The work done in this project has been an integral part of a team effort with NCI and NIAID laboratories to develop new inhibitors of human immunodeficiency virus (HIV) that target the highly conserved nucleocapsid protein (NCp7). The Bioorganic Chemistry Section has had the responsibility to design the actual drug candidates and to carry out their synthesis and characterization. This past year we have carried out a vigorous program of synthesis of N-substituted 2-mercaptobenzamide thioesters, their derivatives and intermediates in order to improve the oral bioavailability profile as predicted for drug-like molecules and to study structure-activity relationships using in vitro assays. A panel of over 250 compounds have been examined in order to optimize the structure of candidate drugs and explore their mechanism of action. These studies have allowed us to design and successfully test above-type thioesters that show optimal stability toward serum enzyme-promoted hydrolysis. These data revealed that there was no significant correlation between thioester stability and antiviral activity; however, a slight inverse correlation between stability in serum and virucidal activity was noted. Based on this virucidal capability and our ability to select lead compounds to inhibit virus expression from latenly infected TNF-alpha-induced U1 cells, we determined if these compounds could prevent HIV cell-to-cell transmission. Several thioesters demonstrated potent inhibition of HIV cell-to-cell transmission in the 80 to 100 nanomolar range. Thus, these selected compounds show important potential for development as topical microbicides. Many of these compounds are active against HIV-1, HIV-2 and simian immunodeficiency virus (SIV), indicating a highly conserved target. A radiolabeled thioester was studied for its action on HIV-infected human cells in culture. Visualization of electrophoretic patterns of intracellular proteins by autoradiography and by Western blotting with antibody specific for NCp7 revealed clearly that NCp7 is indeed a target for the drug and that it is inactivated by an acylation reaction from a portion of the thioester. Additional biochemical experiments and NMR structural studies have revealed more details concerning the molecular mechanism of action of thioesters on this target. A carefully updated patent application (PCT), filed on July 24, 2002 and covering the uncharged thioesters and corresponding, active free thiols, has entered national phase filings.
