Guangyan Zhou

Development of indole compounds as small molecule fusion inhibitors targeting HIV-1 glycoprotein-41.

Nonpeptide inhibition of fusion remains an important goal in anti-HIV research, due to its potential for low cost prophylaxis or prevention of cell-cell transmission of the virus. We report here on a series of indole compounds that have been identified as fusion inhibitors of gp41 through a structure-based drug design approach. Experimental binding affinities of the compounds for the hydrophobic pocket were strongly correlated to fusion inhibitory data (R(2) = 0.91), and corresponding inhibition of viral replication confirmed the hydrophobic pocket as a valid target for low molecular weight fusion inhibitors. The most active compound bound to the hydrophobic pocket and inhibited cell-cell fusion and viral replication at submicromolar levels. A common binding mode for the inhibitors in this series was established by carrying out docking studies using structures of gp41 in the Protein Data Bank. The molecules were flexible enough to conform to the contours of the pocket, and the most active compound was able to adopt a structure mimicking the hydrophobic contacts of the D-peptide PIE7. The results enhance our understanding of indole compounds as inhibitors of gp41.

Paramagnetic relaxation assisted docking of a small indole compound in the HIV-1 gp41 hydrophobic pocket

The hydrophobic pocket contained within the gp41 coiled coil is an important target for small molecules designed to inhibit HIV-1 fusion. While various screening experiments have identified molecules purported to bind in this pocket, few have confirmed details of the interaction, instead relying on computational docking to predict the binding mode. This is made more challenging by the fact that residues lining the hydrophobic pocket are highly flexible, as is typical for a protein-protein interaction site, limiting the predictive power of computational tools. In this study, we report on an NMR method to define the binding mode of 1-5i, a compound in a series of newly developed indole inhibitors. We show that paramagnetic relaxation enhancement of ligand protons due to an MTSL group positioned close to the binding pocket could be applied quantitatively to distinguish between more than 30 different computational poses, selecting a single pose that agreed with the NMR data. In this pose, important hydrophobic and polar contacts occur with pocket lysine, tryptophan, and glutamine residues, including putative hydrogen bonds between the ligand carboxylate and the lysine ε-amino group. A study of the ligand orientation suggests directions for optimization.

Design, Synthesis and Evaluation of Indole Compounds as Novel Inhibitors targeting Gp41

A series of indole ring containing compounds were designed based on the structure of the gp41 complex in the region of the hydrophobic pocket. These compounds were synthesized using a Suzuki Coupling reaction, and evaluated using a fluorescence binding assay and cell-cell fusion assay. The observed inhibition constant of compound 7 was 2.1microM, and the IC(50) for cell-cell fusion inhibition was 1.1microM. Assay data indicated that 7 is a promising lead compound for optimization into an effective low molecular weight fusion inhibitor.

NMR Second Site Screening for Structure Determination of Ligands Bound in the Hydrophobic Pocket of HIV-1 gp41

The development of nonpeptide fusion inhibitors through rational drug design has been hampered by the limited accessibility of the gp41 coiled coil target, which is highly hydrophobic, and the absence of structural data defining details of small molecule interactions. Here we describe a new approach for obtaining structural information on small molecules bound in the hydrophobic pocket of gp41, using a paramagnetic probe peptide which binds adjacent to the pocket along an extended coiled coil. Ligand binding in the pocket leads to paramagnetic relaxation effects or pseudocontact shifts of ligand protons. These effects are distance and/or orientation dependent, permitting determination of ligand pose in the pocket. The method is demonstrated with a fast-exchanging ligand. Multiple measurements at different coiled coil and probe peptide ratios enabled accurate determination of the NMR parameters. Use of a labeled probe peptide stabilizes an otherwise aggregation-prone coiled coil and also enables modulation of the paramagnetic effect to study ligands of various affinities. Ultimately, this technique can provide essential information for structure-based design of nonpeptide fusion inhibitors.

Structure−Activity Relationship Studies of Indole-Based Compounds as Small Molecule HIV‑1 Fusion Inhibitors Targeting Glycoprotein 41

We previously described indole-containing compounds with the potential to inhibit HIV-1 fusion by targeting the hydrophobic pocket of transmembrane glycoprotein gp41. Here we report optimization and structure–activity relationship studies on the basic scaffold, defining the role of shape, contact surface area, and molecular properties. Thirty new compounds were evaluated in binding, cell–cell fusion, and viral replication assays. Below a 1 μM threshold, correlation between binding and biological activity was diminished, indicating an amphipathic requirement for activity in cells. The most active inhibitor 6j exhibited 0.6 μM binding affinity and 0.2 μM EC50 against cell–cell fusion and live virus replication and was active against T20 resistant strains. Twenty-two compounds with the same connectivity displayed a consensus pose in docking calculations, with rank order matching the biological activity. The work provides insight into requirements for small molecule inhibition of HIV-1 fusion and demonstrates a potent low molecular weight fusion inhibitor.

Amphipathic Properties of HIV-1 gp41 Fusion Inhibitors

Small molecule inhibition of HIV fusion has been an elusive goal, despite years of effort by both pharmaceutical and academic laboratories. In this review, we will discuss the amphipathic properties of both peptide and small molecule inhibitors of gp41-mediated fusion. Many of the peptides and small molecules that have been developed target a large hydrophobic pocket situated within the grooves of the coiled coil, a potential hotspot for inhibiting the trimer of hairpin formation that accompanies fusion. Peptide studies reveal molecular properties required for effective inhibition, including elongated structure and lipophilic or amphiphilic nature. The characteristics of peptides that bind in this pocket provide features that should be considered in small molecule development. Additionally, a novel site for small molecule inhibition of fusion has recently been suggested, involving residues of the loop and fusion peptide. We will review the small molecule structures that have been developed, evidence pointing to their mechanism of action and strategies towards improving their affinity. The data points to the need for a strongly amphiphilic character of the inhibitors, possibly as a means to mediate the membrane - protein interaction that occurs in gp41 in addition to the protein - protein interaction that accompanies the fusion-activating conformational transition.

Large scale enzymatic synthesis of oligosaccharides and a novel purification process

Herein we report the practical chemo enzymatic synthesis of trisaccharide and derivatives of iGb3 and Gb3, and a novel purification process using immobilized yeast to remove the monosaccharide from the reaction mixture. High purity oligosaccharide compounds were achieved in large scale. This study represents a facile enzymatic synthesis of and novel purification process of oligosaccharide.

Synthesis and evaluation of the diarylthiourea analogs as novel anti-cancer agents

Ten p-nitrodiarylthiourea analogs were designed, synthesized and evaluated in breast (MCF-7, T-47D, MDA-MB-453) and prostate (DU-145, PC-3, LNCaP) cancer cell lines for their anticancer activities. The majority of the compounds were able to inhibit the growth of these six cancer cell lines at low micromolar concentrations. Compound 7 was found to be the most potent anticancer agent in this series with GI50 values of 3.16μM for MCF-7, 2.53μM for T-47D, 4.77μM for MDA-MB-453 breast cancer lines and 3.54μM for LNCaP prostate cancer cell line. These GI50 values were comparable to the parent compound, SHetA2.

SHetA2, a New Cancer-Preventive Drug Candidate

SHetA2 (NSC 721689) is a novel synthetic flexible heteroarotinoid that has promising cancer-preventive activity, and has exhibited growth inhibition on 60 cancer cell lines in vitro, along with ovarian, lung, and kidney cancers in vivo. It binds and interferes with the function of a molecular chaperone, mortalin, leading to mitochondrial swelling and mitophagy that induce apoptosis in cancer cells without harming normal cells. It showed minimal toxicity in preclinical studies and thus is now in Phase-0 clinical trial. This chapter summarizes its evolution, synthesis, structure-activity relationship, mechanism of action, pharmacokinetics, and potential clinical applications in last 12 years. It also provides insights into designing more potent and safer SHetA2 analogs for future cancer-preventive drug development.