Optimization of HIV-1 Zinc Finger Inhibitors (ZFI) as Topical Microbicides and Mechanism of Action of S-acyl-2-mercaptobenzamide Thioesters.Our efforts have focused on the evaluation of the therapeutic potential of S-acyl-2- mercaptobenzamide thioester (SAMT) compounds targeting the zinc fingers of the HIV-1 nucleocapsid protein (NCp7). These structures are an excellent target for the development of new antiviral and microbicidal agents, based on their structural conservation and broad range of function in the viral life cycle. Alteration of any one of the zinc-chelating amino acids or any of a number of surrounding residues results in the production of noninfectious virus. Our previous work has identified three SAMT compounds that were shown to be virucidal and to inhibit cell-to-cell associated transmission of HIV-1 in co-culture systems; therefore, they have the potential to act as topical microbicides to prevent the transmission of HIV-1. Recently, we have worked closely with Dr. Robin Shattock at St. Georges Medical College, London, UK using his human cervical and rectal explant models for inhibition of HIV-1 infection and transmission. The three SAMT compounds, when tested in the cervical explant model, were able to block infection by cell-free virus from migratory cells. Importantly, SAMT treatment resulted in production of non-infectious virus from cervical explants and also inhibited dissemination of HIV-1 by cervical explant migratory cells. Recently, we have tested one thioester, SAMT-247, in the Simian/Human Immunodeficiency Virus (SHIV)/rhesus macaque model of HIV-1 transmission in collaboration with Dr. Cecilia Cheng-Mayer at the Aaron Diamond AIDS Research Center in New York. These studies have been performed using a mixture of pathogenic CXCR4 (X4) SHIVSF33A and CCR5 (R5) SHIVSF162P3 viruses. Initial experiments have been designed to address the efficacy and safety of SAMT-247 using single applications of the formulated compound. By using a mixture of X4- and R5-SHIV for infection, the relative proportion of the two viruses in the plasma could be evaluated by real-time PCR. In a pilot study, six rhesus macaques treated with Depo-Provera 5 weeks prior to challenge were inoculated once with a mixed inoculum containing 150 TCID50 each of X4 SHIVSF33A and R5 SHIVSF162P3. A 2 ml SAMT-247 microbicide (1%) formulated in the universal placebo gel HEC (hydroxyethyl-cellulose) was applied 20 minutes prior to challenge. SAMT-247 protected five of six macaques against transmission of both X4 and R5 SHIV. When compared to data in which five of five Depo-treated macaques challenged in the presence of placebo gel with the same virus stocks and dose were found to be infected, protection conferred by SAMT-247 reached statistical significance (p = 0.0152, Fisher's exact test). In addition to studies on the therapeutic potential of the SAMT compounds, we have also continued our efforts to understand the mechanism of action of these compounds. Previously, we proposed a general mechanism in which the thiol of a cysteine of NCp7 undergoes a nucleophilic attack on the thioester carbonyl carbon of SAMT, resulting in an acyl transfer from the SAMT to the cysteine sulfur, thiol release, and loss of NCp7 zinc coordination. Recently, we have used NCp7 mutational studies to investigate the target structure requirements for the SAMT reaction. We determined that position x+1 (where x is Cys36, the first cysteine residue in the carboxyl-terminal zinc-binding domain), needs to be an aromatic residue for reactivity, with tryptophan preferred over phenylalanine and the latter preferred over tyrosine. Based upon these mutational studies, we suggest that the SAMT compounds use the aromatic side chain of tryptophan at positon x+1 to dock on NCp7. This properly orients the thioester bond for optimal transacylation to the cysteine sulfur.
