RESUMEN
Inorganic pyrophosphate (PPi) mimetics designed on a basis of methylenediphosphonic acid backbone are promising inhibitors of two key HIV replication enzymes, IN [1] and RT [2]. Herein, we present chemical synthesis of eleven methylenebisphosphonates (BPs) with their NMR and HRMS analysis synthesized via five different ways. Also, we present data on inhibition of HIV RT catalyzed phosphorolysis and polymerization by synthesized BPs using two methods based on denaturing urea PAGE. Tests were also performed for thymidine analogue mutations reverse transcriptase (TAM RT), which was expressed and purified for that. Structure-activity relationships and inhibitory activity data of synthesized BPs are presented in "Methylene bisphosphonates as the inhibitors of HIV RT phosphorolytic activity" [2].
RESUMEN
The structure-function analysis of 36 methylenebisphosphonates (BPs) as inhibitors of the phosphorolytic activity of native and drug-resistant forms of HIV-1 reverse transcriptase (RT) was performed. It was shown that with the increase of the inhibitory potential of BPs towards the phosphorolytic activity raises their ability to inhibit the RT-catalyzed DNA elongation. Herein, we report the impact of the thymidine analog mutations (TAM) on the activity of bisphosphonates, as well as some structural features of the BPs, allowing them to maintain the inhibitory activity on the enzyme resistant to nucleoside analog therapy. We estimated the Mg(2+)-coordinating group structure, the linker and the aromatic pharmacophore influence on the inhibitory potential of the BPs. Based on the 31 BPs SAR, several BPs with improved inhibitory properties were designed and synthesized.
Asunto(s)
Difosfonatos/farmacología , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Transcriptasa Inversa del VIH/metabolismo , VIH-1/enzimología , Adenosina Trifosfato/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Transcriptasa Inversa del VIH/química , Transcriptasa Inversa del VIH/genética , Magnesio/metabolismo , Modelos Moleculares , Mutación , Conformación de Ácido Nucleico , Fosforilación/efectos de los fármacos , Conformación Proteica , ARN Viral/química , ARN Viral/metabolismo , Relación Estructura-ActividadRESUMEN
As has been shown previously, phosphite of acycloguanosine (Hp-ACG) exhibits equal efficacy against ACV-sensitive and ACV-resistant HSV-1 strains in cell culture. Intraperitoneal administration of Hp-ACG to model mice with herpetic encephalitis caused by HSV-1 infection was shown to be effective in protecting against death. In the present work, we continue the study of the antiviral efficiency of Hp-ACG against HSV administered non-invasively; namely in vivo, orally and in the form of ointment formulations. It has been first shown that oral administration of Hp-ACG twice daily for five days prevents systemic infection in mice caused by HSV-1. Mortality in the control group of animals was 57%. Administration of Hp-ACG at doses of 600, 800 and 1,000 mg/kg per day significantly increased the survival and median day of death of the animals compared to the placebo-treated control group. A comparative evaluation of the therapeutic efficacy parameters of polyethylene glycol-based ACV ointment and Hp-ACG ointment was carried out after a 5-day course in the model of an experimental cutaneous infection of HSV-1 in guinea pigs. It was found that Hp-ACG has a significant therapeutic effect resulting in a statistically significant reduction in the lesion's surface area and the amount of vesicular structures. The exhibited therapeutic effect of 10% Hp-ACG in ointment form compares well with that of 5% ACG ointment.
RESUMEN
The activity of the phosphite of acycloguanosine (P-ACG) and six antivirals was tested individually and in double and triple combinations on two strains of the herpes simplex virus (HSV) type 1 (sensitive to acyclovir and resistant to acyclovir) using the CPE inhibition method in the Vero E6 cell microcultures. These are: phosphite of acycloguanosine (P-ACG), Ara-A, cidofovir (CDV), ribavirin (Rib), phosphonoformate (PFA), glycyrrhizic acid (GLN) and alpha-interferon (alpha-IFN). All studied double combinations and triple combinations including P-ACV inhibited replication of both HSV strains more effectively than any drug alone. Various types of interactions depending on the virus type were observed in both viral models: synergistic (double combinations P-ACG with PFA, CDV, Rib, alpha-IFN and triple combinations P-ACG with alpha-IFN +PFA, alpha-IFN +AraA, alpha-IFN +CDV, PFA+CDV, PFA+Rib, CDV+AraA, CDV+Rib, CDV+GLN,PFA+AraA) and additive (P-ACG with AraA and PFA+GLN). Neither antagonism nor interference was noted for any combinations. Adduced results suggest that these combinations might be clinically useful for the treatment of certain herpes simplex virus type 1 infections.
Asunto(s)
Aciclovir/farmacología , Antivirales/farmacología , Herpesvirus Humano 1/efectos de los fármacos , Animales , Chlorocebus aethiops , Cidofovir , Citosina/análogos & derivados , Citosina/farmacología , Farmacorresistencia Viral/fisiología , Sinergismo Farmacológico , Quimioterapia Combinada , Foscarnet/farmacología , Ácido Glicirrínico/farmacología , Herpesvirus Humano 1/fisiología , Humanos , Interferón-alfa/farmacología , Organofosfonatos/farmacología , Fosfitos , Ribavirina/farmacología , Células Vero , Vidarabina/farmacología , Replicación Viral/efectos de los fármacosRESUMEN
Substrate specificities of three viral replicative polymerases of different origins (HIV reverse transcriptase, hepatitis C virus RNA polymerase, and herpes virus DNA polymerase) towards 2'F-NTP were studied. Activated DNA, polyA-oligoUs and (2'F-A)20-oligoU6-complexes were used as templates. It was shown that all DNA polymerases studied can incorporate 2'F-NMP into the 3'-end of primer-template complexes. HIV reverse transcriptase and herpes virus DNA polymerase can elongate synthesis with both dNTP and 2'F-NTP. Homopolymer (2'F-A)20 can serve as a template for polymerization of both UTP and 2'F-UTP,-catalyzed by hepatitis C virus polymerase although with efficacy about 5 to 10-fold lower in comparison with natural primertemplate complex. Pyrophosphorolysis reaction of 2'F-CMP residue at 3'-end of primer catalyzed with HIV reverse transcriptase is going by two orders of magnitude less effective if compared with natural dNMP residue at the same system.
Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , Exodesoxirribonucleasas/metabolismo , Transcriptasa Inversa del VIH/metabolismo , Nucleósidos/metabolismo , Proteínas Virales/metabolismo , Catálisis , ADN Nucleotidilexotransferasa/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Flúor/química , Hepatitis C/enzimología , Nucleósidos/química , Oligonucleótidos/química , Oligonucleótidos/metabolismo , Poli A , Especificidad por SustratoRESUMEN
WHO reports that 90% of human population is infected by different types of herpesviruses, which develop latency or cause oral and genital herpes, conjunctivitis, eczema herpeticum, and other diseases. Herpesvirus almost always accompanies HIV-infection and complicates AIDS treatment. Herpes simplex virus type 1 is one of the most wide spread viruses from the Herpesviridae family. HSV virion, genome structure, replication mechanisms, antiherpes drug development strategies, including design of prodrugs, and mutations causing ACV-resistance in clinical HSV isolates are discussed in this review.
Asunto(s)
Antivirales/uso terapéutico , Herpesvirus Humano 1/fisiología , Replicación Viral , Antivirales/química , Farmacorresistencia Viral , Genoma Viral , Herpesvirus Humano 1/efectos de los fármacos , Herpesvirus Humano 1/genética , Humanos , Virión/ultraestructuraRESUMEN
Antiherpetic activity of the double and triple combinations, including original connections 15Lys-bis-Nt and phosphate of acycloguanosine (P-ACG), was studied in vitro. For the first time, it was demonstrated that in case of their combined use with known antiherpetic agents, whose activity does not depend on TK of HSV (PFA, AraA, CDV, Rib, GLN, αa-IFN), synergistic or additive effects of interaction was observed. The antiviral effect of the tested combinations was studied on the model of ACG-resistant viral strain. The tested combinations could be of interest for practical medicine.
Asunto(s)
Herpes Simple/tratamiento farmacológico , Simplexvirus/efectos de los fármacos , Simplexvirus/genética , Replicación Viral/efectos de los fármacos , Aciclovir/administración & dosificación , Animales , Antivirales/administración & dosificación , Chlorocebus aethiops , Farmacorresistencia Viral/efectos de los fármacos , Farmacorresistencia Viral/genética , Sinergismo Farmacológico , Herpes Simple/genética , Herpes Simple/virología , Simplexvirus/crecimiento & desarrollo , Células VeroRESUMEN
This review provides data on the design of antiretroviral drugs based on nucleoside analogs. About 30 drugs were approved for the treatment of HIV-infected patients over 25 years. Seven nucleoside drugs are inhibitors of HIV reverse transcriptase and clinically used in combination with inhibitors of other viral enzymes, integrase and protease, and non-nucleoside inhibitors of reverse transcriptase. Toxicity of nucleoside drugs and approaches to obtaining of safe anti-HIV drugs are discussed. The results of developing of domestic anti-HIV drugs are presented. The future prospects of anti-HIV investigations are considered.
Asunto(s)
Fármacos Anti-VIH , Diseño de Fármacos , Infecciones por VIH , Nucleósidos , Animales , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/química , Fármacos Anti-VIH/historia , Fármacos Anti-VIH/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/genética , Infecciones por VIH/historia , Infecciones por VIH/metabolismo , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Nucleósidos/síntesis química , Nucleósidos/química , Nucleósidos/historia , Nucleósidos/uso terapéuticoRESUMEN
A study of the antiherpesviral activity of acycloguanosine (ACG) H-phosphonate (ACG-P) on a model of fatal herpesvirus infection in inbred BALB/c albino mice has established that ACG-P reduces death rates in the animals, considerably increases their average lifespan, and significantly decreases brain virus titers with both 60% mortality in the control and 92% mortality in the control group. There was also a significant inhibition of herpes simplex virus type 1 (HSV-1) replication in the brain tissue of animals receiving ACG-P on a model of ACG-resistant HSV-1/L2/RACG(TK-).
Asunto(s)
Aciclovir/análogos & derivados , Herpes Simple/tratamiento farmacológico , Herpesvirus Humano 1/efectos de los fármacos , Organofosfonatos/farmacología , Aciclovir/farmacología , Aciclovir/uso terapéutico , Animales , Antivirales/farmacología , Antivirales/uso terapéutico , Encéfalo/virología , Chlorocebus aethiops , Farmacorresistencia Viral , Humanos , Concentración 50 Inhibidora , Pulmón/virología , Masculino , Ratones , Ratones Endogámicos BALB C , Organofosfonatos/uso terapéutico , Células Vero , Ensayo de Placa ViralRESUMEN
A new class of inhibitors of herpes simplex virus replication was found. The compounds under study are derived from condensed 1,2,4-triazolo[5,1-c][1,2,4]triazines and 1,2,4-triazolo[1,5-a]pyrimidines, structural analogues of natural nucleic bases. Antiherpetic activity and cytotoxicity of the compounds were studied. The corresponding triphosphates of several active compounds were prepared and tested as inhibitors of DNA synthesis catalyzed by herpes simplex virus polymerase. The potential mechanism of their action is blocking of DNA dependent DNA polymerase, a key enzyme of viral replication.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Herpes Simple/tratamiento farmacológico , Herpesvirus Humano 1/efectos de los fármacos , Triazinas/química , Triazinas/farmacología , Triazoles/química , Triazoles/farmacología , Animales , Línea Celular , Supervivencia Celular , Chlorocebus aethiops , ADN Polimerasa Dirigida por ADN/metabolismo , Herpesvirus Humano 1/enzimología , Humanos , Modelos Moleculares , Inhibidores de la Síntesis del Ácido Nucleico , Polifosfatos/química , Polifosfatos/farmacología , Células Vero , Replicación Viral/efectos de los fármacosRESUMEN
The primary structures of DNA-polymerase (ul30) and thymidine kinase (ul23) genes from several herpes simplex virus type 1 (HSV-1) clinical isolates which differed in sensitivity for a number of antiherpetic drugs were determined and compared with those for two laboratory HSV-1 strains one of which was ACV-sensitive (L2), while the another was resistant (L2) to ACV. The phylogenetic analysis of the sequences showed that conserved regions of ul30 gene of HSV-1 clinical isolates and L2 strain were homologous with the exception of point mutations and degenerated substitutions. Several new mutations in the HSV-1 DNA-polymerase and thymidine kinase functional domains were established and identified as the substitutions associated with the strain-resistance to ACV and other drugs.
Asunto(s)
Aciclovir/farmacología , Antivirales/farmacología , ADN Polimerasa Dirigida por ADN/genética , Farmacorresistencia Viral/genética , Exodesoxirribonucleasas/genética , Herpes Simple/genética , Herpesvirus Humano 1/genética , Mutación Puntual , Timidina Quinasa/genética , Proteínas Virales/genética , Línea Celular , ADN Polimerasa Dirigida por ADN/metabolismo , Exodesoxirribonucleasas/metabolismo , Herpes Simple/tratamiento farmacológico , Herpes Simple/enzimología , Herpesvirus Humano 1/enzimología , Herpesvirus Humano 1/aislamiento & purificación , Humanos , Timidina Quinasa/metabolismo , Proteínas Virales/metabolismoRESUMEN
The ability of acycloguanosine H-phosphate to inhibit the reproduction of herpes simplex virus type 1 (HSV-1) variants, including its acycloguanosine (acyclovir)-resistant ones, was studied. Acycloguanosine H-phosphate-resistant HSV-1 variants were obtained. It was found that these variants were cross-resistant to thymidine kinase-dependent HSV reproduction inhibitors, but preserved sensitivity to Apa-A and phosphonoacetic acid.
Asunto(s)
Aciclovir/análogos & derivados , Aciclovir/farmacología , Antivirales/farmacología , Farmacorresistencia Viral , Herpes Simple/virología , Organofosfonatos/farmacología , Simplexvirus/efectos de los fármacos , Animales , Chlorocebus aethiops , Pruebas de Sensibilidad Microbiana , Simplexvirus/fisiología , Células Vero , Replicación Viral/efectos de los fármacosRESUMEN
New non-nucleoside esters of phosphoric acid containing various hydrophobic groups, namely (1) N-(2-tripticencarbonyl)-4-aminobutyl; (2) 5-phenylsubstituted N-(2,4-dinitrophenyl)-4-aminobutyl; (3) N-(4-phenylbenzoyl)- and N-(4-(N-benzylamino)benzoyl)-2-aminoethyl groups, as well as (4) diphenylmethyl and fluorenyl groups were synthesized and studied as substrates of terminal deoxynucleotidyl transferase. With the exception of the two latter derivatives, all the analogues displayed substrate properties and could incorporate into the deoxyoligonucleotide 3'-end. As it was shown in biochemical experiments and by computer modeling, a linker joining the triphosphate and hydrophobic fragments of the molecule was necessary for these compounds to display substrate properties.
Asunto(s)
ADN Nucleotidilexotransferasa/química , Polifosfatos/química , Dominio Catalítico , Ésteres , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Oligodesoxirribonucleótidos/química , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
New phosphonate depot forms of AZT and 3TC were synthesized and their anti-HIV properties in cell systems, cellular uptake, intracellular transformations and some pharmacokinetic and toxicological data were studied.
Asunto(s)
Antivirales/síntesis química , Lamivudine/análogos & derivados , Zidovudina/análogos & derivados , Animales , Antivirales/farmacocinética , Antivirales/farmacología , Línea Celular , Perros , Lamivudine/farmacocinética , Lamivudine/farmacología , Ratones , Organofosfonatos/química , Zidovudina/farmacocinética , Zidovudina/farmacologíaRESUMEN
A method has been developed for obtaining a full-length protein NS3 of hepatitis C virus with the yield of 6.5 mg/liter of cell culture, and conditions for measuring its NTPase and helicase activities have been optimized. The helicase reaction can proceed in two modes depending on the enzyme and substrate concentration ratio: it can be non-catalytic in the case of enzyme excess and catalytic in the case of tenfold substrate excess. In the latter case, helicase activity is coupled with NTPase and is stimulated by ATP. A number of NTP and inorganic pyrophosphate analogs were studied as substrates and/or inhibitors of NS3 NTPase activity, and it was found that the structure of nucleic base and ribose fragment of NTP molecule has a slight effect on its inhibitory (substrate) properties. Among the nucleotide derivatives, the most efficient inhibitor of NTPase activity is 2 -deoxythymidine 5 -phosphoryl-beta,gamma-hypophosphate, and among pyrophosphate analogs imidodiphosphate exhibited maximal inhibitory activity. These compounds were studied as inhibitors of the helicase reaction, and it was shown that imidodiphosphate efficiently inhibited the ATP-dependent helicase reaction and had almost no effect on the ATP-independent duplex unwinding. However, the inhibitory effect of 2 -deoxythymidine 5 -phosphoryl-beta,gamma-hypophosphate was insignificant in both cases, which is due to the possibility of helicase activation by this ATP analog.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Genes Reporteros , Hepacivirus/enzimología , Nucleósido-Trifosfatasa/antagonistas & inhibidores , Nucleósido-Trifosfatasa/genética , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Eficiencia , Regulación de la Expresión Génica , Hepacivirus/genética , Magnesio/farmacología , Modelos Biológicos , Nucleósido-Trifosfatasa/metabolismo , Nucleósido-Trifosfatasa/fisiología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
A new group of terminal deoxynucleotidyltransferase (TDT) substrates, namely, non-nucleoside triphosphates (NNTP) bearing 5-substituted 2,4-dinitrophenyl fragments instead of nucleoside residues was synthesized.
Asunto(s)
ADN Nucleotidilexotransferasa/metabolismo , Ésteres/metabolismo , Polifosfatos/metabolismo , Especificidad por SustratoRESUMEN
A series of bicyclic [2,3-d]furano- and pyrrolopyrimidine ribonucleosides were synthesized and converted chemically into corresponding 5'-O-triphosphates. Substrate properties of the triphosphates toward some RNA and DNA polymerases are reported.
Asunto(s)
Nucleósidos de Pirimidina/síntesis química , Nucleósidos de Pirimidina/metabolismo , Nucleótidos de Pirimidina/síntesis química , Nucleótidos de Pirimidina/metabolismo , Animales , Antivirales/síntesis química , Antivirales/química , Antivirales/metabolismo , Antivirales/farmacología , Compuestos Bicíclicos con Puentes/síntesis química , Compuestos Bicíclicos con Puentes/química , Compuestos Bicíclicos con Puentes/metabolismo , Bovinos , ADN Polimerasa Dirigida por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Diseño de Fármacos , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Hepacivirus/efectos de los fármacos , Hepacivirus/enzimología , Estructura Molecular , Nucleósidos de Pirimidina/química , Nucleósidos de Pirimidina/farmacología , Nucleótidos de Pirimidina/química , Nucleótidos de Pirimidina/farmacología , Especificidad por SustratoRESUMEN
The total fraction of aminoacyl-tRNA synthases from Escherichia coli has been shown to catalyze the synthesis of the bis(5'-nucleosidyl) oligophosphates Ap4AZT, Ap4d4T, Ap43TC, and Ap4ACV, as well as Ap3AZT and Ap3d4T, from [alpha-32P]ATP and the corresponding nucleoside-5'-tri(or di)phosphate. The resulting compounds, characterized by HPLC, are resistant to alkaline phosphatase. Ap4AZT, Ap4d4T, and Ap43TC are formed with approximately equal efficiency, whereas the efficiencies of the synthesis of Ap4ACV, Ap3AZT, and Ap3d4T are three- to fivefold lower. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 6; see also http://www.maik.ru.
Asunto(s)
Aminoacil-ARNt Sintetasas/química , Fosfatos de Dinucleósidos/síntesis química , Adenosina Trifosfato/química , Fosfatasa Alcalina/química , Catálisis , Cromatografía Líquida de Alta Presión , Escherichia coli/enzimologíaRESUMEN
Substrate properties of nucleoside 5'-triphosphate (NTP) analogs, namely, 5'-triphosphates of L- and D-arabinonucleosides (D-FIAUTP, D-FMAUTP, and L-FMAUTP), D- and L-enantiomers of ddCTP analogs (D-ddCTP, L-ddCTP, D-FOddCTP, L-OddCTP, and L-SddCTP), and acyclic guanosine analogs (acyclovir and penciclovir) towards terminal deoxynucleotidyltransferase (TdT, EC 2.7.7.31) were studied. TdT can polymerize 5'-triphosphates of arabinonucleoside analogs (D-FIAUTP and D-FMAUTP). In contrast, L-FMAUTP is not recognized by TdT as a substrate. Kinetic parameters of D- and L-enantiomers of ddCTP analogs and 5'-triphosphates of acyclic nucleosides were evaluated. It is shown that stereospecificity of dNTP analogs and structure of the furanose residue play crucial roles in the interaction with TdT:L-enantiomers are much less potent as substrates compared to their D-counterparts. 5'-Triphosphates of acyclovir (ACVTP) and penciclovir (PCVTP) are about two orders of magnitude less effective as substrates than nucleosides bearing furanose residues, with PCVTP being a better substrate than ACVTP. It can be assumed that the hydroxyl group of PCVTP mimics the 3'-hydroxyl group of the ribose residue and plays an important role in the interaction with TdT.
Asunto(s)
ADN Nucleotidilexotransferasa/metabolismo , Nucleótidos/metabolismo , Catálisis , Cinética , Estructura Molecular , Nucleótidos/química , Especificidad por Sustrato , Factores de TiempoRESUMEN
N-(9-Fluorenylmethoxycarbonyl)-omega-aminoalkyl, N-(9-fluorenylmethoxycarbonyl)-8-amino-3,6-dioxaoctyl, and N-(9-fluorenylmethoxycarbonyl)-6-aminohexanoyl]-2-aminoethyl triphosphates were synthesized. All of them were shown to be the substrates of the calf thymus terminal deoxynucleotidyl transferase. Their substrate properties depend on the length and structure of linker between the 9-fluorenylmethoxycarbonyl and triphosphate moieties.