Structural and Functional Characterization of Indane-Core CD4-Mimetic Compounds Substituted with Heterocyclic Amines

Abstract

The human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer on the virion surface interacts with the host receptors, CD4 and CCR5/CXCR4, to mediate virus entry into the target cell. CD4-mimetic compounds (CD4mcs) bind the gp120 Env, block CD4 binding, and inactivate Env. Previous studies suggested that a C(5)-methylamino methyl moiety on a lead CD4mc, BNM-III-170, contributed to its antiviral potency. By replacing the C(5) chain with differentially substituted pyrrolidine, piperidine, and piperazine ring systems, guided by structural and computational analyses, we found that the 5-position of BNM-III-170 is remarkably tolerant of a variety of ring sizes and substitutions, both in regard to antiviral activity and sensitization to humoral responses. Crystallographic analyses of representative analogues from the pyrrolidine series revealed the potential for 5-substituents to hydrogen bond with gp120 Env residue Thr 283. Further optimization of these interactions holds promise for the development of CD4mcs with greater potency.

Figure 1

(A) Structure of lead CD4mc bis-TFA salt BNM-III-170 (1) with Indane carbons numbered. (B) Cocrystal structure of BNM-III-170 in a gp120_C1086 core_e monomer with target residues Ala 281, Lys 282, and Thr 283 displayed. A transparent surface was added to show the Phe 43 binding cavity and surrounding vestibule surface.

Scheme 1. Synthesis of Preliminary Heterocyclic Amine Analogues of BNM-III-170 from the Late-Stage Synthetic Intermediate 4

Figure 2

Initial CD4mc heterocyclic amine substitutions and bioactivity that inspired further heterocyclic amine incorporation.

Scheme 2. Synthesis of Substituted Heterocyclic Amine Analogues of BNM-III-170 from Late-Stage Synthetic Intermediate 4

Figure 3

CD4mcs sensitize HIV-1 infected cells to ADCC. Primary CD4 T cells isolated from PBMC were infected with HIV-1_CH58TF for 48 h. (A) For cell surface staining, 1:1000 diluted HIV+ plasma (n = 3) was used in the presence of 50 μM of the different CD4mcs, (+)-BNM-III-170, or with equivalent volume of vehicle (DMSO), and an Alexa Fluor 647-conjugated antihuman IgG secondary Ab was then used for fluorescent labeling. (B) For ADCC, infected cells were used as target cells in a FACS-based ADCC assay that measures the killing of infected (p24+) cells to determine their susceptibility to ADCC mediated by a 1:1000 dilution of plasma from 3 HIV-1-infected individuals in the presence of 50 μM different CD4mcs, (+)-BNM-III-170, or with equivalent volume of vehicle (DMSO). (C) Primary CD4 T cells isolated from PBMC were infected with HIV-1_JRFL for 48 h. Cell surface staining was performed as described in (A), except at varying concentrations of CD4mc. Error bars indicate means ± standard errors of the means (SEM).

Figure 4

Crystal structures of CD4mcs in complex with gp120 core. (A) Solvent-excluded surface of 10j conformer A in a gp120 core. The surfaces of 10j and the gp120 core are shown in blue and beige, respectively. The transparency of the gp120 core surface was set at 50% so that the surface of 10j within the CD4 binding pocket is visible. Thereby, the blue surface of 10j inserted into the CD4 binding pocket, bounded by a thin black line, is seen through the beige gp120 surface; and the solvent-exposed surface of 10j, bounded by a thick line, remains fully blue. (B) A detailed view of 10j conformer A inserted into the CD4 binding pocket. This view has the complex rotated from Figure 4A by −60° about the x-axis followed by +8° about the y-axis, and the surface of 10j is replaced by a stick representation. (C) Crystal structure of 10j conformer A in a gp120 core. This structure was refined at 2.77 Å resolution. (D) Crystal structure of 10h in a gp120 core. This structure was refined at 3.07 Å resolution. (E) Crystal structure of 10j conformer B in a gp120 core. (F) Bottom view of 10j conformer A in a gp120 core to illustrate water-mediated contacts from 10j to Asn 425.