Discovery of novel inhibitors of HIV-1 reverse transcriptase through virtual screening of experimental and theoretical ensembles

Abstract

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are potent anti-HIV chemotherapeutics. Although there are FDA-approved NNRTIs, challenges such as the development of resistance have limited their utility. Here, we describe the identification of novel NNRTIs through a combination of computational and experimental approaches. Based on the known plasticity of the NNRTI binding pocket (NNIBP), we adopted an ensemble-based virtual screening strategy: coupling receptor conformations from 10 X-ray crystal structures with 120 snapshots from a total of 480 ns of molecular dynamics (MD) trajectories. A screening library of 2864 National Cancer Institute (NCI) compounds was built and docked against the ensembles in a hierarchical fashion. Sixteen diverse compounds were tested for their ability to block HIV infection in human tissue cultures using a luciferase-based reporter assay. Three promising compounds were further characterized, using a HIV-1 RT-based polymerase assay, to determine the specific mechanism of inhibition. We found that 2 of the three compounds inhibited the polymerase activity of RT (with potency similar to the positive control, the FDA-approved drug nevirapine). Through a computational approach, we were able to discover two compounds which inhibit HIV replication and block the activity of RT, thus offering the potential for optimization into mature inhibitors.

Keywords: HIV; NNRTI; molecular dynamics; reverse transcriptase; virtual screening.

Figure 1

Six second-generation NNRTIs that were used to generate the “similarity subset” of the virtual screening library. Rilpivirine (Tibotec), RDEA-806 (Ardea), GW678248 (GSK), MIV-150 (Medivir/Chiron), UK-453061 (Pfizer) and MK-4965 (Merck).

Figure 2

Overview of the virtual screening process used to select 16 compounds for experimental testing from a library of 2,864 compounds.

Figure 3

Snapshots of the 10 crystal structures of HIV-1 RT used in the crystallographic ensemble, with the co-crystallized bound NNRTI. The NNIBP is depicted in molecular surface representation, colored by residue index. NNRTIs are shown in blue stick representation. Note the heterogeneity of the chemical structures that bind to this allosteric site.

Figure 4

Snapshots of the MD simulations, comprising the complete HIV-1 RT heterodimer complexed with 4 diverse NNRTIs and bathed in a solvent of explicit water molecules and counter-ions to neutralize the net charge. HIV-1 RT shown in a ribbon representation, with the p66 and p51 subunits colored blue and red, respectively. A closeup view of the NNIBP, with the 4 NNRTIs superposed, in stick representation is shown in the inset.

Figure 5

The 20 NCI compounds selected for experimental testing. Compounds are labeled according to the screening subset they came from: Diversity Set (D) and Similarity Set (S). Compounds marked with an asterisk were not available.

Figure 6

Effects of 16 compounds on HIV-1 infectivity. (A) The effects of the compounds (5 μM) on HIV-1 infectivity were determined by measuring firefly luciferase activity at 24 hours post-infection (hpi) in human 293T cells infected with a VSVg-pseudotyped HIV-1 vector encoding the luciferase reporter. DMSO was used as the control. (B) Cytotoxicity of the compounds was measured in mock-infected cells after treatment of the cells with the compounds for 24 hours. The experiments represent the mean of at least three independent experiments each performed in triplicate. The error bars represent the standard errors of the mean.

Figure 7

Three compounds block an early step of HIV-1 replication. (A) Effect of three compounds (5 μM) in PMA differentiated THP-1 monocytes challenged with the NL4-3 Nef⁺ IRES rluc vector encoding renilla luciferase. Luciferase activity was measured at 24 or 48 hpi. Nevirapine (NVP, 10 μM) and DMSO were used as controls. Error bars show standard error mean from three independent experiments each performed in triplicate. (B) Effect of compounds on late HIV-1 reverse transcription products at 24 or 48 hpi. The values represent amounts of viral DNA relative to DMSO treated cell populations and were normalized to cellular GAPDH DNA levels. The data shown are the mean average values obtained from 3 independent experiments that were each performed in triplicate. The error bars represent the standard error.

Figure 8

Compounds had no effect on gene expression from integrated HIV-1. 293T cells that contain an integrated vector DNA that carries and expresses firefly luciferase were treated with 5 μM of the compounds or 10 μM of NVP. Virus encoded firefly luciferase or late viral DNA was measured at 24 hpi. Error bars represent SEM of three independent luciferase or RT-PCR measurements.

Figure 9

Polymerase inhibition assay. As described in the Materials and Methods section, the three compounds that showed inhibitory activity in the cell-based assays were tested for their ability to inhibit the polymerase activity of HIV-1 RT. A radioactive primer annealed to a long template was extended by HIV-1 RT in the presence of varying concentrations of the compounds (the amount of DMSO was constant in all of the reactions), appropriate buffer, and 0.5 μM each dNTP. Nevirapine was included as a positive control for NNRTI inhibition. The reactions were allowed to proceed at 37° for 60 min and were then halted by the addition of EDTA. The samples were fractionated by electrophoresis on a 6.0% polyacrylamide gel, and the gel was autoradiographed. Phosphoimaging was used to determine the amount of signal in each lane. Primer extension products >90 nt in length were considered full length product. The percentage of the full length produced in each of the reaction conditions was calculated, then plotted. Reactions were done in duplicate.

Figure 10

Proposed binding modes of the 2 confirmed HIV-1 RT inhibitors that were active in enzyme and cell-based assays. Active compounds are shown in predicted poses based on docking into 1RT4 (top). Protein backbone is depicted as ribbons, and residues within 5 Å of the binding site are depicted as sticks. Intermolecular and intramolecular hydrogen bonding are denoted with black dashed lines. Two-dimensional ligand interaction diagrams (bottom) indicate predicted proximal residues for each of the inhibitors.