RESUMEN
RNase H inhibitors (RNHIs) have gained attention as potential HIV-1 therapeutics. Although several RNHIs have been studied in the context of HIV-1 reverse transcriptase (RT) RNase H, there is no information on inhibitors that might affect the RNase H activity of other RTs. We performed biochemical, virological, crystallographic, and molecular modeling studies to compare the RNase H function and inhibition profiles of the gammaretroviral xenotropic murine leukemia virus-related virus (XMRV) and Moloney murine leukemia virus (MoMLV) RTs to those of HIV-1 RT. The RNase H activity of XMRV RT is significantly lower than that of HIV-1 RT and comparable to that of MoMLV RT. XMRV and MoMLV, but not HIV-1 RT, had optimal RNase H activities in the presence of Mn²âº and not Mg²âº. Using hydroxyl-radical footprinting assays, we demonstrated that the distance between the polymerase and RNase H domains in the MoMLV and XMRV RTs is longer than that in the HIV-1 RT by â¼3.4 Å. We identified one naphthyridinone and one hydroxyisoquinolinedione as potent inhibitors of HIV-1 and XMRV RT RNases H with 50% inhibitory concentrations ranging from â¼0.8 to 0.02 µM. Two acylhydrazones effective against HIV-1 RT RNase H were less potent against the XMRV enzyme. We also solved the crystal structure of an XMRV RNase H fragment at high resolution (1.5 Å) and determined the molecular details of the XMRV RNase H active site, thus providing a framework that would be useful for the design of antivirals that target RNase H.
Asunto(s)
Fármacos Anti-VIH/farmacología , ADN Polimerasa Dirigida por ARN/química , Inhibidores de la Transcriptasa Inversa/farmacología , Ribonucleasa H/antagonistas & inhibidores , Ribonucleasa H/química , Ribonucleasa H/fisiología , Virus Relacionado con el Virus Xenotrópico de la Leucemia Murina/enzimología , Secuencia de Aminoácidos , Supervivencia Celular/efectos de los fármacos , Cristalografía por Rayos X , Huella de ADN , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Hidrazonas/síntesis química , Hidrazonas/farmacología , Indicadores y Reactivos , Isoquinolinas/síntesis química , Isoquinolinas/farmacología , Magnesio/farmacología , Manganeso/farmacología , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Datos de Secuencia Molecular , Virus de la Leucemia Murina de Moloney/efectos de los fármacos , Virus de la Leucemia Murina de Moloney/enzimología , Naftiridinas/síntesis química , Naftiridinas/farmacología , Plásmidos/genéticaRESUMEN
BACKGROUND: Foot-and-Mouth Disease Virus (FMDV) is a picornavirus that infects cloven-hoofed animals and leads to severe losses in livestock production. In the case of an FMD outbreak, emergency vaccination requires at least 7 days to trigger an effective immune response. There are currently no approved inhibitors for the treatment or prevention of FMDV infections. METHODOLOGY/PRINCIPAL FINDINGS: Using a luciferase-based assay we screened a library of compounds and identified seven novel inhibitors of 3Dpol, the RNA-dependent RNA polymerase of FMDV. The compounds inhibited specifically 3Dpol (IC(50)s from 2-17 µM) and not other viral or bacterial polymerases. Enzyme kinetic studies on the inhibition mechanism by compounds 5D9 and 7F8 showed that they are non-competitive inhibitors with respect to NTP and nucleic acid substrates. Molecular modeling and docking studies into the 3Dpol structure revealed an inhibitor binding pocket proximal to, but distinct from the 3Dpol catalytic site. Residues surrounding this pocket are conserved among all 60 FMDV subtypes. Site directed mutagenesis of two residues located at either side of the pocket caused distinct resistance to the compounds, demonstrating that they indeed bind at this site. Several compounds inhibited viral replication with 5D9 suppressing virus production in FMDV-infected cells with EC(50)â= 12 µM and EC(90)â= 20 µM). SIGNIFICANCE: We identified several non-competitive inhibitors of FMDV 3Dpol that target a novel binding pocket, which can be used for future structure-based drug design studies. Such studies can lead to the discovery of even more potent antivirals that could provide alternative or supplementary options to contain future outbreaks of FMD.