title: Identification of highly conserved residues involved in inhibition of HIV-1 RNase H function by Diketo acid derivatives.
abstract: HIV-1 reverse transcriptase (RT)-associated RNase H activity is an essential function in viral genome retrotranscription. RNase H is a promising drug target for which no inhibitor is available for therapy. Diketo acid (DKA) derivatives are active site Mg(2+)-binding inhibitors of both HIV-1 RNase H and integrase (IN) activities. To investigate the DKA binding site of RNase H and the mechanism of action, six couples of ester and acid DKAs, derived from 6-[1-(4-fluorophenyl)methyl-1H-pyrrol-2-yl)]-2,4-dioxo-5-hexenoic acid ethyl ester (RDS1643), were synthesized and tested on both RNase H and IN functions. Most of the ester derivatives showed selectivity for HIV-1 RNase H versus IN, while acids inhibited both functions. Molecular modeling and site-directed mutagenesis studies on the RNase H domain demonstrated different binding poses for ester and acid DKAs and proved that DKAs interact with residues (R448, N474, Q475, Y501, and R557) involved not in the catalytic motif but in highly conserved portions of the RNase H primer grip motif. The ester derivative RDS1759 selectively inhibited RNase H activity and viral replication in the low micromolar range, making contacts with residues Q475, N474, and Y501. Quantitative PCR studies and fluorescence-activated cell sorting (FACS) analyses showed that RDS1759 selectively inhibited reverse transcription in cell-based assays. Overall, we provide the first demonstration that RNase H inhibition by DKAs is due not only to their chelating properties but also to specific interactions with highly conserved amino acid residues in the RNase H domain, leading to effective targeting of HIV retrotranscription in cells and hence offering important insights for the rational design of RNase H inhibitors.
authors.affiliation: Department of Life and Environmental Sciences, University of Cagliari, Monserrato, Italy.
authors.forename: Angela
authors.initials: A
authors.lastname: Corona
authors.affiliation: Department of Drug Discovery and Development, Italian Institute of Technology, Genoa, Italy.
authors.forename: Francesco Saverio
authors.initials: FS
authors.lastname: Di Leva
authors.affiliation: LBPA, ENS Cachan, CNRS, Cachan, France.
authors.forename: Sylvain
authors.initials: S
authors.lastname: Thierry
authors.affiliation: Dipartimento di Chimica e Tecnologie del Farmaco, Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza Università di Roma, Rome, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Pescatori
authors.affiliation: Dipartimento di Chimica e Tecnologie del Farmaco, Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza Università di Roma, Rome, Italy.
authors.forename: Giuliana
authors.initials: G
authors.lastname: Cuzzucoli Crucitti
authors.affiliation: LBPA, ENS Cachan, CNRS, Cachan, France.
authors.forename: Frederic
authors.initials: F
authors.lastname: Subra
authors.affiliation: LBPA, ENS Cachan, CNRS, Cachan, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Delelis
authors.affiliation: Department of Life and Environmental Sciences, University of Cagliari, Monserrato, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Esposito
authors.affiliation: Department of Life and Environmental Sciences, University of Cagliari, Monserrato, Italy LBPA, ENS Cachan, CNRS, Cachan, France.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Rigogliuso
authors.affiliation: Dipartimento di Chimica e Tecnologie del Farmaco, Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza Università di Roma, Rome, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Costi
authors.affiliation: DiSTABiF, Seconda Università di Napoli, Caserta, Italy.
authors.forename: Sandro
authors.initials: S
authors.lastname: Cosconati
authors.affiliation: Dipartimento di Farmacia, Università degli Studi di Napoli Federico II, Naples, Italy.
authors.forename: Ettore
authors.initials: E
authors.lastname: Novellino
authors.affiliation: Dipartimento di Chimica e Tecnologie del Farmaco, Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza Università di Roma, Rome, Italy.
authors.forename: Roberto
authors.initials: R
authors.lastname: Di Santo
authors.affiliation: Department of Life and Environmental Sciences, University of Cagliari, Monserrato, Italy tramon@unica.it.
authors.forename: Enzo
authors.initials: E
authors.lastname: Tramontano
mesh_terms: D019380:Anti-HIV Agents / Q000494:pharmacology*; D002460:Cell Line; D015497:HIV-1 / Q000201:enzymology*; D006801:Humans; D016914:Ribonuclease H / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D019380:Anti-HIV Agents; D016914:Ribonuclease H
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