diseases, is nephrotoxic, and is not orally bioavailable. Here, we present the synthesis and evaluation of USC-373, a phosphonate prodrug of HPMPC with activity against VZV and other DNA viruses. In cultured fibroblasts, it was potent against VZV Ellen laboratory strain and was not overtly toxic, with EC 50 of 4 nM and CC 50 of 0.20 μM, producing a selectivity index of 50. In ARPE-19 cells, USC-373 was effective against VZV-ORF57-Luc wild type strain and the acyclovir-resistant isogenic strain. In human skin organ culture, USC-373 formulated in cocoa butter and applied topically prevented VZV-ORF57-Luc spread without toxicity. In NuSkin mice with human skin xenografts, one daily dose of 3 mg/kg was effective by the subcutaneous route, and one daily dose of 10 mg/kg was effective by the oral route. Remarkably, a 10 mg/kg oral dose given every other day was also effective. USC-373 was well tolerated and mice did not lose weight or show signs of distress. The prodrug modifications of USC-373 increase the potency and oral bioavailability compared to its parent nucleoside analog, HPMPC., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The substantial impact of acyclic nucleoside phosphonates (ANPs) on human medicine encourages the synthesis of new ANP analogues with a potentially differentiated antiviral spectrum. Herein, we demonstrate the functionalization of the 2-position of the ( R , S )-3-hydroxy-2-(phosphonomethoxy)propyl side-chain of an inactive ANP with a polar cyano group to generate a thymine analogue with selective inhibition of hepatitis B virus (HBV) replication (SI > 302; EC 50 = 0.33 μM), without significant antiretroviral activity. These findings suggest new strategies to synthesize unique ANPs with a targeted antiviral profile., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Human adenovirus (HAdV) infection is common in the general population and can cause a range of clinical manifestations, among which pneumonia and keratoconjunctivitis are the most common. Although HAdV infections are mostly self-limiting, infections in immunocompromised individuals can be severe. No antiviral drug has been approved for treating adenoviruses. Filociclovir (FCV) is a nucleoside analogue which has successfully completed phase I human clinical safety studies and is now being developed for treatment of human cytomegalovirus (HCMV)-related disease in immunocompromised patients. In this report, we show that FCV is a potent broad-spectrum inhibitor of HAdV types 4 to 8, with 50% effective concentrations (EC 50 s) ranging between 1.24 and 3.6 μM and a 50% cytotoxic concentration (CC 50 ) of 100 to 150 μM in human foreskin fibroblasts (HFFs). We also show that the prophylactic oral administration of FCV (10 mg/kg of body weight) 1 day prior to virus challenge and then daily for 14 days to immunosuppressed Syrian hamsters infected intravenously with HAdV6 was sufficient to prevent morbidity and mortality. FCV also mitigated tissue damage and inhibited virus replication in the liver. The 10-mg/kg dose had similar effects even when the treatment was started on day 4 after virus challenge. Furthermore, FCV administered at the same dose after intranasal challenge with HAdV6 partially mitigated body weight loss but significantly reduced pathology and virus replication in the lung. These findings suggest that FCV could potentially be developed as a pan-adenoviral inhibitor., (Copyright © 2020 American Society for Microbiology.)

A series of novel 2-oxoimidazolidine derivatives were synthesized and their antiviral activities against BK human polyomavirus type 1 (BKPyV) were evaluated in vitro. Bioassays showed that the synthesized compounds 1-{[(4E)-5-(dichloromethylidene)-2-oxoimidazolidin-4-ylidene]sulfamoyl}piperidine-4-carboxylic acid (5) and N-Cyclobutyl-N'-[(4E)-5-(dichloromethylidene)-2-oxoimidazolidin-4-ylidene]sulfuric diamide (4) exhibited moderate activities against BKPyV (EC 50 =5.4 and 5.5 μm, respectively) that are comparable to the standard drug Cidofovir. Compound 5 exhibited the same cytotoxicity in HFF cells and selectivity index (SI 50 ) as Cidofovir. The selectivity index of compound 4 is three times less than that of Cidofovir due to the higher toxicity of this compound. Hence, these compounds may be taken as lead compound for further development of novel ant-BKPyV agents., (© 2019 Wiley-VHCA AG, Zurich, Switzerland.)

Objective: To determine frequency of hospital-acquired viral respiratory infections (HA-VRI) and associated outcomes in a NICU., Study Design: Prospective cohort study conducted from 4 October 2016 to 21 March 2017. Infants hospitalized from birth in the NICU had a weekly nasal swab collected for testing using a multiplex PCR assay capable of detecting 16 different respiratory viruses., Results: Seventy-four infants enrolled, with 5 (6.8%) testing positive for a virus (incidence rate of 1.3/1000 patient days). VRI positive infants had a younger gestational age (median 27 w vs. 32 w, p = 0.048); were hospitalized longer (97 d vs 43 d, p = 0.013); required more antibiotics (8 d vs. 4 d, p = 0.037) and were more likely to be diagnosed with bronchopulmonary dysplasia (p = 0.008) compared to VRI negative infants., Conclusion: Respiratory viruses are a frequent cause of HAI in the NICU and are associated with negative outcomes.

More than 14,000 neonates are infected with herpes simplex virus (HSV) annually. Approximately half display manifestations limited to the skin, eyes, or mouth (SEM disease). The rest develop invasive infections that spread to the central nervous system (CNS disease or encephalitis) or throughout the infected neonate (disseminated disease). Invasive HSV disease is associated with significant morbidity and mortality, but the viral and host factors that predispose neonates to these forms are unknown. To define viral diversity within the infected neonatal population, we evaluated 10 HSV-2 isolates from newborns with a range of clinical presentations. To assess viral fitness independently of host immune factors, we measured viral growth characteristics in cultured cells and found diverse in vitro phenotypes. Isolates from neonates with CNS disease were associated with larger plaque size and enhanced spread, with the isolates from cerebrospinal fluid (CSF) exhibiting the most robust growth. We sequenced complete viral genomes of all 10 neonatal viruses, providing new insights into HSV-2 genomic diversity in this clinical setting. We found extensive interhost and intrahost genomic diversity throughout the viral genome, including amino acid differences in more than 90% of the viral proteome. The genes encoding glycoprotein G (gG; US4), glycoprotein I (gI; US7), and glycoprotein K (gK; UL53) and viral proteins UL8, UL20, UL24, and US2 contained variants that were found in association with CNS isolates. Many of these viral proteins are known to contribute to cell spread and neurovirulence in mouse models of CNS disease. This report represents the first application of comparative pathogen genomics to neonatal HSV disease. IMPORTANCE Herpes simplex virus (HSV) causes invasive disease in half of infected neonates, resulting in significant mortality and permanent cognitive morbidity. The factors that contribute to invasive disease are not understood. This study revealed diversity among HSV isolates from infected neonates and detected the first associations between viral genetic variations and clinical disease manifestations. We found that viruses isolated from newborns with encephalitis showed enhanced spread in culture. These viruses contained protein-coding variations not found in viruses causing noninvasive disease. Many of these variations were found in proteins known to impact neurovirulence and viral spread between cells. This work advances our understanding of HSV diversity in the neonatal population and how it may impact disease outcome., (Copyright © 2019 Akhtar et al.)

This paper describes an access to new nitrogen-containing heterocyclic triterpenoids by the reaction of 2,3-indolotriterpenoids with ozone and dimethyldioxirane. The oxidation of indolo-fused 28-oxo-allobetulin or methyl platanoate with ozone led to a mixture of a quinolone as the major product and a nine-membered 2,3-seco-2-oxolactam and three different types of spiroindoles as byproducts. The formation of quinolone and 2,3-seco-2-oxolactam derivatives could be explained by the standard 1,3-dipolar cycloaddition of ozone to the C2(3)-double bond of the triterpene core similar to the products observed in the ozonolysis of indoles in the Witkop-Winterfeldt oxidation (WWO). The formation of spiroindoles was unexpected and could be explained through the 1,2-cycloaddition of ozone to the C2(3)-double bond with consecutive intramolecular rearrangements of the 2,3-epoxy-intermediate. These spiroindoles seem to be novel structures observed in the WWO reaction. The formation of only two isomeric triterpene spiroindolinones was achieved by the oxidation of 2,3-indolo-28-oxo-allobetulin with dimethyldioxirane that could be explained by the rearrangement of the 2,3-epoxy-intermediate. 19β,28-Epoxy-18α-olean-28-oxo-2-nor-2,3-4'(1H)-quinolone was the most active against HPV-11 with EC50 0.45 μM and SI50 322 in a primary assay and SI90 < 10 against HPV-16 in a secondary assay. The oxidative transformations of indolotriterpenoids have great potential for further modifications towards the preparation of new biologically active compounds.

The recent approval of letermovir marks a new era of therapy for human cytomegalovirus (HCMV) infections, particularly for the prevention of HCMV disease in hematopoietic stem cell transplant recipients. For almost 30 years ganciclovir has been the therapy of choice for these infections and by today's standards this drug exhibits only modest antiviral activity that is often insufficient to completely suppress viral replication, and drives the selection of drug-resistant variants that continue to replicate and contribute to disease. While ganciclovir remains the therapy of choice, additional drugs that inhibit novel molecular targets, such as letermovir, will be required as highly effective combination therapies are developed not only for the treatment of immunocompromised hosts, but also for congenitally infected infants. Sustained efforts, largely in the biotech industry and academia, have identified additional highly active lead compounds that have progressed into clinical studies with varying levels of success and at least two have the potential to be approved in the near future. Some of the new drugs in the pipeline inhibit new molecular targets, remain effective against isolates that have developed resistance to existing therapies, and promise to augment existing therapeutic regimens. Here, we will describe some of the unique features of HCMV biology and discuss their effect on therapeutic needs. Existing drugs will also be discussed and some of the more promising candidates will be reviewed with an emphasis on those progressing through clinical studies. The in vitro and in vivo antiviral activity, spectrum of antiviral activity, and mechanism of action of new compounds will be reviewed to provide an update on potential new therapies for HCMV infections that have progressed significantly in recent years., (Copyright © 2018. Published by Elsevier B.V.)

The search for new compounds with a broad spectrum of antiviral activity is important and requires the evaluation of many compounds against several distinct viruses. Researchers attempting to develop new antiviral therapies for DNA virus infections currently use a variety of cell lines, assay conditions and measurement methods to determine in vitro drug efficacy, making it difficult to compare results from within the same laboratory as well as between laboratories. In this paper, we describe the assessment of antiviral activity of a set of nucleoside analogs against BK polyomavirus, JC polyomavirus, Epstein-Barr virus, human herpesvirus 6B, and human herpesvirus 8 in an automated 384-well format and utilize qPCR assays to measure the accumulation of viral DNA. In an accompanying paper, we present a standardized approach to evaluating antivirals against additional herpesviruses, orthopoxviruses, and adenovirus. Together, they reveal new activities for reference compounds and help to define the spectrum of antiviral activity for a set of nucleoside analogs against a set of 12 DNA viruses that infect humans including representative human herpesviruses, orthopoxviruses, adenoviruses, and polyomaviruses. This analysis helps provide perspective on combinations of agents that would help provide broad coverage of significant pathogens in immunocompromised patients as well as against emerging infections., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The search for new compounds with a broad spectrum of antiviral activity is important and requires the evaluation of many compounds against several distinct viruses. Researchers attempting to develop new antiviral therapies for DNA virus infections currently use a variety of cell lines, assay conditions and measurement methods to determine in vitro drug efficacy, making it difficult to compare results from within the same laboratory as well as between laboratories. In this paper we describe a common assay platform designed to facilitate the parallel evaluation of antiviral activity against herpes simplex virus type 1, herpes simplex virus type 2, varicella-zoster virus, cytomegalovirus, vaccinia virus, cowpox virus, and adenovirus. The automated assays utilize monolayers of primary human foreskin fibroblast cells in 384-well plates as a common cell substrate and cytopathic effects and cytotoxicity are quantified with CellTiter-Glo. Data presented demonstrate that each of the assays is highly robust and yields data that are comparable to those from other traditional assays, such as plaque reduction assays. The assays proved to be both accurate and robust and afford an in depth assessment of antiviral activity against the diverse class of viruses with very small quantities of test compounds. In an accompanying paper, we present a standardized approach to evaluating antivirals against lymphotropic herpesviruses and polyomaviruses and together these studies revealed new activities for reference compounds. This approach has the potential to accelerate the development of broad spectrum therapies for the DNA viruses., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Recent advances in xenotransplantation have produced organs from pigs that are well tolerated in primate models because of genetic changes engineered to delete major antigens from donor animals. To ensure the safety of human transplant recipients, it will be essential to understand both the spectrum of infectious agents in donor pigs and their potential to be transmitted to immunocompromised transplant recipients. Equally important will be the development of new highly sensitive diagnostic methods for use in the detection of these agents in donor animals and for the monitoring of transplant recipients., Methods: Herein, we report the development of a panel of 30 quantitative polymerase chain reaction (qPCR) assays for infectious agents with the potential to be transmitted to the human host. The reproducibility, sensitivity and specificity of each assay were evaluated and were found to exhibit analytic sensitivity that was similar to that of quantitative assays used to perform viral load testing of human viruses in clinical laboratories., Results: This analytical approach was used to detect nucleic acids of infectious agents present in specimens from 9 sows and 22 piglets derived by caesarean section. The most commonly detected targets in adult animals were Mycoplasma species and two distinct herpesviruses, porcine lymphotrophic herpesvirus 2 and 3. A total of 14 piglets were derived from three sows infected with either or both herpesviruses, yet none tested positive for the viruses indicating that vertical transmission of these viruses is inefficient., Conclusions: The data presented demonstrate that procedures in place are highly sensitive and can specifically detect nucleic acids from target organisms in the panel, thus ensuring the safety of organs for transplantation as well as the monitoring of patients potentially receiving them., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Human adenoviruses (AdV) cause generally mild infections of the respiratory and GI tracts as well as some other tissues. However, AdV can cause serious infection in severely immunosuppressed individuals, especially pediatric patients undergoing allogeneic hematopoietic stem cell transplantation, where mortality rates are up to 80% with disseminated disease. Despite the seriousness of AdV disease, there are no drugs approved specifically to treat AdV infections. We report here that USC-087, an N-alkyl tyrosinamide phosphonate ester prodrug of HPMPA, the adenine analog of cidofovir, is highly effective against multiple AdV types in cell culture. USC-087 is also effective against AdV-C6 in our immunosuppressed permissive Syrian hamster model. In this model, hamsters are immunosuppressed by treatment with high dose cyclophosphamide. Injection of AdV-C6 (or AdV-C5) intravenously leads to a disseminated infection that resembles the disease seen in humans, including death. We have tested the efficacy of orally-administered USC-087 against the median lethal dose of intravenously administered AdV-C6. USC-087 completely prevented or significantly decreased mortality when administered up to 4 days post challenge. USC-087 also prevented or significantly decreased liver damage caused by AdV-C6 infection, and suppressed virus replication even when administered 4 days post challenge. These results imply that USC-087 is a promising candidate for drug development against HAdV infections., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Pritelivir, a helicase-primase inhibitor, has excellent in vitro and in vivo activity against human herpes simplex virus (HSV). Mice lethally infected with HSV type 1 or 2, including acyclovir-resistant strains, were treated 72 h after infection for 7 days with pritelivir or acyclovir. Both drugs were administered orally twice daily either alone or in combination. Dosages of pritelivir from 0.3 to 30 mg/kg reduced mortality (P < 0.001) against HSV-1, E-377. With an acyclovir resistant HSV-1, 11360, pritelivir at 1 and 3 mg/kg increased survival (P < 0.005). With HSV-2, MS infected mice, all dosages higher than the 0.3 mg/kg dose of pritelivir were effective (P < 0.005). For acyclovir resistant HSV-2, strain 12247, pritelivir dosages of 1-3 mg/kg significantly improved survival (P < 0.0001). Combination therapies of pritelivir at 0.1 or 0.3 mg/kg/dose with acyclovir (10 mg/kg/dose) were protective (P < 0.0001) when compared to the vehicle treated group against HSV-2, strain MS (in line with previous data using HSV-1). An increased mean days to death (P < 0.05) was also observed and was indicative of a potential synergy. Pharmacokinetic studies were performed to determine pritelivir concentrations and a dose dependent relationship was found in both plasma and brain samples regardless of infection status or time of initiation of dosing. In summary, pritelivir was shown to be active when treatment was delayed to 72 h post viral inoculation and appeared to synergistically inhibit mortality in this model in combination with acyclovir. We conclude pritelivir has potent and resistance-breaking antiviral efficacy with potential for the treatment of potentially life-threatening HSV type 1 and 2 infections, including herpes simplex encephalitis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Cell culture antiviral experiments were conducted in order to understand the relationship between percentage data generated by plaque reduction (PR) and logarithmic data derived by virus yield reduction (VYR) assays, using three-dimensional MacSynergy II software. The relationship between percentage and logarithmic data has not been investigated previously. Interpretation of drug-drug interactions is based on a Volume of Synergy (VS) calculation, which can be positive (synergy), negative (antagonistic), or neutral (no or minimal interaction). Interactions of two known inhibitors of vaccinia virus replication, cidofovir and 6-azauridine, used in combination by PR assay yielded a VS value of 265, indicative of strong synergy. By VYR, the VS value was only 37, or weak synergy using the same criterion, even though profound log 10 reductions in virus titer occurred at multiple drug combinations. These results confirm that the differences in VS values is dependent of the measurement scale, and not that the degree of synergy differed between the assays. We propose that for logarithmic data, the calculated VS values will be lower for significant synergy and antagonism and that volumes of >10 μM 2 log 10  PFU/ml (or other units such as μM 2 log 10 genomic equivalents/ml or μM 2 log 10 copies/ml) and <-10 μM 2 log 10  PFU/ml are likely to be indicative of strong synergy and strong antagonism, respectively. Data presented here show that the interaction of cidofovir and 6-azauridine was strongly synergistic in vitro., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Human papillomavirus (HPV) high-risk genotypes such as HPV-16 and HPV-18 cause the majority of anogenital tract carcinomas, including cervical cancer, the second most common malignancy in women worldwide. Currently there are no approved antiviral agents that reduce or eliminate HPV and reverse virus-associated pathology. We synthesized and evaluated several alkoxyalkyl acyclic nucleoside phosphonate diesters and identified octadecyloxyethyl benzyl 9-[(2-phosphonomethoxy)ethyl]guanine (ODE-Bn-PMEG) as an active compound which strongly inhibited transient amplification of HPV-11, -16, and -18 origin-containing plasmid DNA in transfected cells at concentrations well below its cytotoxic concentrations. ODE-Bn-PMEG demonstrated increased uptake in human foreskin fibroblast cells and was readily converted in vitro to the active antiviral metabolite, PMEG diphosphate. The P-chiral enantiomers of ODE-Bn-PMEG were obtained and appeared to have equivalent antiviral activities against HPV. ODE-Bn-PMEG is a promising candidate for the local treatment of HPV-16 and HPV-18 and other high-risk types, an important unmet medical need.

Human polyomaviruses are generally latent but can be reactivated in patients whose immune systems are suppressed. Unfortunately, current therapeutics for diseases associated with polyomaviruses are non-specific, have undefined mechanisms of action, or exacerbate the disease. We previously reported on a class of dihydropyrimidinones that specifically target a polyomavirus-encoded protein, T antigen, and/or inhibit a cellular chaperone, Hsp70, that is required for virus replication. To improve the antiviral activity of the existing class of compounds, we performed Biginelli and modified multi-component reactions to obtain new 3,4-dihydropyrimidin-2(1H)-ones and -thiones for biological evaluation. We also compared how substituents at the N-1 versus N-3 position in the pyrimidine affect activity. We discovered that AMT580-043, a N-3 alkylated dihydropyrimidin-2(1H)-thione, inhibits the replication of a disease-causing polyomavirus in cell culture more potently than an existing drug, cidofovir., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Brincidofovir (BCV) is a broad-spectrum antiviral agent active in vitro against double-stranded DNA viruses including herpesviruses, adenoviruses, polyomaviruses, and poxviruses. We report successful BCV use in management of disseminated acyclovir- and cidofovir-resistant varicella zoster virus in an immunocompromised hematopoietic stem cell transplant patient with chronic graft-versus-host disease who was intolerant to foscarnet., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Brincidofovir is an oral antiviral in development for prevention of cytomegalovirus disease. Cytomegalovirus genotyping results from a phase 2 trial comparing brincidofovir to placebo for prophylaxis against cytomegalovirus infection in hematopoietic cell transplant recipients provided initial data on the clinical resistance profile for brincidofovir. In this study, no known resistance-associated mutations were detected in brincidofovir-treated subjects; identified genotypic substitutions did not confer resistance to cytomegalovirus antivirals in vitro, suggesting that these changes represent polymorphisms unrelated to brincidofovir resistance. Lack of evidence for genotypic resistance during prophylaxis suggests that first-line use of brincidofovir for prevention of cytomegalovirus infection may preserve downstream options for patients., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America.)

Monohydroxymethyl methylenecyclopropane nucleosides (MCPNs) with ether or thioether substituents at the 6-position show promise as broad-spectrum herpes virus inhibitors. Their proposed mechanism of action involves sequential phosphorylation to a triphosphate, which can then inhibit viral DNA polymerase. The inhibition of herpes simplex virus (HSV) by these compounds is not dependent on the viral thymidine kinase (TK), which is known to phosphorylate acyclovir (ACV), a standard treatment for HSV infections. Previous studies on the mechanism of action of these compounds against human cytomegalovirus (HCMV) implicated a host kinase in addition to HCMV UL97 kinase in performing the initial phosphorylation. After first eliminating other candidate HSV-1 encoded kinases (UL13 and US3) as well as potential host nucleoside kinases, using activity-based fractionation, we have now identified the host serine-threonine protein kinase TAOK3 as the kinase responsible for transforming the representative monohydroxymethyl MCPN analog MBX 2168 to its monophosphate., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: The treatment of symptomatic congenital cytomegalovirus (CMV) disease with intravenous ganciclovir for 6 weeks has been shown to improve audiologic outcomes at 6 months, but the benefits wane over time., Methods: We conducted a randomized, placebo-controlled trial of valganciclovir therapy in neonates with symptomatic congenital CMV disease, comparing 6 months of therapy with 6 weeks of therapy. The primary end point was the change in hearing in the better ear ("best-ear" hearing) from baseline to 6 months. Secondary end points included the change in hearing from baseline to follow-up at 12 and 24 months and neurodevelopmental outcomes, with each end point adjusted for central nervous system involvement at baseline., Results: A total of 96 neonates underwent randomization, of whom 86 had follow-up data at 6 months that could be evaluated. Best-ear hearing at 6 months was similar in the 6-month group and the 6-week group (2 and 3 participants, respectively, had improvement; 36 and 37 had no change; and 5 and 3 had worsening; P=0.41). Total-ear hearing (hearing in one or both ears that could be evaluated) was more likely to be improved or to remain normal at 12 months in the 6-month group than in the 6-week group (73% vs. 57%, P=0.01). The benefit in total-ear hearing was maintained at 24 months (77% vs. 64%, P=0.04). At 24 months, the 6-month group, as compared with the 6-week group, had better neurodevelopmental scores on the Bayley Scales of Infant and Toddler Development, third edition, on the language-composite component (P=0.004) and on the receptive-communication scale (P=0.003). Grade 3 or 4 neutropenia occurred in 19% of the participants during the first 6 weeks. During the next 4.5 months of the study, grade 3 or 4 neutropenia occurred in 21% of the participants in the 6-month group and in 27% of those in the 6-week group (P=0.64)., Conclusions: Treating symptomatic congenital CMV disease with valganciclovir for 6 months, as compared with 6 weeks, did not improve hearing in the short term but appeared to improve hearing and developmental outcomes modestly in the longer term. (Funded by the National Institute of Allergy and Infectious Diseases; ClinicalTrials.gov number, NCT00466817.).

Human herpesvirus 6 (HHV-6) infections are typically mild and in rare cases can result in encephalitis. A common theme among all the herpesviruses, however, is the reactivation upon immune suppression. HHV-6 commonly reactivates in transplant recipients. No therapies are approved currently for the treatment of these infections, although small studies and individual case reports have reported intermittent success with drugs such as cidofovir, ganciclovir, and foscarnet. In addition to the current experimental therapies, many other compounds have been reported to inhibit HHV-6 in cell culture with varying degrees of efficacy. Recent advances in the development of new small molecule inhibitors of HHV-6 will be reviewed with regard to their efficacy and spectrum of antiviral activity. The potential for new therapies for HHV-6 infections will also be discussed, and they will likely arise from efforts to develop broad spectrum antiviral therapies for DNA viruses., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Forty years after the discovery of acyclovir (ACV), it remains the mainstay of therapy for herpes simplex virus (HSV) infections. Since then, other antiviral agents have also been added to the armamentarium for these infections but ACV remains the therapy of choice. As the efficacy of ACV is reassessed, however, it is apparent that a therapy with increased efficacy, reduced potential for resistance, and improved pharmacokinetics would improve clinical outcome, particularly in high risk patients. Inhibitors of viral targets other than the DNA polymerase, such as the helicase primase complex, are of particular interest and will be valuable as new therapeutic approaches are conceived. This review focuses on currently approved HSV therapies as well as new systemic therapies in development., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Unlabelled: The human cytomegalovirus (HCMV)-encoded kinase pUL97 is required for efficient viral replication. Previous studies described two isoforms of pUL97, the full-length isoform (M1) and a smaller isoform likely resulting from translation initiation at codon 74 (M74). Here, we report the detection of a third pUL97 isoform during viral infection resulting from translation initiation at codon 157 (isoform M157). The consistent expression of isoform M157 as a minor component of pUL97 during infection with clinical and laboratory-adapted HCMV strains was suppressed when codon 157 was mutagenized. Viral mutants expressing specific isoforms were generated to compare their growth and drug susceptibility phenotypes, as well as pUL97 intracellular localization patterns and kinase activities. The exclusive expression of isoform M157 resulted in substantially reduced viral growth and resistance to the pUL97 inhibitor maribavir while retaining susceptibility to ganciclovir. Confocal imaging demonstrated reduced nuclear import of amino-terminal deletion isoforms compared to isoform M1. Isoform M157 showed reduced efficiency of various substrate protein interactions and autophosphorylation, whereas Rb phosphorylation was preserved. These results reveal differential properties of pUL97 isoforms that affect viral replication, with implications for the antiviral efficacy of maribavir., Importance: The HCMV UL97 kinase performs important functions in viral replication that are targeted by the antiviral drug maribavir. Here, we describe a naturally occurring short isoform of the kinase that when expressed by itself in a recombinant virus results in altered intracellular localization, impaired growth, and high-level resistance to maribavir compared to those of the predominant full-length counterpart. This is another factor to consider in explaining why maribavir appears to have variable antiviral activity in cell culture and in vivo.

Background: Human HBV and HIV integrate their retro-transcribed DNA proviruses into the human host genome. Existing antiretroviral drug regimens fail to directly target these intrachromosomal xenogenomes, leading to persistence of viral genetic information. Retinazone (RTZ) constitutes a novel vitamin A-derived (retinoid) thiosemicarbazone derivative with broad-spectrum antiviral activity versus HIV, HCV, varicella-zoster virus and cytomegalovirus., Methods: The in vitro inhibitory action of RTZ on HIV-1 strain LAI, human HBV strain ayw, HCV-1b strain Con1, enhanced green fluorescent protein-expressing Ebola virus Zaire 1976 strain Mayinga, wild-type Ebola virus Zaire 1976 strain Mayinga, human herpesvirus 6B and Kaposi's sarcoma-associated herpesvirus replication was investigated. The binding of RTZ to human glucocorticoid receptor was determined., Results: RTZ inhibits blood-borne human HBV multiplication in vitro by covalent inactivation of intragenic and intraexonic viral glucocorticoid response elements, and, in close analogy, RTZ suppresses HIV-1 multiplication in vitro. RTZ disrupts the multiplication of blood-borne human HCV and Ebola Zaire virus at nanomolar concentrations in vitro. RTZ has the capacity to bind to human glucocorticoid receptor, to selectively and covalently bind to intraexonic viral glucocorticoid response elements, and thereby to inactivate human genome-integrated proviral DNA of human HBV and HIV., Conclusions: RTZ represents the first reported antiviral agent capable of eradicating HIV and HBV proviruses from their human host. Furthermore, RTZ represents a potent and efficacious small-molecule in vitro inhibitor of Ebola virus Zaire 1976 strain Mayinga replication.

Natural serine protease inhibitors (serpins) elicit sensing of a microbial cell intruder and activate an intrinsic cellular immune response in HIV and HCV infected cells. Here, we demonstrate in vitro inhibition of HSV with serpin antithrombin III (ATIII) early during infection pointing towards inhibition of an entry event. We also found reduction of mortality from 90% to 40% in an abrasion mice model demonstrating a strong reduction of infection in vivo . Our data also indicated that this treatment might be suitable for drug-resistant viruses since high inhibition of an acyclovir-resistant HSV-1 strain was found. Thus, an ATIII tropical treatment might be used for immunocompromised patients where prolonged treatment leads to drug resistant HSV-1 strains. Understanding how ATIII regulates HSV-1 infections may reveal new avenues for therapeutic interventions.

Influenza viruses are a major public health threat worldwide, and options for antiviral therapy are limited by the emergence of drug-resistant virus strains. The influenza virus glycoprotein hemagglutinin (HA) plays critical roles in the early stage of virus infection, including receptor binding and membrane fusion, making it a potential target for the development of anti-influenza drugs. Using pseudotype virus-based high-throughput screens, we have identified several new small molecules capable of inhibiting influenza virus entry. We prioritized two novel inhibitors, MBX2329 and MBX2546, with aminoalkyl phenol ether and sulfonamide scaffolds, respectively, that specifically inhibit HA-mediated viral entry. The two compounds (i) are potent (50% inhibitory concentration [IC50] of 0.3 to 5.9 μM); (ii) are selective (50% cytotoxicity concentration [CC(50)] of >100 μM), with selectivity index (SI) values of >20 to 200 for different influenza virus strains; (iii) inhibit a wide spectrum of influenza A viruses, which includes the 2009 pandemic influenza virus A/H1N1/2009, highly pathogenic avian influenza (HPAI) virus A/H5N1, and oseltamivir-resistant A/H1N1 strains; (iv) exhibit large volumes of synergy with oseltamivir (36 and 331 μM(2) % at 95% confidence); and (v) have chemically tractable structures. Mechanism-of-action studies suggest that both MBX2329 and MBX2546 bind to HA in a nonoverlapping manner. Additional results from HA-mediated hemolysis of chicken red blood cells (cRBCs), competition assays with monoclonal antibody (MAb) C179, and mutational analysis suggest that the compounds bind in the stem region of the HA trimer and inhibit HA-mediated fusion. Therefore, MBX2329 and MBX2546 represent new starting points for chemical optimization and have the potential to provide valuable future therapeutic options and research tools to study the HA-mediated entry process.

Methylenecyclopropane nucleoside (MCPN) analogs are being investigated for treatment of human cytomegalovirus (HCMV) infection because of favorable preclinical data and limited ganciclovir cross-resistance. Monohydroxymethyl MCPNs bearing ether and thioether functionalities at the purine 6 position have antiviral activity against herpes simplex virus (HSV) and varicella-zoster virus (VZV) in addition to HCMV. The role of the HCMV UL97 kinase in the mechanism of action of these derivatives was examined. When tested against a kinase-inactive UL97 K355M virus, a moderate 5- to 7-fold increase in 50% effective concentration (EC50) was observed, in comparison to a 13- to 25-fold increase for either cyclopropavir or ganciclovir. Serial propagation of HCMV under two of these compounds selected for three novel UL97 mutations encoding amino acid substitutions D456N, C480R,and Y617del. When transferred to baseline laboratory HCMV strains, these mutations individually conferred resistance to all of the tested MCPNs, ganciclovir, and maribavir. However, the engineered strains also demonstrated severe growth defects and abnormal cytopathic effects similar to the kinase-inactive mutant. Expressed and purified UL97 kinase showed in vitro phosphorylation of the newly tested MCPNs. Thus, HCMV UL97 kinase is involved in the antiviral action of these MCPNs, but the in vitro selection of UL97-defective viruses suggests that their activity against more typical ganciclovir-resistant growth-competent UL97 mutants may be relatively preserved.

Dihydroxymethyl and monohydroxymethyl methylenecyclopropane nucleosides are effective inhibitors of both variants of human herpesvirus 6 (HHV-6). We investigated involvement of HHV-6 U69 protein kinase in their mechanism of action. Phosphorylation of the dihydroxymethyl analogue cyclopropavir and monohydroxymethyl nucleosides with either a 6-ether moiety (MBX 2168) or a 6-thioether moiety (MBX 1616) with purified U69 was examined. All three compounds were substrates of this viral kinase and had similar Michaelis-Menten kinetic parameters.

Methylenecyclopropane nucleosides have been reported to be active against many of the human herpesviruses. The most active compound of this class is cyclopropavir (CPV), which exhibits good antiviral activity against human cytomegalovirus (HCMV), Epstein-Barr virus, both variants of human herpesvirus 6, and human herpesvirus 8. CPV has two hydroxymethyl groups on the methylenecyclopropane ring, but analogs with a single hydroxymethyl group, such as the prototypical (S)-synguanol, are also active and exhibit a broader spectrum of antiviral activity that also includes hepatitis B virus and human immunodeficiency virus. Here, a large set of monohydroxymethyl compounds with ether and thioether substituents at the 6 position of the purine was synthesized and evaluated for antiviral activity against a range of human herpesviruses. Some of these analogs had a broader spectrum of antiviral activity than CPV, in that they also inhibited the replication of herpes simplex viruses 1 and 2 and varicella-zoster virus. Interestingly, the antiviral activity of these compounds appeared to be dependent on the activity of the HCMV UL97 kinase but was relatively unaffected by the absence of thymidine kinase activity in HSV. These data taken together indicate that the mechanism of action of these analogs is distinct from that of CPV. They also suggest that they might be useful as broad-spectrum antiherpesvirus agents and may be effective in the treatment of resistant virus infections.

CMX001 is an orally available lipid acyclic nucleotide phosphonate that delivers high intracellular levels of cidofovir (CDV)-diphosphate and exhibits enhanced in vitro antiviral activity against a wide range of double-stranded DNA viruses, including cytomegalovirus (CMV). Mutations in the DNA polymerase of CMV that impart resistance to CDV also render the virus resistant to CMX001. Here, we report a novel resistance mutation that arose under the selective pressure of CMX001. The wild-type CMV strain AD169 was propagated in human foreskin fibroblasts under increasing concentrations of CMX001 over 10 months, and the resulting strain (named CMX001(R)) was less susceptible to CDV and CMX001 in a plaque reduction assay. Genotypic analysis of virus strain CMX001(R) via conventional sequencing of the genes encoding the CMV DNA polymerase (UL54) and UL97 kinase (UL97) demonstrated one mutation that changed the wild-type aspartate to glutamate at position 542 in UL54. A recombinant virus with this novel D542E mutation was generated via bacterial artificial chromosome-mediated marker transfer experiments. Subsequent phenotypic resistance analysis of the D542E mutant demonstrated reductions in susceptibility of greater than 10-fold to CMX001 and CDV, but no resistance to foscarnet (FOS) or ganciclovir (GCV). Analysis of replicative fitness showed that both strain CMX001(R) and the D542E mutant viruses demonstrated a smaller plaque phenotype and slower replication kinetics than their respective parent viruses. These data describe the first resistance mutation generated under the selective pressure of CMX001 and suggest that CMX001 may have a unique resistance profile associated with reduced viral replication and maintenance of sensitivity to FOS and GCV.

Background: Children <2 years of age are at high risk of influenza-related mortality and morbidity. However, the appropriate dose of oseltamivir for children <2 years of age is unknown., Methods: The National Institute of Allergy and Infectious Diseases Collaborative Antiviral Study Group evaluated oseltamivir in infants aged <2 years in an age-de-escalation, adaptive design with a targeted systemic exposure., Results: From 2006 to 2010, 87 subjects enrolled. An oseltamivir dose of 3.0 mg/kg produced drug exposures within the target range in subjects 0-8 months of age, although there was a greater degree of variability in infants <3 months of age. In subjects 9-11 months of age, a dose of 3.5 mg/kg produced drug exposures within the target range. Six of 10 subjects aged 12-23 months receiving the Food and Drug Administration-approved unit dose for this age group (ie, 30 mg) had oseltamivir carboxylate exposures below the target range. Virus from 3 subjects developed oseltamivir resistance during antiviral treatment., Conclusions: The appropriate twice-daily oral oseltamivir dose for infants ≤8 months of age is 3.0 mg/kg, while the dose for infants 9-11 months old is 3.5 mg/kg.

The UL97 protein kinase is a serine/threonine kinase expressed by human cytomegalovirus (CMV) that phosphorylates ganciclovir. An investigation of the subcellular localization of pUL97 in infected cells indicated that, early in infection, pUL97 localized to focal sites in the nucleus that transitioned to subnuclear compartments and eventually throughout the entire nucleus. When UL97 kinase activity was eliminated with a K355M mutation or pharmacologically inhibited with maribavir, the expansion and redistribution of pUL97 foci within the nucleus was delayed, nuclear reorganization did not occur and assembly complexes in the cytoplasm failed to form normally. As UL97 kinase and its homologues appear to be functionally related to CDK1, a known regulator of nuclear structural organization, the effects of the UL97 kinase on CDK1 were investigated. Expression of CDK1 in infected cells appeared to be induced by UL97 kinase activity at the level of transcription and was not tied to other virus life-cycle events, such as viral DNA replication or virion assembly. These results suggest that, in addition to phosphorylating CDK1 targets, the UL97 kinase modifies G₂/M cell-cycle checkpoint regulators, specifically CDK1, to promote virus replication.

A second-generation series of substituted methylenecyclopropane nucleosides (MCPNs) has been synthesized and evaluated for antiviral activity against a panel of human herpesviruses, and for cytotoxicity. Although alkylated 2,6-diaminopurine analogs showed little antiviral activity, the compounds containing ether and thioether substituents at the 6-position of the purine did demonstrate potent and selective antiviral activity against several different human herpesviruses. In the 6-alkoxy series, antiviral activity depended on the length of the ether carbon chain, with the optimum chain length being about four carbon units long. For the corresponding thioethers, compounds containing secondary thioethers were more potent than those with primary thioethers., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Investments in the development of new drugs for orthopoxvirus infections have fostered new avenues of research, provided an improved understanding of orthopoxvirus biology and yielded new therapies that are currently progressing through clinical trials. These broad-based efforts have also resulted in the identification of new inhibitors of orthopoxvirus replication that target many different stages of viral replication cycle. This review will discuss progress in the development of new anti-poxvirus drugs and the identification of new molecular targets that can be exploited for the development of new inhibitors. The prototype of the orthopoxvirus group is vaccinia virus and its replication cycle will be discussed in detail noting specific viral functions and their associated gene products that have the potential to serve as new targets for drug development. Progress that has been achieved in recent years should yield new drugs for the treatment of these infections and might also reveal new approaches for antiviral drug development with other viruses., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Synthesis of 6-deoxycyclopropavir (10), a prodrug of cyclopropavir (1) and its in vitro and in vivo antiviral activity is described. 2-Amino-6-chloropurine methylenecyclopropane 13 was transformed to its 6-iodo derivative 14 which was reduced to prodrug 10. It is converted to cyclopropavir (1) by the action of xanthine oxidase and this reaction can also occur in vivo. Compound 10 lacked significant in vitro activity against human cytomegalovirus (HCMV), human herpes virus 1 and 2 (HSV-1 and HSV-2), human immunodeficiency virus type 1 (HIV-1), human hepatitis B virus (HBV), Epstein-Barr virus (EBV), vaccinia virus and cowpox virus. In contrast, prodrug 10 given orally was as active as cyclopropavir (1) reported previously [Kern, E. R.; Bidanset, D. J.; Hartline, C. B.; Yan, Z.; Zemlicka, J.; Quenelle, D. C. et al. Antimicrob. Agents Chemother. 2004, 48, 4745] against murine cytomegalovirus (MCMV) infection in mice and against HCMV in severe combined immunodeficient (SCID) mice., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Subtilosin is a cyclical antimicrobial peptide produced by Bacillus amyloliquefaciens that has antimicrobial activity against the bacterial vaginosis-associated human pathogen Gardnerella vaginalis. The ability of subtilosin to inhibit G. vaginalis alone and in combination with the natural antimicrobial agents glycerol monolaurate (Lauricidin), lauric arginate, and ε-poly-L-lysine was tested using a checkerboard approach. Subtilosin was found to act synergistically with all of the chosen antimicrobials. These promising results indicate that lower concentrations of subtilosin in combination with other compounds could effectively be used to inhibit growth of the pathogen, thereby decreasing the risk of developed antimicrobial resistance. This is the first report on the effects of subtilosin combined with other natural antimicrobials against G. vaginalis.

Resurgent interest in antiviral drugs for the treatment of herpesvirus has led to the development of new compounds that are progressing through clinical trials. This is important because there are few therapeutic options for resistant infections and some viruses such as human cytomegalovirus remain underserved. New compounds include conventional DNA polymerase inhibitors such as valomaciclovir and cyclopropavir, as well as CMX001 that has a broad spectrum of antiviral activity that includes all the herpesviruses. It also includes compounds with new molecular targets such as maribavir (MBV), FV-100, AIC361, and AIC246. Recent advances with each of these compounds will be reviewed including their virus specificity, mechanism of action, and stage of development. The potential of these new compounds to improve clinical outcome will also be discussed.

Poxvirus uracil DNA glycosylase D4 in association with A20 and the catalytic subunit of DNA polymerase forms the processive polymerase complex. The binding of D4 and A20 is essential for processive polymerase activity. Using an AlphaScreen assay, we identified compounds that inhibit protein-protein interactions between D4 and A20. Effective interaction inhibitors exhibited both antiviral activity and binding to D4. These results suggest that novel antiviral agents that target the protein-protein interactions between D4 and A20 can be developed for the treatment of infections with poxviruses, including smallpox.

Although acyclovir (ACV) has proven to be of value in the therapy of certain herpes simplex virus (HSV) infections, there is a need for more effective therapies, particularly for serious infections in neonates and immunocompromised individuals, where resistance to this drug can be problematic. CMX001 is an orally bioavailable lipid conjugate of cidofovir that is substantially less nephrotoxic than the parent drug and has excellent antiviral activity against all the human herpesviruses. This compound retains full antiviral activity against ACV-resistant laboratory and clinical isolates. The combined efficacy of CMX001 and ACV was evaluated in a new real-time PCR combination assay, which demonstrated that the combination synergistically inhibited the replication of HSV in cell culture. This was also confirmed in murine models of HSV infection, where the combined therapy with these two drugs synergistically reduced mortality. These results suggest that CMX001 may be effective in the treatment of ACV-resistant HSV infections and as an adjunct therapy in individuals with suboptimal responses to ACV.

Cyclopropavir (CPV) is active against human cytomegalovirus (CMV), as well as both variants of human herpesvirus 6 and human herpesvirus 8. The mechanism of action of CPV against CMV is similar to that of ganciclovir (GCV) in that it is phosphorylated initially by the CMV UL97 kinase, resulting in inhibition of viral DNA synthesis. Resistance to CPV maps to the UL97 kinase but is associated primarily with H520Q mutations and thus retains good antiviral activity against most GCV-resistant isolates. An examination of CMV-infected cultures treated with CPV revealed unusual cell morphology typically associated with the absence of UL97 kinase activity. A surrogate assay for UL97 kinase activity confirmed that CPV inhibited the activity of this enzyme and that its action was similar to the inhibition seen with maribavir (MBV) in this assay. Combination studies using real-time PCR indicated that, like MBV, CPV also antagonized the efficacy of GCV and were consistent with the observed inhibition of the UL97 kinase. Deep sequencing of CPV-resistant laboratory isolates identified a frameshift mutation in UL27, presumably to compensate for a loss of UL97 enzymatic activity. We conclude that the mechanism of action of CPV against CMV is complex and involves both the inhibition of DNA synthesis and the inhibition of the normal activity of the UL97 kinase.

Infections due to resistant human cytomegalovirus (CMV) are an emerging problem, particularly in immunocompromised hosts. When managing such patients, clinicians should be aware of the possibility of developing CMV antiviral resistance, especially while on prolonged therapy or if severe immunosuppression is present. CMV resistance to current antiviral agents is mediated by alterations in either the UL97 kinase or DNA polymerase, encoded by the UL97 and UL54 genes, respectively. UL97 mutations are capable of conferring resistance to ganciclovir, while UL54 mutations can impart resistance to ganciclovir, cidofovir, and foscarnet. If treatment failure is suspected to be due to antiviral resistance, CMV resistance analysis should be obtained. Phenotypic resistance assays performed on clinical isolates measure antiviral susceptibilities directly, but are laborious and time-consuming. Therefore, genotypic resistance analysis has become the more common means of diagnosing CMV resistance. Mutations in UL97 or UL54 may be clinically associated with resistance, but their effect on antiviral susceptibility must be confirmed by marker transfer techniques such as recombinant phenotyping.

We reported previously that octadecyloxyethyl 9-(S)-[3-hydroxy-2-(phosphonomethoxy)-propyl]adenine (ODE-(S)-HPMPA) was active against genotype 1b and 2a hepatitis C virus (HCV) replicons. This is surprising because acyclic nucleoside phosphonates have been regarded as having antiviral activity only against double stranded DNA viruses, HIV and HBV. We synthesized octadecyloxyethyl 9-(S)-[3-methoxy-2-(phosphonomethoxy)propyl]-adenine and found it to be active in genotype 1b and 2a HCV replicons with EC₅₀ values of 1-2 μM and a CC₅₀ of > 150 μM. Analogs with substitutions at the 3'-hydroxyl larger than methyl or ethyl, or with other purine bases were less active but most compounds had significant antiviral activity against HIV-1 in vitro. The most active anti-HIV compound was octadecyloxyethyl 9-(R)-[3-methoxy-2-(phosphonomethoxy)propyl]guanine with an EC₅₀ < 0.01 nanomolar and a selectivity index of > 4.4 million., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Several benzimidazole nucleoside analogs, including 1H-β-D-ribofuranosyl-2-bromo-5,6-dichlorobenzimidazole (BDCRB) and 1H-β-L-ribofuranosyl-2-isopropylamino-5,6-dichlorobenzimidazole (maribavir [MBV]), inhibit the replication of human cytomegalovirus. Neither analog inhibited the related betaherpesvirus human herpesvirus 6 (HHV-6). Additional analogs of these compounds were evaluated against both variants of HHV-6, and two L-analogs of BDCRB had good antiviral activity against HHV-6A, as well as more modest inhibition of HHV-6B replication.

Alkoxyalkyl esters of cidofovir (CDV) are orally active agents which inhibit the replication of a variety of double stranded DNA (dsDNA) viruses including variola, vaccinia, ectromelia, herpes simplex virus, cytomegalovirus, adenovirus and others. One of these compounds, hexadecyloxypropyl-CDV (HDP-CDV, CMX001) is in clinical development for prevention and treatment of poxvirus infection, vaccination complications, and for infections caused by cytomegalovirus, adenovirus, herpesviruses and other dsDNA viruses. This class of lipid analogs is potentially prone to undergo omega oxidation of the alkyl moiety which can lead to a short chain carboxylic acid lacking antiviral activity. To address this issue, we synthesized a series of alkoxyalkyl or alkyl glycerol esters of CDV and (S)-HPMPA having modifications in the structure of the alkyl residue. Antiviral activity was assessed in cells infected with vaccinia, cowpox or ectromelia viruses. Metabolic stability was determined in S9 membrane fractions from rat, guinea pig, monkey and human liver. All compounds had substantial antiviral activity in cells infected with vaccinia, cowpox or ectromelia. Metabolic stability was lowest in monkey liver S9 incubations where rapid disappearance of HDP-CDV and HDP-(S)-HPMPA was noted. Metabolic stability in monkey preparations increased substantially when a ω-1 methyl group (15-methyl-HDP-CDV) or a terminal cyclopropyl residue (14-cyclopropyl-tetradecyloxypropyl-CDV) was present in the alkyl chain. The most stable compound was 1-O-octadecyl-2-O-benzyl-sn-glycero-3-CDV (ODBG-CDV) which was not metabolized extensively by monkey liver S9. In rat, guinea pig or human liver S9 incubations, most of the modified antiviral compounds were considerably more stable., (Published by Elsevier Ltd.)