Putative HIV-1 reverse transcriptase inhibitors: design, synthesis, in vitro evaluation and in silico analysis.
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One of the most significant treatments for HIV-1 infection has been the combination of drugs targeting the HIV life cycle with the aim of preventing further destruction of the host immune system. This study addresses the design, synthesis, in vitro evaluation, and in silico analysis of putative HIV-1 reverse transcriptase (RT) inhibitors. The inhibitors comprise two structurally diverse components which are intended to bind separately to the enzyme allosteric site and to a location at, or close to, the polymerase active site. Therefore, the hydrophobic N-tritylated phalo-DL-phenylalanine derivatives (fluoro, chloro, bromo, iodo) have been coupled to 8-(6- aminohexyl) amino-adenosine-3',5'-cyclic monophosphate through N-hydroxysuccinimidecarbodiimide chemistry. Compounds were characterized by thin layer chromatography, UV spectroscopy, MALDI-TOF mass spectrometry and proton NMR spectrometry. A reverse transcriptase colorimetric assay kit, which features a sandwich ELISA protocol, based on biotin-avidin and digoxygenin-anti DIG interactions, was used for quantitative determination of the inhibitory effect of synthesized compounds on recombinant HIV-1 reverse transcriptase activity in vitro. Molecular docking simulations of the chimeric inhibitors within the allosteric site of HIV-1 RT, were performed using AutoDock Vina. The predicted binding associations were compared with laboratory findings on HIV-1 RT inhibition. Two dimensional representations of protein-ligand interactions were generated using LigPlot. The non-halogenated N-trityl-L-phenylalanine-8-(6-aminohexyl)amino-adenosine-3',5'-cyclic monophosphate derivative (4a) inhibited RT activity down to 57 % at 10-4 M, while the Ntrityl-para-fluoro-DL-phenylalanine-8-(6-aminohexyl)aminoadenosine-3′,5′-cyclic monophosphate derivative (4b) was the strongest RT inhibitor reducing RT activity to 69 % at 10-7 M (IC50 = 29.2 μM). In the same assay, Nevirapine, a first-line anti-retroviral drug, showed a decline in RT activity down to 43% at 10-5 M (IC50 = 3.03 μM). Ranking of inhibitors according to estimated docking energies obtained from in silico docking was in excellent agreement with potencies calculated from experimental studies. The docking score of N-trityl-para-fluoro-DL-phenylalaline-8-(6-aminohexyl)amino-adenosine-3',5'-cyclic monophosphate was -8.8 kcal/mol, while that of Nevirapine was -9.9 kcal/mol. The benzene rings of the N-trityl-fluoro-DL-phenylalanine-8-(6-aminohexyl) amino-adenosine-3',5'-cyclic monophosphate derivative formed hydrophobic interactions with hydrophobic, non-aromatic amino acid residues Pro176 and Val179 in the allosteric site. Nevirapine, on the other hand showed strong van der Waals interactions with Val106 ,Val179 and Tyr188 due to the aromatic properties of the pyridine ring. Possible π-π stacking between phenyl rings of Nevirapine and Tyr 181/Tyr188 aromatic side chains may also be present. Other HIV-1 RT large subunit residues in the allosteric site common to the binding of Nevirapine and the active para-fluoro derivative include Lys101, Tyr318, Leu 100, Trp229 and Phe227. Apparent binding to the allosteric site suggests that compounds may be acting primarily as non-nucleoside reverse transcriptase inhibitors (NNRTIs).