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.

Inhibition of HIV‐1 Reverse Transcriptase Dimerization by Small Molecules

“…It is possible to determine, by differential scanning fluorimetry, if RT inhibitors can stabilize or destabilize the RT heterodimer, thereby causing an increase or decrease in the melting temperature ( T m ) . In particular, compounds reported to bind close to the interface between the two subunits have been shown to reduce RT T m . Other RT inhibitors (NNRTIs such as EFV or allosteric RNase H and polymerase dual inhibitors) did not affect the RT melting profile .…”

“…Hence, we performed differential scanning fluorimetry analysis in the presence of the compounds, with the RNase H active‐site inhibitor BTP and MAS0 ( 1 ) as controls. In accordance with previous studies, we observed a T m increase of 1.16 °C in the presence of Mg 2+ and BTP and, as expected, we observed a T m decrease of 0.56 °C in the presence of MAS0. Similarly, a T m decrease of 0.5–0.72 °C was observed in the presence of 2 – 6 , thus confirming the hypothesis of interaction at the p66/p51 interface (Table ).…”

From cycloheptathiophene-3-carboxamide to oxazinone-based derivatives as allosteric HIV-1 ribonuclease H inhibitors

“…Heterodimeric RT was expressed essentially as described 19 , 39 . Using a BioLogic LP system (Biorad), using a combination of immobilised metal affinity (Ni-Sepharose high performance, Healthcare Lifescience) and ion exchange chromatography (Hi-trap heparin HP GE).…”

Natural Product Kuwanon‐L Inhibits HIV‐1 Replication through Multiple Target Binding

“…However, not all drugs that act on “more than one target” have the same modality of action. There are compounds that are able to bind different pockets of the same target, others that are able to interact with the same target but at different moments in the infection process, and some compounds that are even able to bind different viral enzymes…”

“…All these findings confirm our initial hypothesis of multiple target binding. Unlike the known molecules capable of dual IN/RNase H inhibition or dual RDDP/RNase H inhibition, kuwanon‐L represents the first compound able to inhibit both the activity associated with HIV‐1 IN and the two activities associated with HIV‐1 RT. This new approach can be considered a solid starting point that demonstrates that the multiple‐target‐binding strategy can be successfully applied against HIV‐1.…”