The work done in this project has been an integral part of a team effort to develop new inhibitors of HIV that target the highly conserved nucleocapsid protein (NCp7) zinc finger structures of the virion. Other team members are Drs. Ettore Appella and Yongsheng Song (Laboratory of Cell Biology, NCI), Drs. William Rice, Jim Turpin and Mingjun Huang (Laboratory of Antiviral Drug Mechanisms, NCI-Frederick), and Drs. David Covell, Andrew Maynard and Anders Wallqvist (Laboratory of Mathematical Biology, NCI- Frederick). This Section (JKI) has the responsibility to design the actual drug candidates and to direct and participate in their synthesis. The nucleocapsid proteins of virtually all human retroviruses contain one or two copies of the zinc finger motif, Cys-(Xaa)2- Cys-(Xaa)4-His-(Xaa)4-Cys (or """"""""CCHC"""""""" motif), which play a number of essential roles in both early and late phases of the replication cycle. It has been shown that these structures are thereby mutationally intolerant. Thus, a drug that inactivates nucleocapsid zinc finger function would not be likely to induce drug resistance and should be active on all strains of HIV-1, HIV-2 and possibly other retroviruses. It was shown by several groups of investigators that 2,2?- dithiobis(benzamides) and several other classes of compounds with oxidizing potential can induce the ejection of Zn ions from one or both zinc fingers of HIV-1 NCp7. Mechanistic studies demonstrated that this event followed covalent (electrophilic) attack on one or more of the crucial cysteine (Cys) residues by which the zinc is chelated. Release of zinc is accompanied by loss of structural integrity and formation of disulfide cross- links within and between the disrupted peptide loop structures, which is an irreversible process. As reported last year, we began exploring a number of variants of the dithiobis(benzamides) with regard to zinc-ejection capacity, in vitro (HIV) antiviral potency, and cellular toxicity. We also prepared, and tested in these assays, other chemotypes and discovered that certain thiolesters showed considerable promise as antiviral agents. Some of these compounds, because of their low toxicity, exhibited very good therapeutic indices (low toxicity coupled with high potency). What is especially interesting is that an entirely different mechanism must operate for the release of zinc; the thiolester sulfur atoms, being in their lowest oxidation state, cannot directly form disrupting disulfide bonds to the NCp7 cysteines. Instead, we postulate attack on a cysteine sulfur via a trans-thioacylation reaction, which underscores the novelty of this class of compounds as zinc finger inhibitors. This mechanism of action is under investigation in Dr. Appella?s lab using mass spectrometric techniques. We have synthesized and screened a panel of the thiolesters, exploring various aspects of structure in relation to function. In particular, we identified a subclass of thiolesters that bear a pyridinium cationic group. These compounds which we dub, """"""""PATEs"""""""" (for pyridinioalkanoyl thiolesters), are noteworthy for their good potency (low micromolar EC50s), very low toxicity, and appreciable water solubility. Their low molecular weight and stability toward glutathione highlight potential advantages over the dithiobis(benzamides) and derivative benzoisothiazolones, especially in regard to bioavailability and retention of in vivo activity. Antiviral (HIV-1) action and toxicity of several selected thiolesters are currently being tested in an infected mouse model. We have submitted a manuscript and filed a patent. The patent covers the thiolester chemotype and its various possible applications, for treatment of HIV infection and for preparing inactivated virus vaccines and diagnostic products.
