Primuline Derivatives That Mimic RNA to Stimulate Hepatitis C Virus NS3 Helicase-catalyzed ATP Hydrolysis

Noreena L Sweeney,William R Shadrick,Sourav Mukherjee,Kelin Li,Kevin J Frankowski,Frank J Schoenen,David N Frick

doi:10.1074/jbc.m113.463166

Noreena L Sweeney, William R Shadrick + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m113.463166

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Abstract

ATP hydrolysis fuels the ability of helicases and related proteins to translocate on nucleic acids and separate base pairs. As a consequence, nucleic acid binding stimulates the rate at which a helicase catalyzes ATP hydrolysis. In this study, we searched a library of small molecule helicase inhibitors for compounds that stimulate ATP hydrolysis catalyzed by the hepatitis C virus (HCV) NS3 helicase, which is an important antiviral drug target. Two compounds were found that stimulate HCV helicase-catalyzed ATP hydrolysis, both of which are amide derivatives synthesized from the main component of the yellow dye primuline. Both compounds possess a terminal pyridine moiety, which was critical for stimulation. Analogs lacking a terminal pyridine inhibited HCV helicase catalyzed ATP hydrolysis. Unlike other HCV helicase inhibitors, the stimulatory compounds differentiate between helicases isolated from various HCV genotypes and related viruses. The compounds only stimulated ATP hydrolysis catalyzed by NS3 purified from HCV genotype 1b. They inhibited helicases from other HCV genotypes (e.g. 1a and 2a) or related flaviviruses (e.g. Dengue virus). The stimulatory compounds interacted with HCV helicase in the absence of ATP with dissociation constants of about 2 μM. Molecular modeling and site-directed mutagenesis studies suggest that the stimulatory compounds bind in the HCV helicase RNA-binding cleft near key residues Arg-393, Glu-493, and Ser-231.

Highlights

DNA/RNA stimulates the ATPase function of a helicase by an unclear mechanism
Identification of hepatitis C virus (HCV) Helicase Inhibitors That Stimulate Helicase-catalyzed ATP Hydrolysis—This project was initiated to understand how NS3h-catalyzed ATP hydrolysis is influenced by primuline derivatives
To understand how primuline derivatives affect the ability of ATP to fuel unwinding, all compounds in the primuline derivative collection described by Li et al [1] were analyzed for their effect on NS3h-catalyzed ATP hydrolysis using a colorimetric assay (Fig. 1A)

Summary

Background

DNA/RNA stimulates the ATPase function of a helicase by an unclear mechanism. Results: A primuline derivative stimulates the ATPase function of only the HCV genotype 1b NS3 helicase. Because nucleic acids in turn stimulate helicase-catalyzed ATP hydrolysis, it is conceivable that a small molecule might bind the protein to elicit the same effect Such compounds might be useful as tools to design drugs that target helicases or as probes. We report the discovery that a small molecule synthesized from the main component of the yellow dye primuline [1] binds the helicase encoded by the hepatitis C virus (HCV) in such a manner that it mimics RNA to stimulate helicase-catalyzed ATP hydrolysis. We report the surprising discovery that two primuline derivatives stimulate helicase-catalyzed ATP hydrolysis Since such an activity has not yet been reported for any small molecule (other than DNA or RNA oligonucleotides), we report a biochemical characterization of the interaction of stimulatory primuline derivatives with NS3h. The compounds should be useful to design more specific NS3 helicase inhibitors that could be used as molecular probes or as the basis to design antiviral drugs to be used alone, in combination with NS3 protease inhibitors, or with other antiviral drugs

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION