Targeting HIV-1 Reverse Transcriptase Using a Fragment-Based Approach

Abstract

Human immunodeficiency virus type I (HIV-1) is a retrovirus that infects cells of the host's immune system leading to acquired immunodeficiency syndrome and potentially death. Although treatments are available to prevent its progression, HIV-1 remains a major burden on health resources worldwide. Continued emergence of drug-resistance mutations drives the need for novel drugs that can inhibit HIV-1 replication through new pathways. The viral protein reverse transcriptase (RT) plays a fundamental role in the HIV-1 replication cycle, and multiple approved medications target this enzyme. In this study, fragment-based drug discovery was used to optimize a previously identified hit fragment (compound B-1), which bound RT at a novel site. Three series of compounds were synthesized and evaluated for their HIV-1 RT binding and inhibition. These series were designed to investigate different vectors around the initial hit in an attempt to improve inhibitory activity against RT. Our results show that the 4-position of the core scaffold is important for binding of the fragment to RT, and a lead compound with a cyclopropyl substitution was selected and further investigated. Requirements for binding to the NNRTI-binding pocket (NNIBP) and a novel adjacent site were investigated, with lead compound 27-a minimal but efficient NNRTI-offering a starting site for the development of novel dual NNIBP-Adjacent site inhibitors.

Keywords: HIV-1; drug discovery; fragment-based drug design; non-nucleoside reverse transcriptase inhibitors (NNRTIs); reverse transcriptase.

Figure 1

Overview of fragment hit locations in newly identified sites by Bauman et al. [7]. Legend: HIV-1 RT p66 subunit with fingers (blue), palm (red), thumb (green), connection (yellow) and RNase H (orange); p51 shown in gray; rilpivirine bound to the NNRTI-binding pocket (NNIBP) is shown as yellow spheres; bound fragments are shown as cyan spheres. Each binding site is circled and color coded according to its name (i.e., purple corresponds to Knuckles). Site, compound, and potency information in Table S1. Created with PyMOL and BioRender.com.

Figure 2

The compound B-1 and the structural features of the three series synthesized in this study; Series 1 with changes around the core depicted in blue, Series 2 with changes to the ester depicted in orange and Series 3 with various alkyne extensions depicted in green.

Scheme 1

The synthesis of Series 1 analogues. Reagents and conditions: (i) DNPH, MeCN:H₂O (1:1) or THF:H₂O (1:1), 40 °C–60 °C, overnight; (ii) ethyl propiolate, K₂CO₃, DMF, rt, overnight.

Scheme 2

The synthesis of Series 2 analogues. Reagents and conditions: (i) K₂HPO₄, MeOH, 80 °C, overnight; (ii) LiOH. H₂O, THF:MeOH:H₂O (4:1:1), rt, 5 h. (iii) H₂SO₄, propanol, 85 °C, overnight; (iv) RNH₂, HBTU, DIPEA, DMF, rt, 4 h.

Scheme 3

The synthesis of Series 3 analogues. Reagents and conditions: (i) Pd(PPh₃)₄, CuI, diisopropylamine, 80 °C, overnight.

Figure 3

Crystal structure of HIV-1 RT in complex with the elaborated compound 27 (PDB ID 8FFX). Atomic model of 27 (magenta) bound to HIV-1 RT residues (blue) in the NNRTI-binding pocket. Pocket residues forming hydrophobic interactions shows as sticks. Polder mFo–DFc (OMIT) map density (green mesh, 3σ) of 27. Created using PyMOL and BioRender.com.

Figure 4

Comparison of the NNRTI Adjacent site of superposed RT-27 (PDB ID 8FFX, blue) and RT-RPV-B-1 (PDB ID 4KFB, orange) structures. Rilpivirine (RPV) is depicted in the neighboring NNIBP (orange). Nitrogen atoms are blue and oxygen atoms are red.

Figure 5

Surface representations of the NNIBP and NNRTI Adjacent sites with approved NNRTIs and investigational inhibitors. The compactness of the NNRTI Adjacent site in structures decreases from left to right (closed to open). NNRTIs and their protein bound structures are indicated in each panel, with their PDB ID in parenthesis. Lead compound B-1 (purple) is superimposed in the NNRTI Adjacent site (right). Compound in the NNRTI site are shown in a range of colors. Nitrogen atoms are blue and oxygen atoms are red.

Figure 6

Thumb subdomain position of RT in complex with approved or investigational NNRTIs. The structure of RT bound to 27 (PDB: 8FFX) is superimposed in green in each panel. Stars indicate complexes with measurable NNRTI Adjacent Site pocket volume (Table 4). Definitions: ETR—etravirine; DOR—doravirine; NVP—nevirapine; DEL—delavirdine; EFV—efavirenz; RPV—rilpivirine. Created with PyMOL and BioRender.com.

Figure 7

Superposition of WT RT (blue), compound 27 (magenta) and K07-15 compound (green) in the NNIBP. Compound B-1 (orange) is shown in the NNRTI Adjacent site (PDB 4KFB). Created with PyMOL and BioRender.com. Nitrogen atoms are blue and oxygen atoms are red.