GS-CA Compounds: First-In-Class HIV-1 Capsid Inhibitors Covering Multiple Grounds

Abstract

Recently reported HIV-1 capsid (CA) inhibitors GS-CA1 and GS-6207 (an analog of GS-CA1) are first-in-class compounds with long-acting potential. Reportedly, both compounds have greater potency than currently approved anti-HIV drugs. Due to the limited access to experimental data and the compounds themselves, a detailed mechanism of their inhibition is yet to be delineated. Using crystal structures of capsid-hexamers bound to well-studied capsid inhibitor PF74 and molecular modeling, we predict that GS-CA compounds bind in the pocket that is shared by previously reported CA inhibitors and host factors. Additionally, comparative modeling suggests that GS-CA compounds have unique structural features contributing to interactions with capsid. To test their proposed binding mode, we also report the design of a cyclic peptide combining structural units from GS-CA compounds, host factors, and previously reported capsid inhibitors. This peptide (Pep-1) binds CA-hexamer with a docking score comparable to GS-CA compounds. Affinity determination by MicroScale thermophoresis (MST) assays showed that CA binds Pep-1 with a ~7-fold better affinity than well-studied capsid inhibitor PF74, suggesting that it can be developed as a possible CA inhibitor.

Keywords: assembly; capsid; disassembly; human immunodeficiency virus; inhibitors; small molecules; uncoating.

Figure 1

Structure of HIV-1 CA protein and representative CA inhibitors. (A) This figure was generated from the X-ray crystal structure of native HIV-1 capsid protein bound to PF74 (PDB entry 4XFZ) (Gres et al., 2015). NTD, N-terminal domain; CA-CTD, C-terminal domain. (B) Chemical structures of selective CA inhibitors. The structures of CA inhibitors shown here have either been solved in complex with CA, or we have used them in docking protocols.

Figure 2

Molecular model of CA/GS-CA1 complex. (A) Docked pose of GS-CA1 in CA-hexamer (only dimer shown). (B) Close up of predicted GS-CA1 binding site in CA-hexamer. The side chains of CA and GS-CA1 are rendered as ball-and-stick. The backbone of CA is rendered as ribbons. Residues with orange carbons depict GS-CA1 resistance mutation positions (Perrier et al., 2017). (C) A detailed view of these residues and their proximity to GS-CA1. (D) Other residues of CA that interact with GS-CA1. GS-CA1 carbons in this and in subsequent figures are shown as green. The nitrogen, oxygen, sulfur, and fluorine atoms are colored blue, red, yellow, and aquamarine, respectively.

Figure 3

(A) Superposition of GS-CA1 and GS-6207. The switched position of cyclopenta-pyrazole ring is shown by dotted circle. (B) Difference in the side chain conformations of K70 and R173 between CA/GS-CA1 (green carbons) and CA/GS-6207 (gray carbons) complexes. Solid arrow shows the displacement of NZ atom of K73 in two complexes, whereas the dotted line shows the H-bond formed by GS-6207 with K70. This interaction is missing in CA/GS-CA1 complex. (C) Superposition of CA/PF74 crystal structure (PDB entry 4XZF) on the molecular model of CA/GS-CA1. The resistance mutations associated with PF74 close to the binding pocket are shown in cyan carbons. GS-CA1 resistance associated residues are depicted as in Figure 2. (D) Approximately 45° rotated view of Panel A. Dotted circles show superposition of three structural components of GS-CA1 and PF74 (magenta carbons). (E) Superposition of GS-CA1, PF74, and BI-2. The nitrogen, oxygen, sulfur, and fluorine atoms are colored blue, red, yellow, and aquamarine, respectively.

Figure 4

(A) Superposition of CA/CPSF6 crystal structure on the molecular model of CA/GS-CA1. For clarity, residues P313-P316 and backbone atoms of CPSF6 have been omitted. Peptide CPSF6 is shown in yellow carbons, and the backbone of CPSF6 peptide is shown as a yellow tube. This figure shows the superposition of the difluorobenzyl ring of GS-CA1 on F321 of CPSF6. (B) Superposition of CA/NUP153 crystal structure complex on the molecular model of CA/GS-CA1. For clarity, only residues F1415-G1418 are shown. The carbon atoms and backbone (tube) of NUP153 are shown in faded red-orange color. This figure shows the superposition of the difluorobenzyl ring of GS-CA1 on F1417 of NUP153. In addition, F1415 of NUP153 and the methylsulfonyl group of GS-CA1 have a common interaction with P38 of CA-hexamer. The atoms of P38 are not shown for clarity.

Figure 5

Superposition of CA/CAP-1 crystal structure complex on the molecular model of CA/GS-CA1. For clarity, only M66 in the two structures is shown (orange – CA/GS-CA1; teal – CA/CAP-1).

Figure 6

Pep-1 (A) and PF74 (B) binding affinities of CA as determined by MicroScale thermophoresis. This figure shows the change in fluorescence due to thermophoresis at the increasing concentrations of Pep-1 and PF74 (1–2,000 nM) in the presence of 200 nM CA-hexamers.