Compounds based on an S-acyl- 2-mercaptobenzamide thioester (SAMTs) template have been shown to specifically eject zinc from the C-terminal zinc-binding domain (ZD2) of NCp7 via acyl transfer from the thioester to the sulfur of a zinc-coordinating cysteine residue. These zinc-binding domains are excellent targets for the development of new antiretroviral and microbicidal agents because of their structural conservation and the broad range of functions of NCp7 in the viral life cycle. Our previous work identified several lead SAMT compounds that were shown to be virucidal and to inhibit cell-to-cell associated transmission of HIV-1 in co-culture systems. In addition, based on mutational analysis of the NCp7 amino acid sequence, we were able to extend the previously reported mechanism of action of the SAMT compounds to include a secondary S to N intramolecular acyl transfer that occurs after the primary transfer from the thioester to a cysteine side chain in the protein. This essential step is irreversible and results in a permanent disruption of the functional native structure. As a corollary to our in vitro studies on the mechanism of action of the SAMT compounds, we have explored their mechanism of viral inactivation in cells. We have determined that treatment with the SAMT compounds results in covalent modification of the Gag polyprotein, consistent with our in vitro results. Furthermore, we have observed that the NCp7 protein is not properly processed from the Gag polyprotein. Incubation of unprocessed Gag protein with exogenous viral protease did not yield processed NCp7, demonstrating that treatment with the SAMT compounds results in processing defects. Since Gag cleavage in a native structural environment is critical for producing mature infectious virions, these results suggest that the SAMT compounds likely inhibit infectivity by preventing the maturation of the released virions. We are currently evaluating the SAMT compounds for use as vaginal, topical microbicides. As part of these efforts, we are exploring ways to formulate the SAMT compounds for sustained delivery. In collaboration with Patrick Kiser at the University of Utah, we are analyzing the stability of our lead SAMT compounds. This is a critical step in formulation, as the stability of the compound at various pHs will determine how the compound should be formulated. We have determined that SAMT-8, containing a benzoyl acyl group, has better stability in comparison with other compounds tested and will likely be the best candidate for further formulation testing. We are also using mass spectrometry experiments to elucidate the metabolism and pharmacokinetics of the SAMT compounds. In collaboration with investigators at Waters Corporation, we have identified a group of potential metabolites of the SAMT compounds that result from exposure of the compounds to liver microsomes in vitro. Currently we are working to identify metabolites of the SAMT compounds extracted from vaginal biopsy tissue. We will continue to investigate the activity and metabolism of the lead SAMT compounds in cellular model systems. Identification of a potentially safe and efficacious single or combination candidate microbicide in non-human primates, and the elucidation of their pharmacokinetics, will lead the way into studies necessary for preclinical evaluation of the SAMT compounds.
