Azodicarbonamide inhibits HIV-1 replication by targeting the nucleocapsid protein

Rice, William G.; Turpin, Jim A.; Huang, Mingjun; Clanton, David J.; Buckheit, Robert W.; Covell, David G.; Wallqvist, Anders; McDonnell, Nazli B.; DeGuzman, Roberto N.; Summers, Michael F.; Zalkow, Leon H.; Bader, John P.; Haugwitz, R. D.; Sausville, Edward A.

doi:10.1038/nm0397-341

Cited by 104 publications

(84 citation statements)

References 18 publications

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“…The model has been shown to predict accurately ligand binding strengths and assess the relative contributions of atomic interactions within a binding interface (17). Moreover, the model has been extended as an adjunct to computational docking (19), has proven effective for identifying ligands active against NCp7 targets (20), and has been useful in providing testable hypotheses about the modes of action of candidate inhibitors for a variety of enzymes (21)(22)(23).…”

Section: Methodsmentioning

confidence: 99%

Mapping the Binding Site of Colchicinoids on β-Tubulin

Bai¹,

Covell²,

Pei³

et al. 2000

Journal of Biological Chemistry

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2-Chloroacetyl-2-demethylthiocolchicine (2CTC) and 3-chloroacetyl-3-demethylthiocolchicine (3CTC) resemble colchicine in binding to tubulin and react covalently with ␤-tubulin, forming adducts with cysteine residues 239 and 354. The adducts at Cys-239 are less stable than those at Cys-354 during formic acid digestion. Extrapolating to zero time, the Cys-239 to Cys-354 adduct ratio is 77:23 for 2CTC and 27:73 for 3CTC. Using energy minimization modeling to dock colchicinoids into the electron crystallographic model of ␤-tubulin in protofilaments (Nogales, E., Wolf, S. G., and Downing, K. H. (1998) Nature 391, 199 -203), we found two potential binding sites. At one, entirely encompassed within ␤-tubulin, the C2-and C3-oxygen atoms of 2CTC and 3CTC overlapped poorly with those of colchicine and thiocolchicine, but distances from the reactive carbon atoms of the analogs to the sulfur atoms of the cysteine residues were qualitatively consistent with reactivity. The other potential binding site was located at the ␣/␤ interface. Here, the oxygen atoms of the analogs overlapped well with those of colchicine, but relative distances of the reactive carbons to the cysteine sulfur atoms did not correlate with the observed reactivity. A significant conformational change must occur in the colchicine binding site of tubulin in the transition from the unpolymerized to the polymerized state.Despite the interaction of tubulin with a large number of drugs that inhibit or promote its assembly into microtubules, precise definition of drug binding sites on the protein has not been possible. This is a consequence of the lack of success in crystallizing the protein, probably because of its sequence and post-translational heterogeneity, its instability, and its tendency to form oligomers and polymers of highly aberrant morphology in the presence of many of these drugs. The recent electron crystallographic determination of a relatively detailed structure for zinc-induced antiparallel tubulin protofilaments has provided insights into the paclitaxel/docetaxel site on these protofilaments, since docetaxel was used to enhance their stability during data accumulation (1), and provided a scaffold on which to model other drug sites (2). A limitation in such analysis, however, is that drugs that inhibit assembly have limited ability to bind to tubulin polymers containing linear protofilaments.Alternative approaches to obtain preliminary information about drug binding sites have included "direct" photoaffinity labeling (e.g. Ref.3), analog photoaffinity labeling (e.g. Ref. 4), and cross-link formation with chemically reactive analogs that retain biological activity (e.g. Ref. 5). In the first method, a drug-tubulin complex is exposed to light of an appropriate wave-length, and ligand-protein cross-link formation is evaluated. In the second, an active drug analog containing a photoreactive moiety is prepared and bound to tubulin, and crosslink formation is induced by exposure of the complex to light of an appropriate wavelength. In the third metho...

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Section: Methodsmentioning

confidence: 99%

Mapping the Binding Site of Colchicinoids on β-Tubulin

Bai¹,

Covell²,

Pei³

et al. 2000

Journal of Biological Chemistry

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“…1) were selected for study: DIBAs-1, -2, and -3, 2,2 -dithiobisphenol (DIBPH), 2,2 -dithiobistoluene (DIBTL), benzoisothiazolones (BITAs-1, -2, and -1E), azodicarbonamide (ADA), cis-1,2-dithiane-4,5-diol-1,1-dioxide (dithiane), N-ethylmaleimide (NEM), and 3-nitrosobenzamide (NOBA). Excluding NEM, members of each chemotype have shown anti-HIV activity by means of NCp7 inhibition (7)(8)(9)(10). ADA is being evaluated in Europe for patients with advanced AIDS (24).…”

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confidence: 99%

Reactivity of the HIV-1 nucleocapsid protein p7 zinc finger domains from the perspective of density-functional theory

Maynard

Huang²,

Rice³

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

349

294

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The reaction of the human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein p7 (NCp7) with a variety of electrophilic agents was investigated by experimental measurements of Trp 37 fluorescence decay and compared with theoretical measures of reactivity based on density-functional theory in the context of the hard and soft acids and bases principle. Statistically significant correlations were found between rates of reaction and the ability of these agents to function as soft electrophiles. Notably, the molecular property that correlated strongest was the ratio of electronegativity to hardness, 2 /, a quantity related to the capacity of an electrophile to promote a soft (covalent) reaction. Electronic and steric determinants of the reaction were also probed by Fukui function and frontier-orbital overlap analysis in combination with protein-ligand docking methods. This analysis identified selective ligand docking regions within the conserved zinc finger domains that promoted reaction. The Cys 49 thiolate was found overall to be the NCp7 site most susceptible to electrophilic attack.

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“…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.…”

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confidence: 99%

Inactivation of HIV-1 Nucleocapsid Protein P7 by Pyridinioalkanoyl Thioesters

Basrur¹,

Song²,

Mazur³

et al. 2000

Journal of Biological Chemistry

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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...

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Azodicarbonamide inhibits HIV-1 replication by targeting the nucleocapsid protein

Cited by 104 publications

References 18 publications

Mapping the Binding Site of Colchicinoids on β-Tubulin

Mapping the Binding Site of Colchicinoids on β-Tubulin

Reactivity of the HIV-1 nucleocapsid protein p7 zinc finger domains from the perspective of density-functional theory

Inactivation of HIV-1 Nucleocapsid Protein P7 by Pyridinioalkanoyl Thioesters

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