title: Ribonuclease H/DNA Polymerase HIV-1 Reverse Transcriptase Dual Inhibitor: Mechanistic Studies on the Allosteric Mode of Action of Isatin-Based Compound RMNC6.
abstract: The DNA polymerase and ribonuclease H (RNase H) activities of human immunodeficiency virus type 1 (HIV-1) are needed for the replication of the viral genome and are validated drug targets. However, there are no approved drugs inhibiting RNase H and the efficiency of DNA polymerase inhibitors can be diminished by the presence of drug resistance mutations. In this context, drugs inhibiting both activities could represent a significant advance towards better anti-HIV therapies. We report on the mechanisms of allosteric inhibition of a newly synthesized isatin-based compound designated as RMNC6 that showed IC50 values of 1.4 and 9.8 μM on HIV-1 RT-associated RNase H and polymerase activities, respectively. Blind docking studies predict that RMNC6 could bind two different pockets in the RT: one in the DNA polymerase domain (partially overlapping the non-nucleoside RT inhibitor [NNRTI] binding pocket), and a second one close to the RNase H active site. Enzymatic studies showed that RMNC6 interferes with efavirenz (an approved NNRTI) in its binding to the RT polymerase domain, although NNRTI resistance-associated mutations such as K103N, Y181C and Y188L had a minor impact on RT susceptibility to RMNC6. In addition, despite being naturally resistant to NNRTIs, the polymerase activity of HIV-1 group O RT was efficiently inhibited by RMNC6. The compound was also an inhibitor of the RNase H activity of wild-type HIV-1 group O RT, although we observed a 6.5-fold increase in the IC50 in comparison with the prototypic HIV-1 group M subtype B enzyme. Mutagenesis studies showed that RT RNase H domain residues Asn474 and Tyr501, and in a lesser extent Ala502 and Ala508, are critical for RMNC6 inhibition of the endonuclease activity of the RT, without affecting its DNA polymerization activity. Our results show that RMNC6 acts as a dual inhibitor with allosteric sites in the DNA polymerase and the RNase H domains of HIV-1 RT.
authors.affiliation: Department of Life and Environmental Sciences, University of Cagliari, Cagliari, Italy.
authors.forename: Angela
authors.initials: A
authors.lastname: Corona
authors.affiliation: Department of Life and Environmental Sciences, University of Cagliari, Cagliari, Italy.
authors.forename: Rita
authors.initials: R
authors.lastname: Meleddu
authors.affiliation: Department of Life and Environmental Sciences, University of Cagliari, Cagliari, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Esposito
authors.affiliation: Department of Life and Environmental Sciences, University of Cagliari, Cagliari, Italy.
authors.forename: Simona
authors.initials: S
authors.lastname: Distinto
authors.affiliation: Department of Life and Environmental Sciences, University of Cagliari, Cagliari, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Bianco
authors.affiliation: National Cancer Institute, Frederick, Maryland, United States of America.
authors.forename: Takashi
authors.initials: T
authors.lastname: Masaoka
authors.affiliation: Department of Life and Environmental Sciences, University of Cagliari, Cagliari, Italy.
authors.forename: Elias
authors.initials: E
authors.lastname: Maccioni
authors.affiliation: Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas & Universidad Autónoma de Madrid), Madrid, Spain.
authors.forename: Luis
authors.initials: L
authors.lastname: Menéndez-Arias
authors.affiliation: Dipartimento di Scienze della Salute, Università Magna Graecia di Catanzaro, Campus "S. Venuta", Viale Europa, 88100, Catanzaro, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Alcaro
authors.affiliation: National Cancer Institute, Frederick, Maryland, United States of America.
authors.forename: Stuart F J
authors.initials: SF
authors.lastname: Le Grice
authors.affiliation: Department of Life and Environmental Sciences, University of Cagliari, Cagliari, Italy.|Istituto di Ricerca Genetica e Biomedica, Consiglio Nazionale delle Ricerche (CNR), Monserrato, Cagliari, Italy.
authors.forename: Enzo
authors.initials: E
authors.lastname: Tramontano
mesh_terms: D000480:Alkynes; D019380:Anti-HIV Agents / Q000494:pharmacology*; D048588:Benzoxazines / Q000037:antagonists & inhibitors / Q000494:pharmacology; D001665:Binding Sites; D003521:Cyclopropanes; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors*; D015497:HIV-1 / Q000201:enzymology; D006801:Humans; D006835:Hydrazones / Q000494:pharmacology*; D007510:Isatin / Q000031:analogs & derivatives* / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D062105:Molecular Docking Simulation; D017434:Protein Structure, Tertiary; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D054309:Ribonuclease H, Human Immunodeficiency Virus / Q000037:antagonists & inhibitors*; D014779:Virus Replication / Q000187:drug effects
chemical_list: C000609417:4-(2-(2-(2-oxoindolin-3-ylidene)hydrazinyl)thiazol-4-yl)benzonitrile; D000480:Alkynes; D019380:Anti-HIV Agents; D048588:Benzoxazines; D003521:Cyclopropanes; D004791:Enzyme Inhibitors; D006835:Hydrazones; D018894:Reverse Transcriptase Inhibitors; D007510:Isatin; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D054309:Ribonuclease H, Human Immunodeficiency Virus; C098320:efavirenz