Inactivation of HIV-1 Nucleocapsid Protein P7 by Pyridinioalkanoyl Thioesters

Abstract

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...

Targeting the Viral Nucleocapsid Protein in Anti-HIV-1 Therapy

“…As mentioned above, NCp7 zinc depletion begins at the level of the C-terminal ZF, which is consistent with its lower thermal stability and lower zinc affinity in comparison with the N-terminal ZF [27,32,54,55]. The Cys49 thiolate appears to be the major reaction site with DIBAs [40,43], PATES [57] and NEM [56] while Cys 39 seems to be the primary target of the SAMT analogues [58]. (3)) with the formation of either a disulfide bridge with DI-BAs [43] or a thioester bond with SAMTs [48] or a thioether link with NEM [56].…”

Targeting Cysteine Residues of Biomolecules: New Approaches for the Design of Antiviral and Anticancer Drugs

“…In all these cases a modified cysteine residue is obtained, which possesses completely different physico-chemical properties (reaction (5), Scheme 1) [3,15]. The S-alkylation/arylation, S-acylation or Ssulfonylation of Cys residues due to the last property mentioned above, is indeed of exceptional importance of all retroviruses are indeed essential for the viral life cycle since they select viral RNA from cellular RNA for dimerization and packaging, promote binding of the essential t-RNA primer to the primer site, stimulate reverse transcription and protect the viral RNA from nucleases, and their mutation/modification are not tolerated [23][24][25][26][27][28]. Rice et al [20,21] showed that it is possible to target NCp7 by chemically modifying the nucleophilic sulfur atoms coordinating the Zn(II) ions in the above-mentioned zinc fingers, with 3-nitrosobenzamide derivatives 1, disulfide benzamides (DIBAs) 2, dithianes of type 3 or azodicarbonamide 4 among others, the process being followed by zinc extrusion from the zinc finger and production of non-infective virions.…”

“…This process leads to the destabilization of the Zn(II) ion coordination sphere, and is followed by ejection of the metal ion from the zinc finger. The initial process may then be followed by the formation of intramolecular disulfides within the protein as well as disulfide bond rearrangements, but anyhow, the final result is the denaturation of the zinc finger protein, which is lethal for the virus, leading to the formation of non-infective virions [24,28].…”

“…The mechanism of action of such zinc finger targeting agents of types 1-4 has been investigated in details [24,28], being shown that the zinc is ejected in a stoichiometric, oxidative manner. Initially, the antiviral agent (i.e., a dithio- [25,27] by the XTT cytoprotection assay.…”

Anticancer and Antiviral Sulfonamides

“…Some of the most active derivatives are shown in Table 2 together with second generation compounds, belonging to the pyridinioalkanoyl thiolesters 112 (PATEs) type of such derivatives [137]. It may be seen that among the most effective antiviral derivatives are some sulfonamides of types 109a, 109c and 112a.…”