RESUMEN
Infection by human papillomaviruses (HPV) can cause warts and tumors. So far, no small molecule antiviral has been approved for the treatment of infections with this DNA virus, although preclinical studies show activity for nucleosidic compounds, such as 9-(2-phosphonylmethoxy)ethylguanine (PMEG) or cidofovir. This prompted us to test new prodrug versions of the nucleoside analog 3'-azido-2',3'-dideoxythymidine (AZT), known to be active against reverse transcriptases and approved for the treatment of HIV. Here we report the synthesis of an ethylbutyl alaninyl ester phosphosphoramidate prodrug of AZT, dubbed AZAEB, and its activity against HPV, a target not known to be sensitive to AZT. A methyl ester derivative was found to be inactive against this and three other DNA viruses, while the phosphoramidate prodrug AZAEB showed a modest inhibitory effect against HPV types 6, 11, 18 and 31. Our results open up new avenues of study for the treatment of diseases caused by members of the papillomaviridae family.
Asunto(s)
Infecciones por Papillomavirus , Profármacos , Humanos , Zidovudina/farmacología , ProTides , Virus del Papiloma Humano , Nucleósidos , Profármacos/farmacología , Ésteres , Antivirales/farmacologíaRESUMEN
Varicella zoster virus (VZV) infection causes severe disease such as chickenpox, shingles, and postherpetic neuralgia, often leading to disability. Reactivation of latent VZV is associated with a decrease in specific cellular immunity in the elderly and in patients with immunodeficiency. However, due to the limited efficacy of existing therapy and the emergence of antiviral resistance, it has become necessary to develop new and effective antiviral drugs for the treatment of diseases caused by VZV, particularly in the setting of opportunistic infections. The goal of this work is to identify potent oxazole derivatives as anti-VZV agents by machine learning, followed by their synthesis and experimental validation. Predictive QSAR models were developed using the Online Chemical Modeling Environment (OCHEM). Data on compounds exhibiting antiviral activity were collected from the ChEMBL and uploaded in the OCHEM database. The predictive ability of the models was tested by cross-validation, giving coefficient of determination q2 = 0.87-0.9. The validation of the models using an external test set proves that the models can be used to predict the antiviral activity of newly designed and known compounds with reasonable accuracy within the applicability domain (q2 = 0.83-0.84). The models were applied to screen a virtual chemical library with expected activity of compounds against VZV. The 7 most promising oxazole derivatives were identified, synthesized, and tested. Two of them showed activity against the VZV Ellen strain upon primary in vitro antiviral screening. The synthesized compounds may represent an interesting starting point for further development of the oxazole derivatives against VZV. The developed models are available online at OCHEM http://ochem.eu/article/145978 and can be used to virtually screen for potential compounds with anti-VZV activity.
RESUMEN
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 ß-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 EC50 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.
Asunto(s)
Infecciones por Citomegalovirus , Organofosfonatos , Profármacos , Ratones , Humanos , Animales , Antivirales/farmacocinética , Disponibilidad Biológica , Profármacos/farmacología , Citosina , CidofovirRESUMEN
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.
Asunto(s)
Adenoviridae/efectos de los fármacos , Antivirales/farmacología , Herpesvirus Humano 1/efectos de los fármacos , Orthopoxvirus/efectos de los fármacos , Ensayo de Placa Viral/normas , Células Cultivadas , Citomegalovirus/efectos de los fármacos , Efecto Citopatogénico Viral , Infecciones por Virus ADN/tratamiento farmacológico , Fibroblastos , Herpesvirus Humano 2/efectos de los fármacos , Herpesvirus Humano 3/efectos de los fármacos , HumanosRESUMEN
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.
Asunto(s)
Antivirales/farmacología , Descubrimiento de Drogas/normas , Herpesviridae/efectos de los fármacos , Nucleósidos/farmacología , Poliomavirus/efectos de los fármacos , Automatización de Laboratorios , ADN Viral/análisis , Descubrimiento de Drogas/métodos , Humanos , Replicación Viral/efectos de los fármacosRESUMEN
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.
Asunto(s)
Antivirales/farmacología , ADN Viral/efectos de los fármacos , Guanina/análogos & derivados , Técnicas de Amplificación de Ácido Nucleico , Organofosfonatos/farmacología , Papillomaviridae/efectos de los fármacos , Antivirales/síntesis química , Antivirales/química , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Guanina/síntesis química , Guanina/química , Guanina/farmacología , Células HEK293 , VIH/efectos de los fármacos , Herpesvirus Humano 2/efectos de los fármacos , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/virología , Estructura Molecular , Organofosfonatos/síntesis química , Organofosfonatos/química , Papillomaviridae/genética , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacosRESUMEN
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.
Asunto(s)
Antivirales/farmacología , Hemaglutininas Virales/metabolismo , Virus de la Influenza A/efectos de los fármacos , Gripe Aviar/virología , Gripe Humana/virología , Enfermedades de las Aves de Corral/virología , Bibliotecas de Moléculas Pequeñas/farmacología , Internalización del Virus/efectos de los fármacos , Animales , Antivirales/química , Línea Celular , Pollos , Hemaglutininas Virales/genética , Humanos , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/fisiología , Subtipo H3N2 del Virus de la Influenza A/efectos de los fármacos , Subtipo H3N2 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/fisiología , Subtipo H5N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H5N1 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/fisiología , Virus de la Influenza A/genética , Virus de la Influenza A/fisiología , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
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.
Asunto(s)
Antivirales/síntesis química , Antivirales/farmacología , Ciclopropanos/farmacología , Fibroblastos/efectos de los fármacos , Herpesviridae/efectos de los fármacos , Nucleósidos/farmacología , Antivirales/química , Línea Celular , Ciclopropanos/síntesis química , Ciclopropanos/química , Relación Dosis-Respuesta a Droga , Fibroblastos/virología , Herpesviridae/aislamiento & purificación , Humanos , Pruebas de Sensibilidad Microbiana , Conformación Molecular , Nucleósidos/síntesis química , Nucleósidos/química , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Antivirales/farmacología , Virus Vaccinia/efectos de los fármacos , Proteínas Virales/metabolismo , Línea Celular , ADN Glicosilasas/metabolismo , Humanos , Unión ProteicaRESUMEN
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.
Asunto(s)
Adenina/análogos & derivados , Antivirales/síntesis química , Citosina/análogos & derivados , Organofosfonatos/química , Adenina/síntesis química , Adenina/química , Adenina/farmacología , Animales , Antivirales/química , Antivirales/farmacología , Cidofovir , Virus de la Viruela Vacuna/efectos de los fármacos , Citosina/síntesis química , Citosina/química , Citosina/farmacología , Virus de la Ectromelia/efectos de los fármacos , Ésteres , Cobayas , Haplorrinos , Humanos , Hígado/metabolismo , Organofosfonatos/síntesis química , Organofosfonatos/farmacología , Ratas , Virus Vaccinia/efectos de los fármacosRESUMEN
Our previous studies showed that esterification of 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine (HPMPA) or 1-(S)-[3-hydroxy-2-(phosphonomethoxy)-propyl]cytosine (HPMPC) with alkoxyalkyl groups such as hexadecyloxypropyl (HDP) or octadecyloxyethyl (ODE) resulted in large increases in antiviral activity and oral bioavailability. The HDP and ODE esters of HPMPA were shown to be active in cells infected with human immunodeficiency virus, type 1 (HIV-1), while HPMPA itself was virtually inactive. To explore this approach in greater detail, we synthesized four new compounds in this series, the ODE esters of 9-(S)-[3-hydroxy-2-(phosphonomethoxy)-propyl]guanine (HPMPG), 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]thymine (HPMPT), 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-2,6-diaminopurine (HPMPDAP) and 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-2-amino-6-cyclopropylaminopurine (HPMP-cPrDAP) and evaluated their antiviral activity against herpes simplex virus, type 1 (HSV-1), human cytomegalovirus (HCMV), and vaccinia, cowpox and ectromelia. Against HSV-1, subnanomolar EC(50) values were observed with ODE-HPMPA and ODE-HPMPC while ODE-HPMPG had intermediate antiviral activity with an EC(50) of 40 nM. In HFF cells infected with HCMV, the lowest EC(50) values were observed with ODE-HPMPC, 0.9 nM. ODE-HPMPA was highly active with an EC(50) of 3 nM, while ODE-HPMPG and ODE-HPMPDAP were also highly active with EC(50)s of 22 and 77 nM, respectively. Against vaccinia and cowpox viruses, ODE-HPMPG and ODE-HPMPDAP were the most active and selective compounds with EC(50) values of 20-60 nM and selectivity index values of 600-3500. ODE-HPMPG was also active against ectromelia virus with an EC(50) value of 410 nM and a selectivity index value of 166. ODE-HPMPG and ODE-HPMPDAP are proposed for further preclinical evaluation as possible candidates for treatment of HSV, HCMV or orthopoxvirus diseases.
Asunto(s)
Antivirales/farmacología , Herpesvirus Humano 1/efectos de los fármacos , Nucleósidos/farmacología , Compuestos Organofosforados/farmacología , Orthopoxvirus/efectos de los fármacos , Animales , Antivirales/síntesis química , Antivirales/química , Línea Celular , Humanos , Pruebas de Sensibilidad Microbiana , Nucleósidos/síntesis química , Nucleósidos/química , Compuestos Organofosforados/síntesis química , Compuestos Organofosforados/químicaRESUMEN
As part of a program to identify new compounds that have activity against orthopoxviruses, a number of 4'-thionucleosides were synthesized and evaluated for their efficacies against vaccinia and cowpox viruses. Seven compounds that were active at about 1 microM against both viruses in human cells but that did not have significant toxicity were identified. The 5-iodo analog, 1-(2-deoxy-4-thio-beta-d-ribofuranosyl)-5-iodouracil (4'-thioIDU), was selected as a representative molecule; and this compound also inhibited viral DNA synthesis at less than 1 microM but only partially inhibited the replication of a recombinant vaccinia virus that lacked a thymidine kinase. This compound retained complete activity against cidofovir- and ST-246-resistant mutants. To determine if this analog had activity in an animal model, mice were infected intranasally with vaccinia or cowpox virus and treatment with 4'-thioIDU was given intraperitoneally or orally twice daily at 50, 15, 5, or 1.5 mg/kg of body weight beginning at 24 to 120 h postinfection and was continued for 5 days. Almost complete protection (87%) was observed when treatment with 1.5 mg/kg was begun at 72 h postinfection, and significant protection (73%) was still obtained when treatment with 5 mg/kg was initiated at 96 h. Virus titers in the liver, spleen, and kidney were reduced by about 4 log(10) units and about 2 log(10) units in mice infected with vaccinia virus and cowpox virus, respectively. These results indicate that 4'-thioIDU is a potent, nontoxic inhibitor of orthopoxvirus replication in cell culture and experimental animal infections and suggest that it may have potential for use in the treatment of orthopoxvirus infections in animals and humans.
Asunto(s)
Antivirales/uso terapéutico , Nucleósidos/uso terapéutico , Orthopoxvirus , Infecciones por Poxviridae/tratamiento farmacológico , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Viruela Vacuna/tratamiento farmacológico , Efecto Citopatogénico Viral/efectos de los fármacos , ADN Viral/biosíntesis , ADN Viral/genética , Farmacorresistencia Viral , Humanos , Ratones , Ratones Endogámicos BALB C , Infecciones por Poxviridae/virología , Relación Estructura-Actividad , Vaccinia/tratamiento farmacológico , beta-Galactosidasa/metabolismoRESUMEN
BACKGROUND: The emergence of drug resistant viruses, together with the possibility of increased virulence, is an important concern in the development of new antiviral compounds. Cidofovir (CDV) is a phosphonate nucleotide that is approved for use against cytomegalovirus retinitis and for the emergency treatment of smallpox or complications following vaccination. One mode of action for CDV has been demonstrated to be the inhibition of the viral DNA polymerase. RESULTS: We have isolated several CDV resistant (CDVR) vaccinia viruses through a one step process, two of which have unique single mutations within the DNA polymerase. An additional resistant virus isolate provides evidence of a second site mutation within the genome involved in CDV resistance. The CDVR viruses were 3-7 fold more resistant to the drug than the parental viruses. The virulence of the CDVR viruses was tested in mice inoculated intranasally and all were found to be attenuated. CONCLUSION: Resistance to CDV in vaccinia virus can be conferred individually by at least two different mutations within the DNA polymerase gene. Additional genes may be involved. This one step approach for isolating resistant viruses without serial passage and in the presence of low doses of drug minimizes unintended secondary mutations and is applicable to other potential antiviral agents.
Asunto(s)
Citosina/análogos & derivados , Farmacorresistencia Viral , Organofosfonatos/farmacología , Virus Vaccinia/efectos de los fármacos , Virus Vaccinia/aislamiento & purificación , Animales , Antivirales/farmacología , Línea Celular , Chlorocebus aethiops , Cidofovir , Citosina/farmacología , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Femenino , Humanos , Ratones , Modelos Moleculares , Mutación , Vaccinia/mortalidad , Vaccinia/virología , Virus Vaccinia/patogenicidad , Virus Vaccinia/fisiología , Células Vero , Ensayo de Placa Viral , VirulenciaRESUMEN
BACKGROUND: The vaccinia virus (VV) F2L gene encodes a functional deoxyuridine triphosphatase (dUTPase) that catalyzes the conversion of dUTP to dUMP and is thought to minimize the incorporation of deoxyuridine residues into the viral genome. Previous studies with with a complex, multigene deletion in this virus suggested that the gene was not required for viral replication, but the impact of deleting this gene alone has not been determined in vitro or in vivo. Although the crystal structure for this enzyme has been determined, its potential as a target for antiviral therapy is unclear. RESULTS: The F2L gene was replaced with GFP in the WR strain of VV to assess its effect on viral replication. The resulting virus replicated well in cell culture and its replication kinetics were almost indistinguishable from those of the wt virus and attained similar titers. The virus also appeared to be as pathogenic as the WR strain suggesting that it also replicated well in mice. Cells infected with the dUTPase mutant would be predicted to affect pyrimidine deoxynucleotide pools and might be expected to exhibit altered susceptibility to pyrimidine analogs. The antiviral activity of cidofovir and four thymidine analogs were evaluated both in the mutant and the parent strain of this virus. The dUTPase knockout remained fully susceptible to cidofovir and idoxuridine, but was hypersensitive to the drug (N)-methanocarbathymidine, suggesting that pyrimidine metabolism was altered in cells infected with the mutant virus. The absence of dUTPase should reduce cellular dUMP pools and may result in a reduced conversion to dTMP by thymidylate synthetase or an increased reliance on the salvage of thymidine by the viral thymidine kinase. CONCLUSION: We confirmed that F2L was not required for replication in cell culture and determined that it does not play a significant role on virulence of the virus in intranasally infected mice. The recombinant virus is hypersensitive to (N)-methanocarbathymidine and may reflect metabolic differences in the mutant virus.
Asunto(s)
Antivirales/farmacología , Pirofosfatasas/metabolismo , Timidina/análogos & derivados , Virus Vaccinia/fisiología , Vaccinia/virología , Replicación Viral/efectos de los fármacos , Animales , Femenino , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Ratones , Ratones Endogámicos BALB C , Pirimidinas/metabolismo , Pirofosfatasas/genética , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , Timidina/farmacología , Virus Vaccinia/efectos de los fármacos , Virus Vaccinia/enzimología , Virus Vaccinia/patogenicidad , Ensayo de Placa Viral , VirulenciaRESUMEN
We have previously reported that (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine, or (S)-HPMPA, is active in vitro against cowpox virus (CV) and vaccinia virus (VV) but is not active orally in animals. However, the ether lipid esters of (S)-HPMPA, hexadecyloxypropyl-[(S)-HPMPA] [HDP-(S)-HPMPA] and octadecyloxyethyl-[(S)-HPMPA] [ODE-(S)-HPMPA], had significantly enhanced activity in vitro and are orally bioavailable in mice. In the current study, HDP-(S)-HPMPA and ODE-(S)-HPMPA were prepared in water and administered once daily by oral gavage to mice at doses of 30, 10, and 3 mg/kg of body weight for 5 days beginning 24, 48, or 72 h after inoculation with CV or VV. Oral HDP-(S)-HPMPA and ODE-(S)-HPMPA were both highly effective (P < 0.001) at preventing mortality due to CV at 30 mg/kg, even when treatments were delayed until up to 72 h postinfection. ODE-(S)-HPMPA or HDP-(S)-HPMPA were also highly effective (P < 0.001) at preventing mortality in mice infected with VV at 30 mg/kg when treatments were delayed until to 48 or 72 h postinfection, respectively. Protection against both viruses was associated with a significant reduction of virus replication in the liver, spleen, and kidney but not in the lung. These data indicate that HDP-(S)-HPMPA and ODE-(S)-HPMPA are active when given orally against lethal CV and VV infections in mice, and further evaluation is warranted to provide additional information on the potential of these orally active compounds for treatment of human orthopoxvirus infection.
Asunto(s)
Adenina/análogos & derivados , Antivirales/uso terapéutico , Viruela Vacuna/tratamiento farmacológico , Organofosfonatos/uso terapéutico , Virus Vaccinia/efectos de los fármacos , Adenina/química , Adenina/farmacocinética , Adenina/uso terapéutico , Administración Oral , Animales , Antivirales/administración & dosificación , Antivirales/farmacocinética , Viruela Vacuna/virología , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/virología , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/virología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/virología , Ratones , Estructura Molecular , Organofosfonatos/química , Organofosfonatos/farmacocinética , Bazo/efectos de los fármacos , Bazo/metabolismo , Bazo/virologíaRESUMEN
The combination of ST-246 and hexadecyloxypropyl-cidofovir or CMX001 was evaluated for synergistic activity in vitro against vaccinia virus and cowpox virus (CV) and in vivo against CV. In cell culture the combination was highly synergistic against both viruses, and the results suggested that combined treatment with these agents might offer superior efficacy in vivo. For animal models, ST-246 was administered orally with or without CMX001 to mice lethally infected with CV. Treatments began 1, 3, or 6 days postinfection using lower dosages than previously used for single-drug treatment. ST-246 was given at 10, 3, or 1 mg/kg of body weight with or without CMX001 at 3, 1, or 0.3 mg/kg to evaluate potential synergistic interactions. Treatment beginning 6 days post-viral inoculation with ST-246 alone only increased the mean day to death at 10 or 3 mg/kg but had no effect on survival. CMX001 alone also had no effect on survival. When the combination of the two drugs was begun 6 days after viral infection using various dosages of the two, a synergistic reduction in mortality was observed. No evidence of increased toxicity was noted with the combination either in vitro or in vivo. These results indicate that combinations of ST-246 and CMX001 are synergistic both in vitro and in vivo and suggest that combination therapy using ST-246 and CMX001 for treatment of orthopoxvirus disease in humans or animals may provide an additional benefit over the use of the two drugs by themselves.
Asunto(s)
Benzamidas/farmacología , Citosina/análogos & derivados , Isoindoles/farmacología , Organofosfonatos/farmacología , Orthopoxvirus/efectos de los fármacos , Administración Oral , Animales , Antivirales/farmacología , Antivirales/uso terapéutico , Benzamidas/administración & dosificación , Benzamidas/uso terapéutico , Células Cultivadas , Chlorocebus aethiops , Virus de la Viruela Vacuna/efectos de los fármacos , Citosina/farmacología , Citosina/uso terapéutico , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Quimioterapia Combinada , Femenino , Humanos , Isoindoles/administración & dosificación , Isoindoles/uso terapéutico , Masculino , Ratones , Ratones Endogámicos BALB C , Organofosfonatos/uso terapéutico , Infecciones por Poxviridae/tratamiento farmacológico , Infecciones por Poxviridae/virología , Resultado del Tratamiento , Virus Vaccinia/efectos de los fármacos , Células Vero , Replicación Viral/efectos de los fármacosRESUMEN
A series of novel, potent orthopoxvirus egress inhibitors was identified during high-throughput screening of the ViroPharma small molecule collection. Using structure--activity relationship information inferred from early hits, several compounds were synthesized, and compound 14 was identified as a potent, orally bioavailable first-in-class inhibitor of orthopoxvirus egress from infected cells. Compound 14 has shown comparable efficaciousness in three murine orthopoxvirus models and has entered Phase I clinical trials.
Asunto(s)
Antivirales/síntesis química , Benzamidas/síntesis química , Indoles/síntesis química , Orthopoxvirus/efectos de los fármacos , Administración Oral , Animales , Antivirales/farmacocinética , Antivirales/farmacología , Benzamidas/farmacocinética , Benzamidas/farmacología , Disponibilidad Biológica , Línea Celular , Cristalografía por Rayos X , Humanos , Técnicas In Vitro , Indoles/farmacocinética , Indoles/farmacología , Isoindoles , Macaca fascicularis , Ratones , Estructura Molecular , Orthopoxvirus/fisiología , Ratas , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Esterification of cidofovir (CDV), an antiviral nucleoside phosphonate, with alkyl or alkoxyalkyl groups increases antiviral activity by enhancing cell uptake and conversion to CDV diphosphate. Hexadecyloxypropyl-CDV (HDP-CDV) has been shown to be 40-100 times more active than CDV in vitro in cells infected with herpes group viruses, variola, cowpox, vaccinia or ectromelia viruses. Since the first phosphorylation of CDV may be rate limiting, we synthesized the hexadecyloxypropyl-phosphate (HDP-P-) and octadecyloxyethyl-phosphate (ODE-P-) conjugates of CDV and phosphonomethoxy-ethyl-adenine (PMEA, adefovir). We tested the CDV analogs in cells infected with human cytomegalovirus, herpes simplex virus, cowpox virus and vaccinia virus; the analogs of PMEA were tested in cells infected with the human immunodeficiency virus, type 1. In general, the alkoxyalkyl-phosphate conjugates of CDV were substantially more active than CDV. HDP-P-CDV and ODE-P-CDV were 4.6-40 times more active against HCMV and 7-30 times more active against cowpox and vaccinia in vitro. Although the compounds of this type were more cytotoxic than the unmodified bases, their selectivity for virally infected cells was generally greater than the parent nucleotides except that HDP-P-PMEA showed little or no selectivity in HIV-1 infected MT-2 cells. Although the new compounds with an interposed phosphate were generally less active than the corresponding alkoxyalkyl esters of CDV and PMEA, the present approach provides a possible alternative method for enhancing the antiviral activity of drugs of this class.
Asunto(s)
Adenina/análogos & derivados , Antivirales/toxicidad , Citosina/análogos & derivados , Organofosfonatos/síntesis química , Organofosfonatos/toxicidad , Adenina/síntesis química , Adenina/química , Adenina/toxicidad , Línea Celular , Cidofovir , Viruela Vacuna/tratamiento farmacológico , Infecciones por Citomegalovirus/tratamiento farmacológico , Citosina/síntesis química , Citosina/química , Citosina/toxicidad , VIH-1/efectos de los fármacos , Herpes Simple/tratamiento farmacológico , Humanos , Técnicas In Vitro , Estructura Molecular , Organofosfonatos/química , Sensibilidad y Especificidad , Vaccinia/tratamiento farmacológicoRESUMEN
The antiviral activity of a new series of thymidine analogs was determined against vaccinia virus (VV), cowpox virus (CV), herpes simplex virus, and varicella-zoster virus. Several compounds were identified that had good activity against each of the viruses, including a set of novel 5-substituted deoxyuridine analogs. To investigate the possibility that these drugs might be phosphorylated preferentially by the viral thymidine kinase (TK) homologs, the antiviral activities of these compounds were also assessed using TK-deficient strains of some of these viruses. Some of these compounds were shown to be much less effective in the absence of a functional TK gene in CV, which was unexpected given the high degree of amino acid identity between this enzyme and its cellular homolog. This unanticipated result suggested that the CV TK was important in the mechanism of action of these compounds and also that it might phosphorylate a wider variety of substrates than other type II enzymes. To confirm these data, we expressed the VV TK and human TK1 in bacteria and isolated the purified enzymes. Enzymatic assays demonstrated that the viral TK could efficiently phosphorylate many of these compounds, whereas most of the compounds were very poor substrates for the cellular kinase, TK1. Thus, the specific phosphorylation of these compounds by the viral kinase may be sufficient to explain the TK dependence. This unexpected result suggests that selective phosphorylation by the viral kinase may be a promising new approach in the discovery of highly selective inhibitors of orthopoxvirus replication.
Asunto(s)
Antivirales/metabolismo , Antivirales/farmacología , Timidina Quinasa/metabolismo , Virus Vaccinia/enzimología , Secuencia de Aminoácidos , Herpesviridae/efectos de los fármacos , Humanos , Cinética , Datos de Secuencia Molecular , Orthopoxvirus/efectos de los fármacos , Fosforilación , Timidina Quinasa/químicaRESUMEN
ST-246 was evaluated for activity against cowpox virus (CV), vaccinia virus (VV), and ectromelia virus (ECTV) and had an in vitro 50% effective concentration (EC50) of 0.48 microM against CV, 0.05 microM against VV, and 0.07 microM against ECTV. The selectivity indices were >208 and >2,000 for CV and VV, respectively. The in vitro antiviral activity of ST-246 was significantly greater than that of cidofovir, which had an EC50 of 41.1 microM against CV and 29.2 microM against VV, with selectivity indices of >7 and >10, respectively. ST-246 administered once daily by oral gavage to mice infected intranasally with CV beginning 4 h or delayed until 72 h postinoculation was highly effective when given for a 14-day duration using 100, 30, or 10 mg/kg of body weight. When 100 mg/kg of ST-246 was administered to VV-infected mice, a duration of 5 days was sufficient to significantly reduce mortality even when treatment was delayed 24 h postinoculation. Viral replication in liver, spleen, and kidney, but not lung, of CV- or VV-infected mice was reduced by ST-246 compared to levels for vehicle-treated mice. When 100 mg/kg of ST-246 was given once daily to mice infected by the intranasal route with ECTV, treatment for 10 days prevented mortality even when treatment was delayed up to 72 h after viral inoculation. Viral replication in target organs of ECTV-infected mice was also reduced.