The synthesis and antiviral properties of pyridinioalkanoyl thioester (PATE) compounds that target nucleocapsid p7 protein (NCp7) of the human immunodeficiency virus type 1 (HIV-1) have been described previously (Turpin, J. A., Song, Y., Inman, J. K., Huang, M., Wallqvist, A., Maynard, A., Covell, D. G., Rice, W. G., and Appella, E. (1999) J. Med. Chem. 42, 67-86). In the present study, fluorescence and electrospray ionizationmass spectrometry were employed to determine the mechanism of modification of NCp7 by two lead compounds, N-[2-(5-pyridiniovaleroylthio)benzoyl]sulfacetamide bromide and N-[2-(5-pyridiniovaleroylthio)-benzoyl]-4-(4-nitrophenylsulfonyl)aniline bromide (compounds 45 and 47, respectively). Although both compounds exhibit antiviral activity in cell-based assays, we failed to detect appreciable ejection of zinc from NCp7 under conditions in which previously described NCp7-active disulfides readily eject zinc. However, upon "activation" by Ag ؉ , compound 45 reacted with NCp7 resulting in the zinc ejection from both zinc fingers. The reaction followed a two-step mechanism in which zinc was ejected from the carboxyl-terminal zinc finger faster than from the amino-terminal zinc finger. Both compounds covalently modified the protein with pyridinioalkanoyl groups. Compound 45 modified cysteines 36 and 49 of the carboxyl-terminal zinc finger.The results obtained herein demonstrate that PATE compounds can be constructed that selectively target only one of the two zinc fingers of NCp7, thus providing an impetus to pursue development of highly selective zinc finger inhibitors.
Development of drug-resistant HIV1 strains in response to therapy with inhibitors of the viral reverse transcriptase (1-3) and protease enzymes (4, 5) has necessitated the search for novel antiretroviral agents that are directed against new molecular targets. The involvement of HIV-1 NCp7 zinc fingers in multiple phases of the HIV-1 replication cycle and their mutationally non-permissive nature has provided incentives for choosing this protein as a target for antiretroviral therapy. Moreover, mutations or modifications of either the conserved zinc chelating or non-chelating residues have resulted in loss of NCp7-mediated activities, including rendering the HIV noninfectious (6 -8). These observations gave impetus to explore several types of organic compounds that selectively target NCp7 protein. First among them being 3-nitrosobenzamide (9) followed by a series of 2,2Ј-dithiobis(benzamide) disulfides (DIBA) (10) and azodicarbonamide (11) that inhibited a wide range of HIV-1 isolates. Even though 2,2Ј-dithiobis(benzamide) disulfides represent a new class of highly specific antiretroviral agents, the disulfide bond is susceptible to reduction in vivo, resulting in the loss of antiviral activity. To circumvent this problem, we synthesized novel pyridinioalkanoyl thioester (PATE) derivatives (12). Of various such compounds synthesized, two of them, compounds 45 and 47 (Fig. 1), showed superior antiviral activity. Both compounds were sp...