HIV-1 transcriptase (RT) is a virus-coded multifunctional enzyme responsible for viral genome replication using a combination of DNA polymerase and ribonuclease H (RNaseH) activities. These combined synthetic and hydrolytic functions are validated targets for drug development. The identification of new scaffolds capable of inhibiting both enzymatic activities is an innovative challenge that involves the interaction of virologists and chemists, with the aim of overcoming the continuing problem of drug resistance. From a previous virtual screening (Distinto et al. Eur. J. Med. Chem. 2012) we individuated an isatine-derivative able to inhibit both RT enzymatic function. Therefore we synthesized 13 new related compounds and tested them against both activities of HIV-1 RT. We found some active compounds with dual inhibitor profile. In order to define the mechanism of action of these molecules we performed a Yonetani-Theorell analysis using compounds previously reported to bind to different RT sites (non-nucleoside RT inhibitors (NNRTIs) and RNaseH inhibitors). Data suggest that isatin derivatives have an allosteric binding mode. Subsequently we performed a blind docking experiment to identify in silico the potential binding sites. Two allosteric pockets were identified to be most favorable for this series of compounds. To validate our hypothesis we performed single site mutagenesis on different residues, potentially critical for the inhibitors binding, namely V106, V108 and A508 of the p66 RT subunit, and we tested their sensitivity to the most active compounds RMNC0 e RMNC6. Inhibitor sensitivity was also examined with HIV-1 RT containing a K103N and Y181C mutations, shown to confer resistance to the NNRTIs Efavirence and Nevirapine. Overall, our biological data show a consistent correspondence between the docking outcome and the site-directed mutagenesis study. These results allow us to propose isatine derivatives as a promising scaffold for HIV-1 RT dual inhibitors development. The allosteric mechanism of action, different from classical NNRTI, highlight the need of further investigation on the role of non-catalytic regions for HIV-1 RT function
SITE DIRECT MUTAGENESIS AND KINETIC STUDIES TO EXPLORE THE MECHANISM OF ACTION OF ISATIN DERIVATIVES AS HIV-1 REVERSE TRANSCRIPTASE DUAL INHIBITORS
CORONA, ANGELA;R. Meleddu;ESPOSITO, FRANCESCA;S. Distinto;MACCIONI, ELIAS;
2012-01-01
Abstract
HIV-1 transcriptase (RT) is a virus-coded multifunctional enzyme responsible for viral genome replication using a combination of DNA polymerase and ribonuclease H (RNaseH) activities. These combined synthetic and hydrolytic functions are validated targets for drug development. The identification of new scaffolds capable of inhibiting both enzymatic activities is an innovative challenge that involves the interaction of virologists and chemists, with the aim of overcoming the continuing problem of drug resistance. From a previous virtual screening (Distinto et al. Eur. J. Med. Chem. 2012) we individuated an isatine-derivative able to inhibit both RT enzymatic function. Therefore we synthesized 13 new related compounds and tested them against both activities of HIV-1 RT. We found some active compounds with dual inhibitor profile. In order to define the mechanism of action of these molecules we performed a Yonetani-Theorell analysis using compounds previously reported to bind to different RT sites (non-nucleoside RT inhibitors (NNRTIs) and RNaseH inhibitors). Data suggest that isatin derivatives have an allosteric binding mode. Subsequently we performed a blind docking experiment to identify in silico the potential binding sites. Two allosteric pockets were identified to be most favorable for this series of compounds. To validate our hypothesis we performed single site mutagenesis on different residues, potentially critical for the inhibitors binding, namely V106, V108 and A508 of the p66 RT subunit, and we tested their sensitivity to the most active compounds RMNC0 e RMNC6. Inhibitor sensitivity was also examined with HIV-1 RT containing a K103N and Y181C mutations, shown to confer resistance to the NNRTIs Efavirence and Nevirapine. Overall, our biological data show a consistent correspondence between the docking outcome and the site-directed mutagenesis study. These results allow us to propose isatine derivatives as a promising scaffold for HIV-1 RT dual inhibitors development. The allosteric mechanism of action, different from classical NNRTI, highlight the need of further investigation on the role of non-catalytic regions for HIV-1 RT function
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