RNA-dependent RNA polymerase of hepatitis C virus: Study on inhibition by α,γ-diketo acid derivatives

• Published in: Biochemistry (Moscow) Vol. 74; no. 8; pp. 834 - 841
• Main Authors: Kozlov, M. V, Polyakov, K. M, Filippova, S. E, Evstifeev, V. V, Lyudva, G. S, Kochetkov, S. N
• Format: Journal Article
• Language: English
• Published: Dordrecht Dordrecht : SP MAIK Nauka/Interperiodica 01-08-2009; SP MAIK Nauka/Interperiodica
• Subjects: Aminobutyrates - chemical synthesis; Aminobutyrates - chemistry; Biochemistry; Biomedical and Life Sciences; Biomedicine; Bioorganic Chemistry; Catalytic Domain; Enzyme Inhibitors - chemical synthesis; Enzyme Inhibitors - chemistry; Hepacivirus - enzymology; Kinetics; Life Sciences; Microbiology; Phenylbutyrates; RNA Replicase - chemistry; RNA Replicase - genetics; RNA Replicase - metabolism; Substrate Specificity; Viral Nonstructural Proteins - chemistry; Viral Nonstructural Proteins - genetics; Viral Nonstructural Proteins - metabolism; hepatitis C virus; inhibition mechanism; α,γ-diketo acid derivatives; molecular modeling; RNA-dependent RNA polymerase
• ISSN: 0006-2979; 1608-3040
• DOI: 10.1134/S0006297909080033

It is supposed that α,γ-diketo acids (DKAs) inhibit the activity of hepatitis C virus RNA-dependent RNA poly-merase (RdRP HCV) via chelation of catalytic magnesium ions in the active center of the enzyme. However, DKAs display noncompetitive mode of inhibition with respect to NTP substrate, which contradicts the proposed mechanism. We have examined the NTP substrate entry channel and the active site of RdRP HCV for their possible interaction with DKAs. The substitutions R48A, K51A, and R222A greatly facilitated RdRP inhibition by DKAs and simultaneously increased K m values for UTP substrate. Interestingly, C223A was the only one of a number of substitutions that decreased K m(UTP) but facilitated the inhibitory action of DKAs. The findings allowed us to model an enzyme-inhibitor complex. According to the proposed model, DKAs introduce an additional Mg²⁺ ion into the active site of the enzyme at a stage of phosphodiester bond formation, which results in displacement of the NTP substrate triphosphate moiety to a catalytically inactive binding mode. This mechanism, in contrast to the currently adopted one, explains the noncompetitive mode of inhibition.