Fig 1.
Chemical structure of the isatin derivative RMNC6.
Fig 2.
Effect of RT inhibitors on the thermal stability of p66/p51 HIV-1 RT.
(A). The melting temperature of HIV-1 RT was measured by differential scanning fluorimetry in the presence of increasing concentrations of different inhibitors: (▼) Efavirenz (EFV), (○) β-thujaplicinol (BTP), (Δ) 2-(3, 4-dihydroxyphenyl)-5, 6-dimethylthieno[2, 3-d]pyrimidin-4(3H)-one (VU) and (●) RMNC6. (B). Maximum HIV-1 RT thermal shift (ΔTm) observed in the presence of 50 μM concentration of compounds. ΔTm values are the average of triplicate analysis, standard deviations are indicated as bars.
Fig 3.
Yonetani-Theorell plot of the combination of RMNC6 and EFV on the HIV-1 RT RNA- dependent DNA polymerase activity.
HIV-1 RT was incubated in the presence of RMNC6 alone (●) or in presence of different concentrations of EFV: 4 nM (Δ), 8 nM (■) and 16 nM (☐).
Table 1.
Susceptibility to RMNC6 of wt and NNRTI-resistant HIV-1 RTs measured in RNase H and RDDP activity assays.
Table 2.
Ensemble docking results: binding free energies of [RMNC•RT] complexes. The most likely binding poses are indicated in bold.
Fig 4.
Binding sites of RMNC6 individuated after blind docking experiments on the whole wt HIV-1 RT structure.
Fig 5.
Putative binding mode of RMC6 and critical residues individuated for RMNC6 binding in the pocket 1.
(A) binding mode; (B) 2D depiction of RMNC6 and its respective interactions with RT residues: pale yellow sphere indicates hydrophobic interactions with lipophilic residues. Red arrow indicates an hydrogen bond (HB) acceptor interaction, green HB donor, while the violet sphere represents the aromatic π-π stacking interaction.
Fig 6.
(A) Putative binding mode of RMNC6 and critical residues individuated for RMNC6 binding in the pocket 2. (B) 2D depiction of RMNC6 and its respective interactions with RT residues.
Table 3.
Effects of selected amino acid substitutions in pocket 1 of HIV-1 RT in the susceptibility to RMNC6 in RNase H and RDDP activity assays.
Table 4.
Effects of selected amino acid substitutions in pocket 2 of HIV-1 RT in the susceptibility to RMNC6 in RNase H and RDDP activity assays.
Table 5.
Kinetic parameters of RNase H cleavage for wt and mutant HIV-1 RTs.