Halogenated benzimidazoles and benzotriazoles as inhibitors of the NTPase/helicase activities of hepatitis C and related viruses

• Published in: European journal of biochemistry Vol. 270; no. 8; pp. 1645 - 1653
• Main Authors: Borowski, Peter, Deinert, Johanna, Schalinski, Sarah, Bretner, Maria, Ginalski, Krzysztof, Kulikowski, Tadeusz, Shugar, David
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01.04.2003
• Subjects: Acid Anhydride Hydrolases - antagonists & inhibitors; Adenosine Triphosphatases - antagonists & inhibitors; Adenosine Triphosphatases - chemistry; Antiviral Agents - pharmacology; Benzimidazoles - pharmacology; Binding Sites; DNA Helicases - antagonists & inhibitors; halogenated benzimidazoles/benzotriazoles; Hepacivirus - drug effects; Hepacivirus - enzymology; hepatitis C and related viruses; inhibitors; Kinetics; Models, Molecular; NTPase/helicases; Nucleoside-Triphosphatase; Protein Conformation; Substrate Specificity; Triazoles - pharmacology; Viral Nonstructural Proteins - antagonists & inhibitors; Viral Nonstructural Proteins - chemistry
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1046/j.1432-1033.2003.03540.x

A search has been initiated for lead inhibitors of the nonstructural protein 3 (NS3)‐associated NTPase/helicase activities of hepatitis C virus, the related West Nile virus, Japanese encephalitis virus and the human mitochondrial Suv3 enzyme. Random screening of a broad range of unrelated low‐molecular mass compounds, employing both RNA and DNA substrates, revealed that 4,5,6,7‐tetrabromobenzotriazole (TBBT) hitherto known as a potent highly selective inhibitor of protein kinase 2, is a good inhibitor of the helicase, but not NTPase, activity of hepatitis C virus NTPase/helicase. The IC50 is approximately 20 µm with a DNA substrate, but only 60 µm with an RNA substrate. Several related analogues of TBBT were enzyme‐ and/or substrate‐specific inhibitors. For example, 5,6‐dichloro‐1‐(β‐d‐ribofuranosyl)benzotriazole (DRBT) was a good, and selective, inhibitor of the West Nile virus enzyme with an RNA substrate (IC50 ≈ 0.3 µm), but much weaker with a DNA substrate (IC50 ≈ 3 µm). Preincubation of the enzymes, but not substrates, with DRBT enhanced inhibitory potency, e.g. the IC50 vs the hepatitis C virus helicase activity was reduced from 1.5 to 0.1 µm. No effect of preincubation was noted with TBBT, suggesting a different mode of interaction with the enzyme. The tetrachloro congener of TBBT, 4,5,6,7,‐tetrachlorobenzotriazole (TCBT; a much weaker inhibitor of casein kinase 2) is also a much weaker inhibitor than TBBT of all four helicases. Kinetic studies, supplemented by comparison of ATP‐binding sites, indicated that, unlike the case with casein kinase 2, the mode of action of the inhibitors vs the helicases is not by interaction with the catalytic ATP‐binding site, but rather by occupation of an allosteric nucleoside/nucleotide binding site. The halogeno benzimidazoles and benzotriazoles included in this study are excellent lead compounds for the development of more potent inhibitors of hepatitis C virus and other viral NTPase/helicases.