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

Abstract

Persistent HIV infection requires lifelong treatment and among the 2.1 million new HIV infections that occur every year there is an increased rate of transmitted drug-resistant mutations. This fact requires a constant and timely effort in order to identify and develop new HIV inhibitors with innovative mechanisms. The HIV-1 reverse transcriptase (RT) associated ribonuclease H (RNase H) is the only viral encoded enzyme 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 the 45 compounds selected for experimental testing, 15 inhibited the RNase H function below 100 μM with three hits exhibiting IC₅₀ values <10 μM. The most active compound, AA, inhibits HIV-1 RNase H with an IC₅₀ of 5.1 μM and exhibits a 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, and Trp (406, 426 and 535) and polar interactions with Arg557 and the highly conserved RNase H primer-grip residue Asn474. The structural insights obtained from this work provide the bases for further lead optimization.

Fig. 1. The HIV-RT associated RNase H structural topology is shown with a bound ligand NHQD (green, ball and stick model). The catalytically important residues and magnesium ions (orange spheres) have been highlighted. The 2D structures of representative molecules belong to the active site and allosteric RNase H inhibitors.

Fig. 2. Overall workflow of structure-based virtual screening strategies applied.

Fig. 3. Initial hit molecules (A–C) from the first screening and compound “B” is highlighted in regions where it shares a common structural pattern with BHMP07. Compounds AQ and AR and analogs of B and C as found by the shape-based screening (see text for details).

Fig. 4. Comparison of the structure and activity of A analogs.

Fig. 5. Comparison of the binding mode of known RNase H inhibitors in an X-ray crystal; important residues are highlighted. Panel A, N-hydroxy quinazolinedione inhibitor (NHQD, active site binder, green) and BHMP07 (allosteric site binder, cyan). Panel B, compound A (magenta). Panel C, compound AA (blue). Panel D, compound AB (cyan).

Fig. 6. Titration graphs of inhibitors with Mg²⁺; panel A: compound AC, panel B: compound A, panel C: compound AA and panel D: compound AB.

Fig. 7. A comparison of the binding modes of compounds A, AA, AB and BPT in the wild type and various clinically relevant mutants.

Fig. 8. A comparison of the various binding modes of compound AA in the RNase H active site; wild type is shown in orange and N474A mutant shown in cyan. Important residues are highlighted.