Structure-Based Design of a Small Molecule CD4-Antagonist with Broad Spectrum Anti-HIV-1 Activity

Abstract

Earlier we reported the discovery and design of NBD-556 and their analogs which demonstrated their potential as HIV-1 entry inhibitors. However, progress in developing these inhibitors has been stymied by their CD4-agonist properties, an unfavorable trait for use as drug. Here, we demonstrate the successful conversion of a full CD4-agonist (NBD-556) through a partial CD4-agonist (NBD-09027), to a full CD4-antagonist (NBD-11021) by structure-based modification of the critical oxalamide midregion, previously thought to be intolerant of modification. NBD-11021 showed unprecedented neutralization breath for this class of inhibitors, with pan-neutralization against a panel of 56 Env-pseudotyped HIV-1 representing diverse subtypes of clinical isolates (IC50 as low as 270 nM). The cocrystal structure of NBD-11021 complexed to a monomeric HIV-1 gp120 core revealed its detail binding characteristics. The study is expected to provide a framework for further development of NBD series as HIV-1 entry inhibitors for clinical application against AIDS.

Figure 1

Structures of NBD series compounds and their CD4-agonist/antagonist characterization by functional (cell-based assay) and biophysical (competitive-SPR) experiments. (a) The chemical structures of NBD series compounds indicating regions I, II, and III and the stereoisomers of NBD-11021. (b) Dose-dependent curve of CD4-positive Cf2Th/CD4–CCR5 cells infected with CD4-dependent HIV-1_ADA (solid line) and CD4-negative Cf2Th–CCR5 cells infected with CD4-independent HIV-1_ADAN197S (dashed line) in the presence of different concentrations of NBD-11021, NBD-556, and NBD-09027. Three independent experiments were performed in triplicate, and the graph is representative of one experiment; the values represent the mean ± standard deviation. (c) CD4-negative Cf2Th–CCR5 cells were infected with CD4-dependent HIV-1_ADA in the presence of increasing concentrations of NBD-11021, NBD-556, NBD-09027, and BMS-378806. Relative virus infectivity specifies the amount of infection detected in the presence of the compounds relative to the infection detected in the absence of the compounds. Three independent experiments were performed in triplicate, and the graph is representative of one experiment; the values represent the mean ± standard deviation. (d) Impact of NBD series compounds on the binding between mAb 17b and YU2 core gp120 protein. The YU2 core gp120 was passed over the anti-human IgG-captured 17b in the absence or presence of saturated NBD series compounds or sCD4 (control). The injection time was 2 min, and the disassociation time was 5 min. The graph is representative of two independent experiments. (e, f) Competitive SPR to probe whether NBD-11021 mimics the binding site of sCD4 and VRC-PG04. The protein alone or in the presence of varied concentrations of NBD-11021 was passed over YU2 core-immobilized surface on the CM5 chip. The association time and the dissociation time were 2 and 5 min, respectively.

Figure 2

Effect of NBD-11021 on cell-to-cell transmission of HIV-1. (a) Antiviral activity of NBD compounds in a CD4-dependent CXCR4-tropic HIV-1 cell–cell transmission assay. Ghost X4/R5 cells and H9/HIV-1_IIIB cells were cocultured in the presence of escalating concentrations of test compounds. (b) Antiviral activity of NBD compounds in a CD4-dependent CCR5-tropic HIV-1 cell–cell transmission assay. Ghost X4/R5 cells and MOLT-4/HIV-1_ADA cells were cocultured in the presence of escalating concentrations of test compounds. The plots represent the percentage of inhibition of p24 in the target cells Ghost X4/R5. Three independent experiments were performed in triplicate, and the graph is representative of one experiment; the values represent the mean ± standard deviation.

Figure 3

Crystal structure of NBD-11021-bound HIV-1 gp120. (a) NBD-11021 is shown in stick representation with its feature-enhanced map (FEM) (blue mesh) contoured at 1.5σ. The nitrogen atom in the piperidine ring makes a hydrogen bond with Asp368 of gp120. The average length of hydrogen bonds between the nitrogen atom and Asp368 is about 3.1 Å (There are four NBD-11021-bound gp120 molecules in the asymmetric unit). (b, c) Superposition of NBD-11021 (purple)-bound gp120 and NBD-09027 (yellow)-bound gp120 structures. The phenyl rings were superimposable (c), but the piperidine rings deviate substantially from each other (b, c), which results in the average distance between the nitrogen in the piperidine ring of NBD-11021 (NBD-09027) and Asp368 being about 3.1 Å (4.5 Å). (d) Detailed interactions between NBD-11021 and gp120.

Scheme 1

Retrosynthetic Analysis of NBD-11021 (15)

Scheme 2

Outline of the Synthesis of NBD-11021 (Diastereomeric Mixture), NBD-11021A (15a), and NBD-11021B (15b)