RESUMEN
A key step in the replication of human cytomegalovirus (HCMV) in the host cell is the generation and packaging of unit-length genomes into preformed capsids. Enzymes required for this process are so-called terminases, first described for double-stranded DNA bacteriophages. The HCMV terminase consists of the two subunits, the ATPase pUL56 and the nuclease pUL89, and a potential third component pUL51. The terminase subunits are essential for virus replication and are highly conserved throughout the Herpesviridae family. Together with the portal protein pUL104 they form a powerful biological nanomotor. It has been shown for tailed dsDNA bacteriophages that DNA translocation into preformed capsid needs an extraordinary amount of energy. The HCMV terminase subunit pUL56 provides the required ATP hydrolyzing activity. The necessary nuclease activity to cleave the concatemers into unit-length genomes is mediated by the terminase subunit pUL89. Whether this cleavage is mediated by site-specific duplex nicking has not been demonstrated, however, it is required for packaging. Binding to the portal is a prerequisite for DNA translocation. To date, it is a common view that during translocation the terminase moves along some domains of the DNA by a binding and release mechanism. These critical structures have proven to be outstanding targets for drugs to treat HCMV infections because corresponding structures do not exist in mammalian cells. Herein we examine the HCMV terminase as a target for drugs and review several inhibitors discovered by both lead-directed medicinal chemistry and by target-specific design. In addition to producing clinically active compounds the research also has furthered the understanding of the role and function of the terminase itself.
Asunto(s)
Antivirales/farmacología , Infecciones por Citomegalovirus/prevención & control , Citomegalovirus/efectos de los fármacos , Citomegalovirus/enzimología , Endodesoxirribonucleasas/antagonistas & inhibidores , Acetatos/uso terapéutico , Animales , Ensayos Clínicos como Asunto , Citomegalovirus/genética , Infecciones por Citomegalovirus/tratamiento farmacológico , Humanos , Quinazolinas/uso terapéutico , Proteínas Virales/antagonistas & inhibidores , Replicación Viral/efectos de los fármacosRESUMEN
BACKGROUND: Benzimidazole D-ribonucleosides are potent and selective inhibitors of CMV infection that have been shown to target the viral terminase, the enzyme complex responsible for viral DNA cleavage into single unit-length genomes and subsequent DNA packaging into procapsids. Here, we evaluated the viral inhibition by benzimidazole D-ribonucleosides against rat cytomegalovirus (RCMV). METHODS: Antiviral activity of compounds Cl4RB and BTCRB against RCMV was quantified by measurement of plaque formation. Yield assays and electron microscopy of thin sections was performed using RCMV-infected cells in the presence or absence of the compounds. The effects of Cl4RB and BTCRB on cleavage of concatemers was analyzed by pulsed-field gel electrophoresis. To characterize the behaviour of the antiviral compounds in a more physiological environment, a 3D cell culture model was employed where cells are embedded in an extracellular matrix using rat-tail collagen I. RESULTS: Both compounds had an inhibitory effect against RCMV-E. Electron microscopy revealed that only few virions were formed in RCMV-E infected cells in the presence of the compounds. Pulsed-field gel electrophoresis showed that DNA concatemers failed to be processed in the presence of the compounds. Yield Assays showed a comparable viral growth in the 3D vs. 2D cell culture as well as inhibition in the presence of Cl4RB or BTCRB for RCMV-E/GFP. CONCLUSIONS: These results demonstrate that the tetrahalogenated benzimidazole D-ribonucleosides are effective against RCMV-E by preventing cleavage of concatemeric DNA and nuclear egress of mature capsids.
Asunto(s)
Antivirales/farmacología , Bencimidazoles/farmacología , Infecciones por Herpesviridae/tratamiento farmacológico , Muromegalovirus/efectos de los fármacos , Nucleósidos/farmacología , Ribonucleósidos/farmacología , Animales , Antivirales/química , Bencimidazoles/química , Técnicas de Cultivo de Célula , Colágeno/química , Empaquetamiento del ADN/efectos de los fármacos , Endodesoxirribonucleasas/efectos de los fármacos , Halogenación , Infecciones por Herpesviridae/virología , Microscopía Electrónica , Modelos Biológicos , Muromegalovirus/ultraestructura , Nucleósidos/química , Ratas , Ribonucleósidos/química , Andamios del Tejido , Ensayo de Placa ViralRESUMEN
Human cytomegalovirus (HCMV) infection can cause severe illnesses, including encephalopathy and mental retardation, in immunocompromised and immunologically immature patients. Current pharmacotherapies for treating systemic HCMV infections include ganciclovir, cidofovir, and foscarnet. However, long-term administration of these agents can result in serious adverse effects (myelosuppression and/or nephrotoxicity) and the development of viral strains with reduced susceptibility to drugs. The deoxyribosylindole (indole) nucleosides demonstrate a 20-fold greater activity in vitro (the drug concentration at which 50% of the number of plaques was reduced with the presence of drug compared to the number in the absence of drug [EC50] = 0.34 µM) than ganciclovir (EC50 = 7.4 µM) without any observed increase in cytotoxicity. Based on structural similarity to the benzimidazole nucleosides, we hypothesize that the indole nucleosides target the HCMV terminase, an enzyme responsible for packaging viral DNA into capsids and cleaving the DNA into genome-length units. To test this hypothesis, an indole nucleoside-resistant HCMV strain was isolated, the open reading frames of the genes that encode the viral terminase were sequenced, and a G766C mutation in exon 1 of UL89 was identified; this mutation resulted in an E256Q change in the amino acid sequence of the corresponding protein. An HCMV wild-type strain, engineered with this mutation to confirm resistance, demonstrated an 18-fold decrease in susceptibility to the indole nucleosides (EC50 = 3.1 ± 0.7 µM) compared to that of wild-type virus (EC50 = 0.17 ± 0.04 µM). Interestingly, this mutation did not confer resistance to the benzimidazole nucleosides (EC50 for wild-type HCMV = 0.25 ± 0.04 µM, EC50 for HCMV pUL89 E256Q = 0.23 ± 0.04 µM). We conclude, therefore, that the G766C mutation that results in the E256Q substitution is unique for indole nucleoside resistance and distinct from previously discovered substitutions that confer both indole and benzimidazole nucleoside resistance (D344E and A355T).
Asunto(s)
Bencimidazoles/farmacología , Citomegalovirus/efectos de los fármacos , Desoxirribonucleósidos/farmacología , Farmacorresistencia Viral/genética , Indoles/farmacología , Ribonucleósidos/farmacología , Proteínas Virales/genética , Secuencia de Aminoácidos , Antivirales/farmacología , Secuencia de Bases , Citomegalovirus/genética , Citomegalovirus/aislamiento & purificación , Datos de Secuencia Molecular , MutaciónRESUMEN
Human cytomegalovirus (HCMV) is a widespread pathogen that can cause severe disease in immunologically immature and immunocompromised patients. The current standard of therapy for the treatment of HCMV infections is ganciclovir (GCV). However, high incidence rates of adverse effects are prevalent and limit the use of this drug. Cyclopropavir (CPV) is 10-fold more effective against HCMV in vitro than GCV (50% effective concentrations [EC50s]=0.46 and 4.1 µM, respectively) without any observed increase in cytotoxicity (S. Zhou, J. M. Breitenbach, K. Z. Borysko, J. C. Drach, E. R. Kern, E. Gullen, Y. C. Cheng, and J. Zemlicka, J. Med. Chem. 47:566-575, 2004, doi:10.1021/jm030316s). We have previously determined that the viral protein kinase pUL97 and endogenous cellular kinases are responsible for the conversion of CPV to a triphosphate (TP), the active compound responsible for inhibiting viral DNA synthesis and viral replication. However, this conversion has not been observed in HCMV-infected cells. To that end, we subjected HCMV-infected cells to equivalently effective concentrations (â¼5 times the EC50) of either CPV or GCV and observed a time-dependent increase in triphosphate levels for both compounds (CPV-TP=121±11 pmol/10(6) cells; GCV-TP=43.7±0.4 pmol/10(6) cells). A longer half-life was observed for GCV-TP (48.2±5.7 h) than for CPV-TP (23.8±5.1 h). The area under the curve for CPV-TP produced from incubation with 2.5 µM CPV was 8,680±930 pmol·h/10(6) cells, approximately 2-fold greater than the area under the curve for GCV-TP of 4,520±420 pmol·h/10(6) cells produced from incubation with 25 µM GCV. We therefore conclude that the exposure of HCMV-infected cells to CPV-TP is greater than that of GCV-TP under these experimental conditions.
Asunto(s)
Ciclopropanos/metabolismo , Citomegalovirus/metabolismo , Ganciclovir/metabolismo , Guanina/análogos & derivados , Línea Celular , Ciclopropanos/farmacología , Citomegalovirus/efectos de los fármacos , Ganciclovir/farmacología , Guanina/metabolismo , Guanina/farmacología , Humanos , Estructura MolecularRESUMEN
Human cytomegalovirus (HCMV) is a widespread pathogen in the human population, affecting many immunologically immature and immunocompromised patients, and can result in severe complications, such as interstitial pneumonia and mental retardation. Current chemotherapies for the treatment of HCMV infections include ganciclovir (GCV), foscarnet, and cidofovir. However, the high incidences of adverse effects (neutropenia and nephrotoxicity) limit the use of these drugs. Cyclopropavir (CPV), a guanosine nucleoside analog, is 10-fold more active against HCMV than GCV (50% effective concentrations [EC50s] = 0.46 and 4.1 µM, respectively). We hypothesize that the mechanism of action of CPV is similar to that of GCV: phosphorylation to a monophosphate by viral pUL97 protein kinase with further phosphorylation to a triphosphate by endogenous kinases, resulting in inhibition of viral DNA synthesis. To test this hypothesis, we isolated a CPV-resistant virus, sequenced its genome, and discovered that bp 498 of UL97 was deleted. This mutation caused a frameshift in UL97 resulting in a truncated protein that lacks a kinase domain. To determine if this base pair deletion was responsible for drug resistance, the mutation was engineered into the wild-type viral genome, which was then exposed to increasing concentrations of CPV. The results demonstrate that the engineered virus was approximately 72-fold more resistant to CPV (EC50 = 25.8 ± 3.1 µM) than the wild-type virus (EC50 = 0.36 ± 0.11 µM). We conclude, therefore, that this mutation is sufficient for drug resistance and that pUL97 is involved in the mechanism of action of CPV.
Asunto(s)
Citomegalovirus/genética , Mutación del Sistema de Lectura , Sistemas de Lectura Abierta , Proteínas Quinasas/genética , Proteínas Virales/genética , Antivirales/farmacología , Secuencia de Bases , Células Cultivadas , Ciclopropanos/farmacología , Citomegalovirus/efectos de los fármacos , Citomegalovirus/crecimiento & desarrollo , Farmacorresistencia Viral/efectos de los fármacos , Farmacorresistencia Viral/genética , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/virología , Ganciclovir/farmacología , Guanina/análogos & derivados , Guanina/farmacología , Humanos , Datos de Secuencia Molecular , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Proteínas Virales/metabolismoRESUMEN
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.
Asunto(s)
Antivirales/síntesis química , Antivirales/farmacología , Ciclopropanos/síntesis química , Ciclopropanos/farmacología , Guanina/análogos & derivados , Profármacos/síntesis química , Profármacos/farmacología , Administración Oral , Animales , Antivirales/administración & dosificación , Virus de la Viruela Vacuna/efectos de los fármacos , Ciclopropanos/química , Citomegalovirus/efectos de los fármacos , Guanina/síntesis química , Guanina/química , Guanina/farmacología , VIH-1/efectos de los fármacos , Virus de la Hepatitis B/efectos de los fármacos , Herpesvirus Humano 1/efectos de los fármacos , Herpesvirus Humano 2/efectos de los fármacos , Herpesvirus Humano 4/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Profármacos/administración & dosificación , Virus Vaccinia/efectos de los fármacosRESUMEN
Eight novel single amino acid (6-11) and dipeptide (12, 13) tyrosine P-O esters of cyclic cidofovir ((S)-cHPMPC, 4) and its cyclic adenine analogue ((S)-cHPMPA, 3) were synthesized and evaluated as prodrugs. In vitro IC(50) values for the prodrugs (<0.1-50 µM) vs vaccinia, cowpox, human cytomegalovirus, and herpes simplex type 1 virus were compared to those for the parent drugs ((S)-HPMPC, 2; (S)-HPMPA, 1; IC(50) 0.3-35 µM); there was no cytoxicity with KB or HFF cells at ≤100 µM. The prodrugs exhibited a wide range of half-lives in rat intestinal homogenate at pH 6.5 (<30-1732 min) with differences of 3-10× between phostonate diastereomers. The tyrosine alkylamide derivatives of 3 and 4 were the most stable. (l)-Tyr-NH-i-Bu cHPMPA (11) was converted in rat or mouse plasma solely to two active metabolites and had significantly enhanced oral bioavailability vs parent drug 1 in a mouse model (39% vs <5%).
Asunto(s)
Adenina/análogos & derivados , Citosina/análogos & derivados , Organofosfonatos/química , Profármacos/química , Tirosina/química , Adenina/química , Adenina/farmacocinética , Adenina/farmacología , Animales , Antivirales/química , Antivirales/farmacocinética , Antivirales/farmacología , Área Bajo la Curva , Disponibilidad Biológica , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cidofovir , Virus de la Viruela Vacuna/efectos de los fármacos , Citomegalovirus/efectos de los fármacos , Citosina/química , Citosina/farmacocinética , Citosina/farmacología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Herpesvirus Humano 1/genética , Humanos , Concentración 50 Inhibidora , Ratones , Modelos Químicos , Estructura Molecular , Organofosfonatos/farmacocinética , Organofosfonatos/farmacología , Profármacos/farmacocinética , Profármacos/farmacología , Ratas , Virus Vaccinia/efectos de los fármacosRESUMEN
We report the synthesis and biological evaluation of Ala-(Val-)l-Ser-CO(2)R prodrugs of 1, where a dipeptide promoiety is conjugated to the P(OH)(2) group of cidofovir (1) via esterification by the Ser side chain hydroxyl group and an ethyl group (4 and 5) or alone (6 and 7). In a murine model, oral administration of 4 or 5 did not significantly increase total cidofovir species in the plasma compared to 1 or 2, but 7 resulted in a 15-fold increase in a rat model and had an in vitro EC(50) value against human cytomegalovirus comparable to 1. Neither 6 nor 7 exhibited toxicity up to 100 µM in KB or HFF cells.
Asunto(s)
Antivirales/síntesis química , Antivirales/farmacología , Citomegalovirus/efectos de los fármacos , Citosina/análogos & derivados , Organofosfonatos/química , Péptidos/síntesis química , Profármacos/síntesis química , Administración Oral , Alanina/química , Alanina/farmacología , Animales , Antivirales/administración & dosificación , Células Cultivadas , Cidofovir , Citosina/sangre , Citosina/química , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Humanos , Concentración 50 Inhibidora , Estructura Molecular , Organofosfonatos/sangre , Péptidos/química , Péptidos/farmacología , Profármacos/administración & dosificación , Profármacos/farmacología , Ratas , Serina/química , Serina/farmacología , Valina/química , Valina/farmacologíaRESUMEN
Several benzimidazole nucleoside analogs, including 1H-ß-D-ribofuranosyl-2-bromo-5,6-dichlorobenzimidazole (BDCRB) and 1H-ß-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.
Asunto(s)
Antivirales/farmacología , Bencimidazoles/farmacología , Herpesvirus Humano 6/efectos de los fármacos , Antivirales/química , Bencimidazoles/química , Citomegalovirus/efectos de los fármacos , Humanos , Replicación Viral/efectos de los fármacosRESUMEN
Many fraudulent nucleosides including the antivirals acyclovir (ACV) and ganciclovir (GCV) must be metabolized to triphosphates to be active. Cyclopropavir (CPV) is a newer, related guanosine nucleoside analog that is active against human cytomegalovirus (HCMV) in vitro and in vivo. We have previously demonstrated that CPV is phosphorylated to its monophosphate (CPV-MP) by the HCMV pUL97 kinase. Consequently, like other nucleoside analogs phosphorylated by viral kinases, CPV most likely must be converted to a triphosphate (CPV-TP) in order to elicit antiviral activity. Once formed by pUL97, we hypothesized that guanosine monophosphate kinase (GMPK) is the enzyme responsible for the conversion of CPV-MP to CPV-DP. Incubation of CPV-MP with GMPK resulted in the formation of CPV-DP and, surprisingly, CPV-TP. When CPV-DP was incubated with GMPK, a time-dependent increase in CPV-TP occurred corresponding to a decrease in CPV-DP thereby demonstrating that CPV-DP is a substrate for GMPK. Substrate specificity experiments revealed that GMP, dGMP, GDP, and dGDP are substrates for GMPK. In contrast, GMPK recognized only acyclovir and ganciclovir monophosphates as substrates, not their diphosphates. Kinetic studies demonstrated that CPV-DP has a K(M) value of 45±15µM. We were, however, unable to determine the K(M) value for CPV-MP directly, but a mathematical model of experimental data gave a theoretical K(M) value for CPV-MP of 332±60µM. We conclude that unlike many other antivirals, cyclopropavir can be converted to its active triphosphate by a single cellular enzyme once the monophosphate is formed by a virally encoded kinase.
Asunto(s)
Antivirales/química , Antivirales/metabolismo , Guanilato-Quinasas/metabolismo , Nucleótidos/química , Nucleótidos/metabolismo , Antivirales/farmacología , Cinética , Modelos Químicos , Estructura Molecular , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Human cytomegalovirus (HCMV) is a widespread pathogen that can cause severe disease in immunologically immature and immunocompromised individuals. Cyclopropavir (CPV) is a guanine nucleoside analog active against human and murine cytomegaloviruses in cell culture and efficacious in mice by oral administration. Previous studies established that the mechanism of action of CPV involves inhibition of viral DNA synthesis. Based upon this action and the structural similarity of CPV to ganciclovir (GCV), we hypothesized that CPV must be phosphorylated to a triphosphate to inhibit HCMV DNA synthesis and that pUL97 is the enzyme responsible for the initial phosphorylation of CPV to a monophosphate (CPV-MP). We found that purified pUL97 phosphorylated CPV 45-fold more extensively than GCV, a known pUL97 substrate and the current standard of treatment for HCMV infections. Kinetic studies with CPV as the substrate for pUL97 demonstrated a Km of 1,750+/-210 microM. Introduction of 1.0 or 10 nM maribavir, a known pUL97 inhibitor, and subsequent Lineweaver-Burk analysis demonstrated competitive inhibition of CPV phosphorylation, with a Ki of 3.0+/-0.3 nM. Incubation of CPV with pUL97 combined with GMP kinase [known to preferentially phosphorylate the (+)-enantiomer of CPV-MP] established that pUL97 stereoselectively phosphorylates CPV to its (+)-monophosphate. These results elucidate the mechanism of CPV phosphorylation and help explain its selective antiviral action.
Asunto(s)
Antivirales/metabolismo , Ciclopropanos/metabolismo , Guanina/análogos & derivados , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Bencimidazoles/metabolismo , Citomegalovirus/metabolismo , Ganciclovir/metabolismo , Guanina/metabolismo , Guanilato-Quinasas/metabolismo , Humanos , Cinética , Fosforilación , Ribonucleósidos/metabolismo , EstereoisomerismoRESUMEN
Triciribine (TCN) is a tricyclic nucleoside that inhibits human immunodeficiency virus type 1 (HIV-1) replication by a unique mechanism not involving the inhibition of enzymes directly involved in viral replication. This activity requires the phosphorylation of TCN to its 5' monophosphate by intracellular adenosine kinase. New testing with a panel of HIV and simian immunodeficiency virus isolates, including low-passage-number clinical isolates and selected subgroups of HIV-1, multidrug resistant HIV-1, and HIV-2, has demonstrated that TCN has broad antiretroviral activity. It was active in cell lines chronically infected with HIV-1 in which the provirus was integrated into chromosomal DNA, thereby indicating that TCN inhibits a late process in virus replication. The selection of TCN-resistant HIV-1 isolates resulted in up to a 750-fold increase in the level of resistance to the drug. DNA sequence analysis of highly resistant isolate HIV-1(H10) found five point mutations in the HIV-1 gene nef, resulting in five different amino acid changes. DNA sequencing of the other TCN-resistant isolates identified at least one and up to three of the same mutations observed in isolate HIV-1(H10). Transfer of the mutations from TCN-resistant isolate HIV-1(H10) to wild-type virus and subsequent viral growth experiments with increasing concentrations of TCN demonstrated resistance to the drug. We conclude that TCN is a late-phase inhibitor of HIV-1 replication and that mutations in nef are necessary and sufficient for TCN resistance.
Asunto(s)
Fármacos Anti-VIH/farmacología , VIH-1/efectos de los fármacos , VIH-1/fisiología , Ribonucleósidos/farmacología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/fisiología , Línea Celular , Farmacorresistencia Viral/genética , Genes nef , Transcriptasa Inversa del VIH/antagonistas & inhibidores , VIH-1/genética , VIH-2/efectos de los fármacos , Humanos , Técnicas In Vitro , Pruebas de Sensibilidad Microbiana , Mutación Puntual , Virus de la Inmunodeficiencia de los Simios/efectos de los fármacos , Ensamble de Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Replicación Viral/genética , Replicación Viral/fisiología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
BACKGROUND: Following the example of L-valine prodrugs of antiviral nucleoside analogues, L-valine ester of cyclopropavir (valcyclopropavir) was synthesized. METHODS: The known tetrahydropyranylcyclopropavir was transformed to N-(tert-butoxycarbonyl)-L-valine ester, which was deprotected to valcyclopropavir. RESULTS: Stability of valcyclopropavir towards hydrolysis at pH 7.0 roughly corresponded to that of valganciclovir. Valcyclopropavir inhibited replication of human cytomegalovirus (HCMV, Towne and AD169 strains) to approximately the same extent as the parent drug cyclopropavir. Pharmacokinetic studies in mice established that the oral bioavailability of valcyclopropavir was 95%. CONCLUSIONS: The prodrug valcyclopropavir offers some improved therapeutic parameters over the parent compound cyclopropavir.
Asunto(s)
Antivirales/química , Ciclopropanos/química , Ciclopropanos/farmacocinética , Guanina/análogos & derivados , Profármacos/síntesis química , Valina/química , Animales , Antivirales/farmacocinética , Disponibilidad Biológica , Citomegalovirus/efectos de los fármacos , Ésteres , Ganciclovir/análogos & derivados , Guanina/química , Guanina/farmacocinética , Humanos , Hidrólisis , Ratones , Profármacos/farmacocinética , Valganciclovir , Replicación Viral/efectos de los fármacosRESUMEN
Recently we characterized two inhibitors targeting the human cytomegalovirus (HCMV) terminase, 2-bromo-4,5,6-trichloro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl) benzimidazole (BTCRB) and 2,4,5,6-tetrachloro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl) benzimidazole (Cl(4)RB). The terminase consists of the ATP-hydrolyzing subunit pUL56 and the subunit pUL89 required for duplex nicking. Because mammalian cell DNA replication does not involve cleavage of concatemeric DNA by a terminase, these compounds represent attractive alternative HCMV antivirals. We now have tested these previously identified benzimidazole ribonucleosides in order to determine if they are active against HCMV clinical isolates as well as those of herpes simplex virus type 1, mouse cytomegalovirus, rat cytomegalovirus (RCMV), and varicella-zoster virus (VZV). Antiviral activity was quantified by measurement of viral plaque formation (plaque reduction) and by viral growth kinetics. Interestingly, both BTCRB and Cl(4)RB had an inhibitory effect in ganciclovir (GCV)-sensitive and GCV-resistant clinical isolates, with the best effect produced by Cl(4)RB. Electron microscopy revealed that in cells infected with GCV-sensitive or GCV-resistant isolates, B capsids and dense bodies were formed mainly. Furthermore, pulsed-field gel electrophoresis showed that cleavage of concatenated DNA was inhibited in clinical isolates. In addition, the antiviral effect on other herpesviruses was determined. Interestingly, in plaque reduction assays, BTCRB was active against all tested herpesviruses. The best effects were observed on VZV- and RCMV-infected cells. These results demonstrate that the new compounds are highly active against GCV-resistant and GCV-sensitive clinical isolates and slightly active against other herpesviruses.
Asunto(s)
Antivirales/farmacología , Bencimidazoles/farmacología , Citomegalovirus/efectos de los fármacos , Hidrocarburos Halogenados/farmacología , Ribonucleósidos/farmacología , Animales , Antivirales/efectos adversos , Antivirales/química , Bencimidazoles/efectos adversos , Bencimidazoles/química , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citomegalovirus/aislamiento & purificación , Citomegalovirus/ultraestructura , Infecciones por Citomegalovirus/virología , Electroforesis en Gel de Campo Pulsado , Herpes Simple/virología , Herpes Zóster/virología , Herpesvirus Humano 1/efectos de los fármacos , Herpesvirus Humano 3/efectos de los fármacos , Humanos , Hidrocarburos Halogenados/efectos adversos , Hidrocarburos Halogenados/química , Ratones , Microscopía Electrónica de Transmisión , Células 3T3 NIH , Ratas , Ribonucleósidos/efectos adversos , Ribonucleósidos/químicaRESUMEN
Z- and E-Phosphonate analogues 12 and 13 derived from cyclopropavir and the corresponding cyclic phosphonates 14 and 15 were synthesized and their antiviral activity was investigated. The 2,2-bis(hydroxymethylmethylenecyclopropane acetate (17) was transformed to tetrahydropyranyl acetate 18. Deacetylation gave intermediate 19 which was converted to bromide 20. Alkylation with diisopropyl methylphosphonate afforded after protecting group exchange (21 to 22) acetylated phosphonate intermediate 22. Addition of bromine gave the dibromo derivative 16 which was used in the alkylation-elimination procedure with 2-amino-6-chloropurine to give Z- and E-isomers 23 and 24. Hydrolytic dechlorination coupled with removal of all protecting groups gave the guanine phosphonates 12 and 13. Cyclization afforded the cyclic phosphonates 14 and 15. Z-Phosphonate 12 was a potent and non-cytotoxic inhibitor of human and murine cytomegalovirus (HCMV and MCMV) with EC(50) 2.2-2.7 and 0.13 microM, respectively. It was also an effective agent against Epstein-Barr virus (EBV, EC(50) 3.1 microM). The cyclic phosphonate 14 inhibited HCMV (EC(50) 2.4-11.5 microM) and MCMV (EC(50) 0.4 microM) but it was ineffective against EBV. Both phosphonates 12 and 14 were as active against two HCMV Towne strains with mutations in UL97 as they were against wild-type HCMV thereby circumventing resistance due to such mutations. Z-Phosphonate 12 was a moderate inhibitor of replication of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) but it was a potent agent against varicella zoster virus (VZV, EC(50) 2.9 microM). The cyclic phosphonate 14 lacked significant potency against these viruses. E-isomers 13 and 15 were devoid of antiviral activity.
Asunto(s)
Antivirales , Ciclopropanos , Citomegalovirus/efectos de los fármacos , Guanina/análogos & derivados , Organofosfonatos , Simplexvirus/efectos de los fármacos , Animales , Antivirales/síntesis química , Antivirales/química , Antivirales/farmacología , Ciclopropanos/síntesis química , Ciclopropanos/química , Ciclopropanos/farmacología , Guanina/síntesis química , Guanina/química , Guanina/farmacología , Humanos , Concentración 50 Inhibidora , Isomerismo , Espectroscopía de Resonancia Magnética , Ratones , Estructura Molecular , Organofosfonatos/síntesis química , Organofosfonatos/química , Organofosfonatos/farmacologíaRESUMEN
Chiral Z- and E-stereoisomers of (1,2-dihydroxyethyl)methylenecyclopropane analogues of 2'-deoxyadenosine and 2'-deoxyguanosine were synthesized, and their antiviral activity was investigated. (S)-Methylenecyclopropylcarbinol (16) was converted in seven steps to reagents 26 and 27, which were used for alkylation-elimination of adenine and 2-amino-6-chloropurine to get ultimately analogues 12a, 12b, 13a, 13b, 14a, 14b, 15a, and 15b. The enantiomeric series ent-12a, ent-12b, ent-13a, ent-13b, ent-14a, ent-14b, ent-15a, and ent-15b was obtained by similar procedures starting from (R)-methylenecyclopropylcarbinol (ent-16). The Z-isomer ent-12b was an inhibitor of two strains of human cytomegalovirus (HCMV) with EC(50) of 6.8 and 7.5 microM and of murine cytomegalovirus (MCMV) with EC(50) of 11.3 microM. It was less active against HCMV with mutated gene UL97. It inhibited Epstein-Barr virus (EBV) with EC(50) of 8 microM. The E-isomers ent-15a, ent-13a, and 15b were less effective. All adenine analogues with the exception of the Z-isomers ent-12a and ent-14a were moderate substrates for adenosine deaminase.
Asunto(s)
Antivirales/síntesis química , Ciclopropanos/síntesis química , Desoxiadenosinas/química , Desoxiguanosina/análogos & derivados , Animales , Antivirales/farmacología , Citomegalovirus/efectos de los fármacos , Desoxiadenosinas/farmacología , Desoxiguanosina/síntesis química , Desoxiguanosina/farmacología , Herpesvirus Humano 4/efectos de los fármacos , Humanos , Ratones , Muromegalovirus/efectos de los fármacos , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Enantiomeric cyclopropavir phosphates (+)-9 and (-)-9 were synthesized and investigated as substrates for GMP kinase. N(2)-Isobutyryl-di-O-acetylcyclopropavir (11) was converted to (+)-monoacetate 12 using hydrolysis catalyzed by porcine liver esterase. Phosphorylation via phosphite 13 gave after deacylation, phosphate (+)-9. Acid-catalyzed tetrahydropyranylation of (+)-monoacetate 12 gave, after deacylation, tetrahydropyranyl derivative 14. Phosphorylation via phosphite 15 furnished, after deprotection, enantiomeric phosphate (-)-9. Racemic diphosphate 16 was also synthesized. The phosphate (+)-9 is a relatively good substrate for GMP kinase with a K(M) value of 57 microM that is similar to that of the natural substrates GMP (61 microM) and dGMP (82 microM). In contrast, the enantiomer (-)-9 is not a good substrate (K(M) 1200 microM) indicating a significant enantioselectivity for the GMP kinase catalyzed reaction of monophosphate to diphosphate.
Asunto(s)
Ciclopropanos/química , Guanina/análogos & derivados , Guanilato-Quinasas/metabolismo , Compuestos Organofosforados/síntesis química , Compuestos Organofosforados/metabolismo , Acilación , Esterasas/metabolismo , Guanina/química , Cinética , Estructura Molecular , Compuestos Organofosforados/química , Fosforilación , Especificidad por SustratoRESUMEN
5'-O-D- and L-amino acid derivatives and 5'-O-(D- and L-amino acid methyl ester phosphoramidate) derivatives of vidarabine (ara-A) were synthesized as vidarabine prodrugs. Some compounds were equi- or more potent in vitro than vidarabine against two pox viruses and their uptake by cultured cells was improved compared to the parent drug.
Asunto(s)
Antivirales/síntesis química , Química Farmacéutica/métodos , Profármacos/síntesis química , Vidarabina/síntesis química , Administración Oral , Antivirales/farmacología , Arabinonucleósidos/química , Células CACO-2 , Células Cultivadas , Diseño de Fármacos , Ésteres , Células HeLa , Humanos , Ácidos Levulínicos/química , Nucleósidos/química , Poxviridae/metabolismo , Profármacos/farmacología , Vidarabina/farmacologíaRESUMEN
5-Alkylaminopyrazole nucleosides underwent nitrosation to give the corresponding N1-ribosylated 5-alkyl-amino-4-nitrosopyrazoles. The intramolecular cyclo-dehydration reactions of these 5-alkylamino-4-nitrosopyrazoles were carried out in pyridine at reflux temperature to afford the ring-closure N-1 ribosylated imidazo[4,5-c]pyrazoles in good yields.
Asunto(s)
Nucleósidos de Purina/síntesis química , Pirazoles/síntesis química , Compuestos Bicíclicos con Puentes/síntesis química , Compuestos Bicíclicos con Puentes/química , Nucleósidos de Purina/química , Pirazoles/químicaRESUMEN
Cidofovir (HPMPC, 1), a broad-spectrum antiviral agent, is currently used to treat AIDS-related human cytomegalovirus (HCMV) retinitis and has recognized therapeutic potential for orthopox virus infections, but is limited by its low oral bioavailability. Cyclic cidofovir (2) displays decreased nephrotoxicity compared to 1, while also exhibiting potent antiviral activity. Here we describe in detail the synthesis and evaluation as prodrugs of four cHPMPC dipeptide conjugates in which the free POH of 2 is esterified by the Ser side chain alcohol group of an X-L-Ser(OMe) dipeptide: 3 (X=L-Ala), 4 (X=L-Val), 5 (X=L-Leu), and 6 (X=L-Phe). Perfusion studies in the rat establish that the mesenteric permeability to 4 is more than 20-fold greater than to 1, and the bioavailability of 4 is increased 6-fold relative to 1 in an in vivo murine model. In gastrointestinal and liver homogenates, the cHPMPC prodrugs are rapidly hydrolyzed to 2. Prodrugs 3, 4, and 5 are nontoxic at 100 microM in HFF and KB cells and in cell-based plaque reduction assays had IC 50 values of 0.1-0.5 microM for HCMV and 10 microM for two orthopox viruses (vaccinia and cowpox). The enhanced transport properties of 3-6, conferred by incorporation of a biologically benign dipeptide moiety, and the facile cleavage of the Ser-O-P linkage suggest that these prodrugs represent a promising new approach to enhancing the bioavailability of 2.