High affinity interaction of HIV-1 integrase with specific and non-specific single-stranded short oligonucleotides.

Retroviral integrase (IN) catalyzes the integration of double-stranded viral DNA into the host cell genome. The reaction can be divided in two steps: 3'-end processing and DNA strand transfer. Here we studied the effect of short oligonucleotides (ODNs) on human immunodeficiency virus type 1 (HIV-1) IN. ODNs were either specific, with sequences representing the extreme termini of the viral long terminal repeats, or nonspecific. All ODNs were found to competitively inhibit the processing reaction with Ki values in the nM range for the best inhibitors. Our studies on the interaction of IN with ODNs also showed that: (i) besides the 3'-terminal GT, the interaction of IN with the remaining nucleotides of the 21-mer specific sequence was also important for an effective interaction of the enzyme with the substrate; (ii) in the presence of specific ODNs the activity of the enzyme was enhanced, a result which suggests an ODN-induced conformational change of HIV-1 IN.     

Ribonuclease H/DNA Polymerase HIV-1 Reverse Transcriptase Dual Inhibitor: Mechanistic Studies on the Allosteric Mode of Action of Isatin-Based Compound RMNC6

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.