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.     

Thioaptamer interactions with prion proteins: sequence-specific and non-specific binding sites

Binding of nucleic acids to the prion protein (PrP) created a conundrum that required distinguishing between non-specific interactions and biologically important polynucleotides. In the process of developing selective ligands for PrP, we found using a single-stranded DNA thioaptamer library that the binding of thioaptamers to PrP occurs on at least two different sites on the protein. Selection against recombinant (rec) PrP of Syrian hamster (SHa) sequence 90-231 folded into an alpha-helical-rich conformation identified a 12-base consensus sequence within a series of 20 thioaptamers, all of which consist of 40 bases. Each thioaptamer was comprised of both normal and thio-dA modified bases. One thioaptamer designated 97 bound to recSHaPrP with affinity of 0.58(+/-0.1) nM; lower affinities for bovine (Bo), and human (Hu) were found, establishing that binding is dependent on the primary structure of PrP. High affinity binding of thioaptamer 97 to PrP was found to be mediated through the dodecyl sequence GACACAAGCCGA within the consensus region with five critical backbone modifications 5' to each dA residue. A control oligonucleotide with an equivalent number of phosphorothioates to thioaptamer 97 and a scrambled consensus sequence could not distinguish among the three PrP sequences. Control oligonucleotides bearing non-selected sequences bound to PrP at a sequence-independent DNA-binding site. In contrast, the high-affinity binding of thioaptamer 97 to PrP depends on (1) backbone modifications, (2) oligonucleotide sequence, and (3) PrP sequence.