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.

An analog of camptothecin inactive against Topoisomerase I is broadly neutralizing of HIV-1 through inhibition of Vif-dependent APOBEC3G degradation.

Camptothecin (CPT) is a natural product discovered to be active against various cancers through its ability to inhibit Topoisomerase I (TOP1). CPT analogs also have anti-HIV-1 (HIV) activity that was previously shown to be independent of TOP1 inhibition. We show that a cancer inactive CPT analog (O2-16) inhibits HIV infection by disrupting multimerization of the HIV protein Vif. Antiviral activity depended on the expression of the cellular viral restriction factor APOBEC3G (A3G) that, in the absence of functional Vif, has the ability to hypermutate HIV proviral DNA during reverse transcription. Our studies demonstrate that O2-16 has low cytotoxicity and inhibits Vif-dependent A3G degradation, enabling A3G packaging into HIV viral particles that results in A3G signature hypermutations in viral genomes. This antiviral activity was A3G-dependent and broadly neutralizing against sixteen HIV clinical isolates from groups M (subtypes A-G), N, and O as well as seven single and multi-drug resistant strains of HIV. Molecular modeling predicted binding near the PPLP motif crucial for Vif multimerization and activity. O2-16 also was active in blocking Vif degradation of APOBEC3F (A3F). We propose that CPT analogs not active against TOP1 have novel therapeutic potential as Vif antagonists that enable A3G-dependent hypermutation of HIV.

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.

A novel anti-HIV active integrase inhibitor with a favorable in vitro cytochrome P450 and uridine 5'-diphospho-glucuronosyltransferase metabolism profile.

Research efforts on the human immunodeficiency virus (HIV) integrase have resulted in two approved drugs. However, co-infection of HIV with Mycobacterium tuberculosis and other microbial and viral agents has introduced added complications to this pandemic, requiring favorable drug-drug interaction profiles for antiviral therapeutics targeting HIV. Cytochrome P450 (CYP) and uridine 5'-diphospho-glucuronosyltransferase (UGT) are pivotal determining factors in the occurrence of adverse drug-drug interactions. For this reason, it is important that anti-HIV agents, such as integrase inhibitors, possess favorable profiles with respect to CYP and UGT. We have discovered a novel HIV integrase inhibitor (compound 1) that exhibits low nM antiviral activity against a diverse set of HIV-1 isolates, and against HIV-2 and the simian immunodeficiency virus (SIV). Compound 1 displays low in vitro cytotoxicity and its resistance and related drug susceptibility profiles are favorable. Data from in vitro studies revealed that compound 1 was not a substrate for UGT isoforms and that it was not an inhibitor or activator of key CYP isozymes.