Targeting a Conserved Lysine in the Hydrophobic Pocket of HIV-1 gp41 Improves Small Molecule Antiviral Activity.

Human Immunodeficiency virus (HIV-1) fusion is mediated by glycoprotein-41, a protein that has not been widely exploited as a drug target. Small molecules directed at the gp41 ectodomain have proved to be poorly drug-like, having moderate efficacy, high hydrophobicity and/or high molecular weight. We recently investigated conversion of a fairly potent hydrophobic inhibitor into a covalent binder, by modifying it to react with a lysine residue on the protein. We demonstrated a 10-fold improvement in antiviral efficacy. Here, we continue this study, utilizing instead molecules with better inherent drug-like properties. Molecules possessing low to no antiviral activity as equilibrium binders were converted into µM inhibitors upon addition of an electrophilic warhead in the form of a sulfotetrafluorophenyl (STP) activated ester. We confirmed specificity for gp41 and for entry. The small size of the inhibitors described here offers an opportunity to expand their reach into neighboring pockets while retaining drug-likeness. STP esterification of equilibrium binders is a promising avenue to explore for inhibiting HIV-1 entry. Many gp41 targeting molecules studied over the years possess carboxylic acid groups which can be easily converted into the corresponding STP ester. It may be worth the effort to evaluate a library of such inhibitors as a way forward to small molecule inhibition of fusion of HIV and possibly other enveloped viruses.

Enhanced potency of bivalent small molecule gp41 inhibitors.

Low molecular weight peptidomimetic inhibitors with hydrophobic pocket binding properties and moderate fusion inhibitory activity against HIV-1 gp41-mediated cell fusion were elaborated by increasing the available surface area for interacting with the heptad repeat-1 (HR1) coiled coil on gp41. Two types of modifications were tested: 1) increasing the overall hydrophobicity of the molecules with an extension that could interact in the HR1 groove, and 2) forming symmetrical dimers with two peptidomimetic motifs that could potentially interact simultaneously in two hydrophobic pockets on the HR1 trimer. The latter approach was more successful, yielding 40-60times improved potency against HIV fusion over the monomers. Biophysical characterization, including equilibrium binding studies by fluorescence and kinetic analysis by Surface Plasmon Resonance, revealed that inhibitor potency was better correlated to off-rates than to binding affinity. Binding and kinetic data could be fit to a model of bidentate interaction of dimers with the HR1 trimer as an explanation for the slow off-rate, albeit with minimal cooperativity due to the highly flexible ligand structures. The strong cooperativity observed in fusion inhibitory activity of the dimers implied accentuated potency due to the transient nature of the targeted intermediate. Optimization of monomer, dimer or higher order structures has the potential to lead to highly potent non-peptide fusion inhibitors by targeting multiple hydrophobic pockets.

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.

Unbiased screening of marine sponge extracts for anti-inflammatory agents combined with chemical genomics identifies girolline as an inhibitor of protein synthesis.

Toll-like receptors (TLRs) play a critical role in innate immunity, but activation of TLR signaling pathways is also associated with many harmful inflammatory diseases. Identification of novel anti-inflammatory molecules targeting TLR signaling pathways is central to the development of new treatment approaches for acute and chronic inflammation. We performed high-throughput screening from crude marine sponge extracts on TLR5 signaling and identified girolline. We demonstrated that girolline inhibits signaling through both MyD88-dependent and -independent TLRs (i.e., TLR2, 3, 4, 5, and 7) and reduces cytokine (IL-6 and IL-8) production in human peripheral blood mononuclear cells and macrophages. Using a chemical genomics approach, we identified Elongation Factor 2 as the molecular target of girolline, which inhibits protein synthesis at the elongation step. Together these data identify the sponge natural product girolline as a potential anti-inflammatory agent acting through inhibition of protein synthesis.

Total synthesis of exiguamines A and B inspired by catecholamine chemistry.

The evolution of a total synthesis of the exiguamines, two structurally unusual natural products that are highly active inhibitors of indolamine-2,3-dioxygenase (IDO), is described. The ultimately successful strategy involves advanced cross-coupling methodology and features a potentially biosynthetic tautomerization/electrocyclization cascade reaction that forms two heterocycles and installs a quaternary ammonium ion in a single synthetic operation.

Biomimetic synthesis of the shimalactones.

A biomimetic synthesis of shimalactone A and B is described. Its key features are an unprecedented acid-catalyzed cyclization of a dienyl beta-ketolactone and a Stille coupling/8pi-6pi electrocyclization cascade to create the oxabicyclo[2.2.1]heptane and bicyclo[4.2.0]octadiene, respectively. The synthesis is convergent and void of protecting groups.