Structure-guided approach identifies a novel class of HIV-1 ribonuclease H inhibitors: binding mode insights through magnesium complexation and site-directed mutagenesis studies

HIV persistent infection requires a life-long treatment and among the 2.1 million new HIV infections that occur every year there is an increased rate of transmitted drugresistant mutations. This fact requires a constant and timely effort in order to identify and develop new HIV inhibitors endowed with innovative mechanisms. The HIV-1 Reverse Transcriptase (RT) associated Ribonuclease H (RNase H) is the only viral encoded enzymatic activity that still lacks an efficient inhibitor despite the fact that it is a well-validated target whose functional abrogation compromises viral infectivity. Identification of new drugs is a long and expensive process that can be speeded up by in silico methods. In the present study, a structure-guided screening is coupled with a similarity-based search on the Specs database to identify a new class of HIV-1 RNase H inhibitors. Out of 45 compounds selected for experimental testing, 15 of these inhibited the RNase H function below 100 μM with three hits possessing IC50 values <10 μM. The most active compound AA inhibits HIV-1 RNase H with an IC50 of 5.1 μM, and possesses Mg-independent mode of inhibition. Site-directed mutagenesis studies provide valuable insight into the binding mode of newly identified compounds, for instance, compound AA involves extensive interactions with a lipophilic pocket formed by Ala502, Lys503, Trp (406, 426 and 535) and polar interactions with Arg557 and with the highly conserved RNase H primer-grip residue Asn474. The structural insights obtained from this work provide the bases for further lead optimization.