Structural Basis of HIV-1 Inhibition by Nucleotide-Competing Reverse Transcriptase Inhibitor INDOPY-1

Abstract

HIV-1 reverse transcriptase (RT) is an essential enzyme, targeting half of approved anti-AIDS drugs. While nucleoside RT inhibitors (NRTIs) are DNA chain terminators, the nucleotide-competing RT inhibitor (NcRTI) INDOPY-1 blocks dNTP binding to RT. Lack of structural information hindered INDOPY-1 improvement. Here we report the HIV-1 RT/DNA/INDOPY-1 crystal structure, revealing a unique mode of inhibitor binding at the polymerase active site without involving catalytic metal ions. The structure may enable new strategies for developing NcRTIs.

Figure 1.

Binding of INDOPY-1 to the HIV-1 RT/DNA complex. (A) Close-up view of INDOPY-1 binding to HIV-1 RT/DNA, with subdomains, motifs, N (nucleotide-binding or pretranslocation) site and P (priming or post-translocation) site indicated. (B) 2D-interaction diagram of INDOPY-1 (blue) with HIV-1 RT/DNA [dashed lines color code: black, hydrogen bonds/polar contacts; green, π–π stacking (dotted lines) and cation-π; yellow, sulfur-π]. (C) Two views of the atomic model of INDOPY-1 (orange) bound to HIV-1 RT active site residues (yellow) and DNA (pink), with difference Polder F_o – F_c map (green mesh, 4.5σ). (D) Zoomed-in view of the Y115, G152, and M184 interactions with INDOPY-1. (E) Zoomed-in view of the stacking module in the structure and in a model where the A:T base pair is switched; interactions as in (B) except for the red dashed line indicating reduced π–π stacking.

Figure 2.

Structural basis of INDOPY-1 preference for P-site complexes. (A) Atomic model of INDOPY-1 binding to HIV-1 RT/DNA, as in Figure 1C, except that the difference Polder F_o – F_c map (green mesh, 5σ) calculation omits nucleotides dA-0 and dA-1. (B) Superposition of HIV-1 RT/DNA/INDOPY-1 with the HIV-1 RT/DNA binary P complex (residues in silver, PDB code 5D3G). (C) Superposition of HIV-1 RT/DNA/INDOPY-1 onto the N-site HIV-1 RT/DNA/AZT triphosphate (AZT-TP) complex (protein and DNA in turquoise sticks, AZT-TP in green sticks, PDB code 5I42); predicted steric clashes (<2 Å) indicated with red dashed lines.

Figure 3.

Modeling of the effects of the most prevalent resistance mutations on INDOPY-1 binding. (A) M184 sulfur-π interaction with INDOPY-1 (yellow dashes, distances for both copies of the complex present in the asymmetric unit) and M184V hydrophobic contact (cyan dashes). (B) Wild-type and K65R RT cation-π interactions (distances in green and in cyan dashes, respectively). (C) Crystallographic overlay of the nucleoside moiety of AZT-TP (PDB code 5I42) and INDOPY-1 (present structure). INDOPY-1 moieties are highlighted.

Figure 4.

Binding of compound A to HIV-1 RT/DNA. (A) Atomic model of compound A (violet) bound to HIV-1 RT (yellow) and DNA (pink), displaying the best pose from molecular docking (dashed lines color code as in Figure 1B). (B) Zoomed-in view of the cation-π interaction between K65 (and K65R, cyan) and the pyrimidone ring of compound A.

Chart 1.

Chemical Structures of the Reported NcRTI Chemotypes