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 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 IC values <10 μM. The most active compound, , inhibits HIV-1 RNase H with an IC 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 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.

Design, synthesis, and biologic evaluation of novel galloyl derivatives as HIV‐1 RNase H inhibitors

“…Although there is only one oxygen anion in the II‐26 structure that interacts with Mg 2+ , multiple additional interactions with Arg557 and His539 of such an oxygen and of the substituted benzene rings with Ala538 and Lys540 are predicted to contribute to increase the inhibitory activity of the compound. In agreement with our modeling studies, previous analyses predicted the involvement of ionic/π interactions between Arg557 and Mg 2+ ‐binding RNase H inhibitors (Poongavanam et al., ; Corona et al., ).…”

Targeting HIV-1 RNase H: N’-(2-Hydroxy-benzylidene)-3,4,5-Trihydroxybenzoylhydrazone as Selective Inhibitor Active against NNRTIs-Resistant Variants

“…The inhibitory activity of compound 13 and RDS1759 was assessed in conditions of competition with the substrate, proving that the compound potency was not affected by substrate displacement, as suggested elsewhere for other classes of compounds [25,63]. The significant loss of potency of compound 13 against all the generated mutant RTs proved the critical role of those amino acid residues for its binding to the RNase H domain and implies a binding orientation different from RDS1759, imidazolidinedione derivatives and beta-thujaplicinol [26,27] that were reported to be fully active against both R448A and R557A HIV-1 RTs.…”

“…Attempts to target highly conserved residues in the RNase H domain have been successfully shown [26][27][28]. Given the structural similarities and overlaps with the HIV-1 IN catalytic core, many of the identified RNase H active-site inhibitors exhibit dual anti-RNase H and anti-integrase (IN) activity [20,29].…”

Biological evaluation of molecules of the azaBINOL class as antiviral agents: Inhibition of HIV-1 RNase H activity by 7-isopropoxy-8-(naphth-1-yl)quinoline

“…Testing for complexation of B#24 with Mg 2+ ions was carried out following previous protocols with adjustments. 50 In brief, a 1 M stock solution of MgCl2 and a 1 M solution of B#24 were prepared in 1:1 ethanol/acetonitrile mixtures. B#24 was diluted to 100 µM and UV absorbance readings recorded using a BioTek synergy plate reader from 200 -400 nm.…”

“…With this in mind, we sought to probe whether azaBINOL B#24 was inhibiting RNase H via the active site using a Mg 2+ ion chelation and absorbance test. Mg 2+ ions present in the active site of RNase H are an integral part of its endonuclease function and various inhibitors have been shown to interfere with their chelatingproperties.8, Compound B#24 was tested for Mg 2+ binding by assessment of its UV absorption under an increasing concentration of Mg 2+ following existing procedures 50. No UV absorbance changes were observed with the addition of Mg 2+ up to 120 mM with B#24 (100 µM)(Figure 4).Therefore, we conclude that B#24 is not interacting with Mg 2+ ions and subsequently is not directly inhibiting RNase H activity through active site binding.…”