The capsid domain of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein is a critical determinant of virus assembly, and is therefore a potential target for developing drugs for AIDS therapy. Recently, a 12-mer alpha-helical peptide (CAI) was reported to disrupt immature- and mature-like capsid particle assembly in vitro; however, it failed to inhibit HIV-1 in cell culture due to its inability to penetrate cells. The same group reported the X-ray crystal structure of CAI in complex with the C-terminal domain of capsid (C-CA) at a resolution of 1.7 A. Using this structural information, we have utilized a structure-based rational design approach to stabilize the alpha-helical structure of CAI and convert it to a cell-penetrating peptide (CPP). The modified peptide (NYAD-1) showed enhanced alpha-helicity. Experiments with laser scanning confocal microscopy indicated that NYAD-1 penetrated cells and colocalized with the Gag polyprotein during its trafficking to the plasma membrane where virus assembly takes place. NYAD-1 disrupted the assembly of both immature- and mature-like virus particles in cell-free and cell-based in vitro systems. NMR chemical shift perturbation analysis mapped the binding site of NYAD-1 to residues 169-191 of the C-terminal domain of HIV-1 capsid encompassing the hydrophobic cavity and the critical dimerization domain with an improved binding affinity over CAI. Furthermore, experimental data indicate that NYAD-1 most likely targets capsid at a post-entry stage. Most significantly, NYAD-1 inhibited a large panel of HIV-1 isolates in cell culture at low micromolar potency. Our study demonstrates how a structure-based rational design strategy can be used to convert a cell-impermeable peptide to a cell-permeable peptide that displays activity in cell-based assays without compromising its mechanism of action. This proof-of-concept cell-penetrating peptide may aid validation of capsid as an anti-HIV-1 drug target and may help in designing peptidomimetics and small molecule drugs targeted to this protein.

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.

In our attempt to optimize the lead HIV-1 entry antagonist, NBD-11021, we present in this study the rational design and synthesis of 60 new analogues and determination of their antiviral activity in a single-cycle and a multicycle infection assay to derive a comprehensive structure-activity relationship (SAR). Two of these compounds, NBD-14088 and NBD-14107, showed significant improvement in antiviral activity compared to the lead entry antagonist in a single-cycle assay against a large panel of Env-pseudotyped viruses. The X-ray structure of a similar compound, NBD-14010, confirmed the binding mode of the newly designed compounds. The in vitro ADMET profiles of these compounds are comparable to that of the most potent attachment inhibitor BMS-626529, a prodrug of which is currently undergoing phase III clinical trials. The systematic study presented here is expected to pave the way for improving the potency, toxicity, and ADMET profile of this series of compounds with the potential to be moved to the early preclinical development.

We previously identified two small-molecule CD4 mimetics-NBD-556 and NBD-557-and synthesized a series of NBD compounds that resulted in improved neutralization activity in a single-cycle HIV-1 infectivity assay. For the current investigation, we selected several of the most active compounds and assessed their antiviral activity on a panel of 53 reference HIV-1 Env pseudoviruses representing diverse clades of clinical isolates. The selected compounds inhibited tested clades with low-micromolar potencies. Mechanism studies indicated that they act as CD4 agonists, a potentially unfavorable therapeutic trait, in that they can bind to the gp120 envelope glycoprotein and initiate a similar physiological response as CD4. However, one of the compounds, NBD-09027, exhibited reduced agonist properties, in both functional and biophysical studies. To understand the binding mode of these inhibitors, we first generated HIV-1-resistant mutants, assessed their behavior with NBD compounds, and determined the X-ray structures of two inhibitors, NBD-09027 and NBD-10007, in complex with the HIV-1 gp120 core at ϳ2-Å resolution. Both studies confirmed that the NBD compounds bind similarly to NBD-556 and NBD-557 by inserting their hydrophobic groups into the Phe43 cavity of gp120. The basic nitrogen of the piperidine ring is located in close proximity to D368 of gp120 but it does not form any H-bond or salt bridge, a likely explanation for their nonoptimal antagonist properties. The results reveal the structural and biological character of the NBD series of CD4 mimetics and identify ways to reduce their agonist properties and convert them to antagonists.

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