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Thermodynamics of binding of a low-molecular-weight CD4 mimetic to human immunodeficiency virus (HIV-1) gp120 is studied. Results reveal that low-molecular-weight compounds can induce conformational changes in the HIV-1 gp120 glycoprotein similar to those in CD4 binding, revealing distinct strategies for inhibiting the function of the HIV-1 gp120 envelope glycoprotein.

Research indicates that the CD4 residue Phe43 maintains the high affinity of gp120 ligands but is not needed for initiating the conformational isomerization in gp120, leading to exposure of the binding sites for 17b antibody and CCR5 receptor.

Binding of the antibody epitope 17b to gp120 protein alters V1/V2 loop position thereby blocking the CD4 binding site and weakening CD4 interaction. Data also indicate that the neutralizing effect of the epitope 17b may be due to its blocking of the coreceptor CCR5 binding site on the gp120 protein.

The CD4-induced conformational change of glycoprotein gp120 as seen through envelope binding to the neutralizing human antibody 17b has been studied. The gp120 mediates the fusion of viral membrane of HIV-1 with the target cell membrane. A dextran sensor surface was used in immobilizing the 17b Fab fragment while the gp120 binding kinetics were studied by global and linear transformation analyses. Results indicate that the association rate of gp120 is increased 25 times when soluble CD4 is added. This is consistent with the greater exposure of the 17b binding epitope on gp120 when CD4 is bound.

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., Competing Interests: The authors declare no competing financial interest., (© 2022 American Chemical Society.)

The optimization, based on computational, thermodynamic, and crystallographic data, of a series of small-molecule ligands of the Phe43 cavity of the envelope glycoprotein gp120 of human immunodeficiency virus (HIV) has been achieved. Importantly, biological evaluation revealed that the small-molecule CD4 mimics (4-7) inhibit HIV-1 entry into target cells with both significantly higher potency and neutralization breadth than previous congeners, while maintaining high selectivity for the target virus. Their binding mode was characterized via thermodynamic and crystallographic studies.

The design, synthesis, thermodynamic and crystallographic characterization of a potent, broad spectrum, second-generation HIV-1 entry inhibitor that engages conserved carbonyl hydrogen bonds within gp120 has been achieved. The optimized antagonist exhibits a sub-micromolar binding affinity (110 nM) and inhibits viral entry of clade B and C viruses (IC 50 geometric mean titer of 1.7 and 14.0 μM, respectively), without promoting CD4-independent viral entry. thermodynamic signatures indicate a binding preference for the ( R,R )-over the ( S,S )-enantiomer. The crystal structure of the small molecule-gp120 complex reveals the displacement of crystallographic water and the formation of a hydrogen bond with a backbone carbonyl of the bridging sheet. Thus, structure-based design and synthesis targeting the highly conserved and structurally characterized CD4:gp120 interface is an effective tactic to enhance the neutralization potency of small molecule HIV-1 entry inhibitors.

Chemokine receptor CXCR4 is one of two principal coreceptors for the entry of HIV-1 into target cells. CXCR4 is known to form homodimers. We previously demonstrated that the amino terminus of viral macrophage protein II (vMIP-II) is the major determinant for CXCR4 recognition, and that V1 peptide derived from the N-terminus of vMIP-II (1-21 residues) showed significant CXCR4 binding. Interestingly, an all-d-amino acid analogue of V1 peptide, DV1 peptide, displayed an even higher binding affinity and strong antiviral activity in inhibiting the replication of CXCR4-dependent HIV-1 strains. In this study, we synthetically linked two DV1 peptides with the formation of a disulfide bond between the two cysteine residues present in the peptide sequence to generate a dimeric molecule potentially capable of interacting with two CXCR4 receptors. DV1 dimer exhibited enhanced binding affinity and antiviral activity compared with those of DV1 monomer. Ligand binding site mapping experiments showed that DV1 dimer overlaps with HIV-1 gp120 on CXCR4 binding sites, including several transmembrane (TM) residues located close to the extracellular side and the N-terminus of CXCR4. This finding was supported by the molecular modeling of CXCR4 dimer-DV1 dimer interaction based on the crystal structure of CXCR4, which showed that DV1 dimer is capable of interacting with the CXCR4 dimeric structure by allowing the N-terminus of each DV1 monomer to reach into the binding pocket of CXCR4 monomer. The development of this bivalent ligand provides a tool for further probing the functions of CXCR4 dimerization and studying CXCR4 heterodimerization with other receptors.

Cellular infection by HIV-1 is initiated with a binding event between the viral envelope glycoprotein gp120 and the cellular receptor protein CD4. The CD4-gp120 interface is dominated by two hotspots: a hydrophobic gp120 cavity capped by Phe43(CD4) and an electrostatic interaction between residues Arg59(CD4) and Asp368(gp120). The CD4 mimetic small-molecule NBD-556 (1) binds within the gp120 cavity; however, 1 and related congeners demonstrate limited viral neutralization breadth. Herein, we report the design, synthesis, characterization, and X-ray structures of gp120 in complex with small molecules that simultaneously engage both binding hotspots. The compounds specifically inhibit viral infection of 42 tier 2 clades B and C viruses and are shown to be antagonists of entry into CD4-negative cells. Dual hotspot design thus provides both a means to enhance neutralization potency of HIV-1 entry inhibitors and a novel structural paradigm for inhibiting the CD4-gp120 protein-protein interaction.

The distinctive properties of graphene sheets may be significantly influenced by the presence of corrugation structures. Our understanding of these graphene structures has been limited to the mesoscopic scale. Here we characterize angstrom-scale periodic buckling structures in free-standing graphene bilayers produced by liquid-phase processing in the absence of specific substrates. Monochromated, aberration-corrected transmission electron microscopy with sub-angstrom resolution revealed that the unit structures in the major buckling direction consist of only two and three unit cells of graphene's honeycomb lattice, resulting in buckling wavelengths of 3.6 ± 0.5 and 6.4 ± 0.8 Å, respectively. The buckling shows a strong preference of chiral direction and spontaneously chooses the orientation of the lowest deformation energy, governed by simple geometry rules agreeing with Euler buckling theory. Unexpectedly, the overall buckled structures demonstrate geometric complexity with cascaded features. First-principles calculations suggest that significant anisotropic changes in the electronic structure of graphene are induced by the buckling.

Structure-activity correlations were investigated for substituted peptide conjugates that function as dual receptor site antagonists of HIV-1 gp120. A series of peptide conjugates were constructed via click reaction of both aryl and alkyl acetylenes with an internally incorporated azidoproline 6 derived from the parent peptide 1 (12p1, RINNIPWSEAMM). Compared to 1, many of these conjugates were found to exhibit several orders of magnitude increase in both affinity for HIV-1 gp120 and inhibition potencies at both the CD4 and coreceptor binding sites of gp120. We sought to determine structural factors in the added triazole grouping responsible for the increased binding affinity and antiviral activity of the dual inhibitor conjugates. We measured peptide conjugate potencies in both kinetic and cell infection assays. High affinity was sterically specific, being exhibited by the cis- but not the trans-triazole. The results demonstrate that aromatic, hydrophobic, and steric features in the residue 6 side-chain are important for increased affinity and inhibition. Optimizing these features provides a basis for developing gp120 dual inhibitors into peptidomimetic and increasingly smaller molecular weight entry antagonist leads.

Human immunodeficiency virus type 1 (HIV-1) uses a chemokine receptor, usually CXCR4 or CCR5, for entry into the target cells. Here, we used a chemical biology approach to demonstrate that binding and signaling domains in CXCR4 are possibly distinct and separate, as the new analogue, D(1-10)-vMIP-II-(9-68)-SDF-1alpha (RCP222), could not activate CXCR4 despite the fact that its binding activity was comparable to that of stromal cell-derived factor (SDF)-1alpha, the only natural ligand of CXCR4.