Monitoring Binding of HIV‐1 Capsid Assembly Inhibitors Using ¹⁹F Ligand‐and ¹⁵N Protein‐Based NMR and X‐ray Crystallography: Early Hit Validation of a Benzodiazepine Series

Abstract

The emergence of resistance to existing classes of antiretroviral drugs underlines the need to find novel human immunodeficiency virus (HIV)-1 targets for drug discovery. The viral capsid protein (CA) represents one such potential target. Recently, a series of benzodiazepine inhibitors was identified via high-throughput screening using an in vitro capsid assembly assay (CAA). Here, we demonstrate how a combination of NMR and X-ray co-crystallography allowed for the rapid characterization of the early hits from this inhibitor series. Ligand-based (19)F NMR was used to confirm inhibitor binding specificity and reversibility as well as to identify the N-terminal domain of the capsid (CA(NTD)) as its molecular target. Protein-based NMR ((1)H and (15)N chemical shift perturbation analysis) identified key residues within the CA(NTD) involved in inhibitor binding, while X-ray co-crystallography confirmed the inhibitor binding site and its binding mode. Based on these results, two conformationally restricted cyclic inhibitors were designed to further validate the possible binding modes. These studies were crucial to early hit confirmation and subsequent lead optimization.

Functional conservation of HIV-1 Gag: implications for rational drug design

“…To reveal the ideal drug target, we described 4 crystalized drug binding pockets in capsid (Figure  4, Additional file 2: Figure S4). Inhibitors that target pockets 1–3 have shown promising antiviral activity against capsid multimerization in different subtype strains by altering NTD-CTD interaction (pockets 1 and 3) or NTD-NTD interaction (pocket 2) [15,20,21]. Pocket 4 is less conserved and its polymorphic residues make direct contact with inhibitors, hindering the development of inhibitors that target this pocket [13].…”

19F-Modified Proteins and 19F-Containing Ligands as Tools in Solution NMR Studies of Protein Interactions

“…In an elegant study, Goudreau and colleagues characterized a series of benzodiazepine inhibitors that target the viral capsid protein (CA) of HIV-1, using a multipronged approach employing X-ray crystallography, and NMR (Goudreau et al, 2013). In particular, they used 19 F NMR to assess binding of inhibitors to HIV-1 Gag and subfragments such as, CA-Nucleocapsid (CA-NC).…”

“…The use of 19 F-containing ligands to probe protein-ligand interactions by NMR is also well established (Chen et al, 2013;Fielding, 2007;Ghitti et al, 2014;Hortin & Boime, 1983). 19 F-containing ligand-observe experiments have been used to provide information on ligand-binding affinity, specificity, and reversibility and to obtain ligandprotein structural constraints (Clore, Gronenborn, Birdsall, Feeney, & Roberts, 1984;Goudreau, Coulombe, & Faucher, 2013;Kim et al, 1990;Matei et al, 2013;Yu, Hajduk, Mack, & Olejniczak, 2006).…”

Allosteric modulation of protein oligomerization: an emerging approach to drug design

“…Structures from pdb files 1VUU (Zhao et al, 2013) and 2JPR (Kelly et al, 2007). The formulas of CAP-1 and a representative Benzimidazole (Goudreau et al, 2013) and Benzodiazepine (Tremblay et al, 2012) are given at the bottom. (C) Formula of CA-binding compound described by Lemke et al (2013).…”

“…In later studies, the compounds were modified and improved - Tremblay et al systematically modified the functional groups in a BM scaffold, yielding CA-inhibitors with IC 50 values below 0.1 μM (Tremblay et al, 2012). Goudreau et al developed BD-based compounds with IC 50 values below 1 μM (Goudreau et al, 2013). …”