Detecting allosteric sites of HIV-1 reverse transcriptase by X-ray crystallographic fragment screening

Abstract

HIV-1 reverse transcriptase (RT) undergoes a series of conformational changes during viral replication and is a central target for antiretroviral therapy. The intrinsic flexibility of RT can provide novel allosteric sites for inhibition. Crystals of RT that diffract X-rays to better than 2 Å resolution facilitated the probing of RT for new druggable sites using fragment screening by X-ray crystallography. A total of 775 fragments were grouped into 143 cocktails, which were soaked into crystals of RT in complex with the non-nucleoside drug rilpivirine (TMC278). Seven new sites were discovered, including the Incoming Nucleotide Binding, Knuckles, NNRTI Adjacent, and 399 sites, located in the polymerase region of RT, and the 428, RNase H Primer Grip Adjacent, and 507 sites, located in the RNase H region. Three of these sites (Knuckles, NNRTI Adjacent, and Incoming Nucleotide Binding) are inhibitory and provide opportunities for discovery of new anti-AIDS drugs.

Figure 1

Overview of fragment screening. (a) Color-coded cartoon of the RT-rilpivirine complex with 20% d₆-DMSO present. Rilpivirine (brown space filling) is bound at the NNRTI-binding pocket. The p66 subdomains are color-coded fingers (blue), palm (red), thumb (green), connection (yellow), RNase H (red), and the p51 subunit (cyan). Ordered waters are shown as blue dots and d₆-DMSO molecules are shown as green, yellow, and red spheres. (b) Flowchart for fragment screening by X-ray crystallography with approximate completion times indicated. Hit to lead development is the current focus of this project. (c) Schematic drawing depicting the JDAM activity assay. (d) Drawing representing p66 (dark green), p51 (cyan), and rilpivirine (yellow spheres) with all fragments discovered in this study (orange spheres). The sites of fragment binding described in this report are circled.

Figure 2

The NNRTI Adjacent, Knuckles, Incoming Nucleotide Binding, and 399 sites. Residues that shift due to ligand binding are shown in magenta in their non-bound positions and dark green in their bound position except p51 residues shown in cyan. (a) 4 (gray) bound at the NNRTI Adjacent site (dark green). Rilpivirine is shown on the right side of the panel (yellow). Electron density is shown for 1 from a F_o-F_c omit map (3.5σ contours). (b) Top-down view of panel (A) showing the new hydrogen-bonding network as green dashes. (c) Structure of RT-rilpivirine-1. (d) Structure turned 90 degrees. (e) Structure of RT-rilpivirine-3 with the position of 2 shown at the bottom of the panel. Electron density is shown for 2 from an F_o-F_c omit map (2σ contours). (f) Incoming nucleotide binding site with 5 bound. Q151 shifts from the unbound position (magenta) to the bound position (green). Electron density is shown for 5 from an F_o-F_c omit map (3.0σ contours). (g)6 bound at the 399 site. An ordered d₆-DMSO molecule is shown at the bottom of the panel. Electron density is shown for 6 from an F_o-F_c omit map (1.5σ contours).

Figure 3

428, 507, and RNase H Primer Grip Adjacent sites. Residues that shift due to ligand binding are shown in magenta in their non-bound positions and dark green in their bound position. (a)7 (gray) bound at the 428 site. A bound d₆-DMSO molecule is in the bottom right of the panel. Electron density is shown for 7 from an F_o-F_c omit map (2.5σ contours). (b)8 (gray) bound at the 507 site. Electron density is shown for 8 from an F_o-F_c omit map (2.5σ contours). (c)8 (gray) at the NNRTI-binding pocket without rilpivirine present. The normal position of rilpivirine at the NNRTI-binding pocket is shown in magenta. Electron density is shown for 8 from an F_o-F_c omit map (4.0σ contours). (d)9 (gray) bound at the RNase H primer grip adjacent site. Electron density is shown for 9 from an F_o-F_c omit map (3.0σ contours).