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.

Recently protein kinases have emerged as some of the most promising drug targets; and therefore, pharmaceutical strategies have been developed to inhibit kinases in the treatment of a variety of diseases. CK2 is a serine/threonine-protein kinase that has been implicated in a number of cellular processes, including maintenance of cell viability, protection of cells from apoptosis, and tumorigenesis. Elevated CK2 activity has been established in a number of cancers where it was shown to promote tumorigenesis via the regulation of the activity of various oncogenes and tumor suppressor proteins. Consequently the development of CK2 inhibitors has been ongoing in preclinical studies, resulting in the generation of a number of CK2-directed compounds. In the present study, an unbiased evaluation of CK2 inhibitors 4,5,6,7-tetrabromo-1H-benzotriazole (TBB), 4,5,6,7-tetrabromo-1H-benzimidazole (TBBz), and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) was carried out to elucidate the mechanism of action as well as inhibitor specificity of these compounds. Utilizing a chemoproteomics approach in conjunction with inhibitor-resistant mutant studies, CK2α and CK2α′ were identified as bona fide targets of TBB, TBBz, and DMAT in cells. However, inhibitor-specific cellular effects were observed indicating that the structurally related compounds had unique biological properties, suggesting differences in inhibitor specificity. Rescue experiments utilizing inhibitor-resistant CK2 mutants were unable to rescue the apoptosis associated with TBBz and DMAT treatment, suggesting the inhibitors had off-target effects. Exploitation of an unbiased chemoproteomics approach revealed a number of putative off-target inhibitor interactions, including the discovery of a novel TBBz and DMAT (but not TBB) target, the detoxification enzyme quinone reductase 2 (QR2). The results described in the present study provide insight into the molecular mechanism of action of the inhibitors as well as drug specificity that will assist in the development of more specific next generation CK2 inhibitors.

The nucleoside triphosphatase (NTPase)/helicase associated with nonstructural protein 3 of West Nile (WN) virus was purified from cell culture medium harvested from virus-infected Vero cells. The purification procedure included sequential chromatography on Superdex-200 and Reactive Red 120 columns, followed by a concentration step on an Ultrogel hydroxyapatite column. The nature of the purified protein was confirmed by immunoblot analysis using a WN virus-positive antiserum, determination of its NH₂ terminus by microsequencing, and a binding assay with 5′-[¹⁴C]fluorosulfonylbenzoyladenosine. Under optimized reaction conditions the enzyme catalyzed the hydrolysis of ATP and the unwinding of the DNA duplex with k _cat values of 133 and 5.5 × 10⁻³ s⁻¹, respectively. Characterization of the NTPase activity of the WN virus enzyme revealed that optimum conditions with respect to the Mg²⁺requirement and the monovalent salt or polynucleotide response differed from those of other flavivirus NTPases. Initial kinetic studies demonstrated that the inhibition (or activation) of ATPase activity by ribavirin-5′-triphosphate is not directly related to changes in the helicase activity of the enzyme. Further analysis using guanine andO ⁶-benzoylguanine derivatives revealed that the ATPase activity of WN virus NTPase/helicase may be modulated, i.e., increased or reduced, with no effect on the helicase activity of the enzyme. On the other hand the helicase activity could be modulated without changing the ATPase activity. Our observations show that the number of ATP hydrolysis events per unwinding cycle is not a constant value.