6-(1-(4-fluorophenyl)methyl-1H-pyrrol-2-yl))-2,4-dioxo-5-hexenoic acid ethyl ester a novel diketo acid derivative which selectively inhibits the HIV-1 viral replication in cell culture and the ribonuclease H activity in vitro

The HIV-1 reverse transcriptase (RT) is a multifunctional enzyme which displays DNA polymerase activity, which recognizesRNAandDNAtemplates, and a degradative ribonucleaseH(RNase H) activity. While bothRT functions are required for retroviral replication, until now only the polymerase function has been widely explored as drug target. We have identified a novel diketo acid derivative, 6-[1-(4-fluorophenyl)methyl-1H-pyrrol-2-yl)]-2,4-dioxo-5-hexenoic acid ethyl ester (RDS 1643), which inhibits in enzyme assays the HIV-1 RT-associated polymerase-independent RNase H activity but has no effect on the HIV-1 RT-associated RNA-dependent DNA polymerase (RDDP) activity and on the RNase H activities displayed by the Avian Myeloblastosis Virus and E. coli. Time-dependence studies revealed that the compound is active independently on the order of its addition to the reaction mixture, and inhibition kinetics studies demonstrated that RDS 1643 inhibits the RNase H activity noncompetitively, with a KI value of 17M. When RDS 1643 was combined with non-nucleoside RT inhibitors (NNRTI), such as efavirenz and nevirapine, results indicated that RDS 1643 does not affect the NNRTIs anti-RDDP activity and that, vice versa, the NNRTIs do not alter the RNase H inhibition by RDS 1643. When assayed on the viral replication in cell-based assays, RDS 1643 inhibited the HIV-1IIIB strain with an EC50 of 14M. Similar results were obtained against the Y181C and Y181C/K103N HIV-1NNRTI resistant mutant strains. RDS 1643 may be the first HIV-1 inhibitor selectively targeted to the viral RT-associated RNase-H function.