Fluorescent primuline derivatives inhibit hepatitis C virus NS3-catalyzed RNA unwinding, peptide hydrolysis and viral replicase formation

Abstract

The hepatitis C virus (HCV) multifunctional nonstructural protein 3 (NS3) is a protease that cleaves viral and host proteins and a helicase that separates DNA and RNA structures in reactions fueled by ATP hydrolysis. Li et al. (J. Med. Chem. 2012, 55:3319-3330) recently synthesized a series of new NS3 helicase inhibitors from the benzothiazole dimer component of the fluorescent yellow dye primuline. This study further characterizes a subset of these primuline derivatives with respect to their specificity, mechanism of action, and effect on cells harboring HCV subgenomic replicons. All compounds inhibited DNA and RNA unwinding catalyzed by NS3 from different HCV genotypes, but only some inhibited the NS3 protease function, and few had any effect on HCV NS3 catalyzed ATP hydrolysis. A different subset were potent inhibitors of RNA stimulated ATP hydrolysis catalyzed by the related NS3 protein from Dengue virus. In assays monitoring intrinsic protein fluorescence in the absence of nucleic acids, the compounds cooperatively bound NS3 with Kd’s that reflect their potency in assays. The fluorescent properties of the primuline derivatives both in vitro and in cells are also described. The primuline derivative that was the most active against subgenomic replicons in cells caused a 14-fold drop in HCV RNA levels (IC50 = 5 ± 2 µM). In cells, the most effective primuline derivative did not inhibit the cellular activity of NS3 protease but disrupted HCV replicase structures.

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

“…To this end, we examined the effect of each compound on NS3h intrinsic protein fluorescence. Ndjomou et al recently showed that primuline derivatives quench intrinsic protein fluorescence with apparent dissociation constants in the low micromolar range similar to the IC 50 values seen in helicase assays (13). Similar results were obtained here for compounds 1, 2, 3, and titan yellow, with helicases isolated from either genotype 1b or 2a (Fig.…”

“…These results suggest that primuline derivatives do not act nonspecifically (e.g. through protein aggregation), and that they do not inhibit helicase action simply by binding in place of ATP (13).…”

Protease Inhibitors Block Multiple Functions of the NS3/4A Protease-Helicase during the Hepatitis C Virus Life Cycle

“…Previous biochemical studies have shown that the protease domain of NS3 is required for efficient unwinding of double-stranded RNA (dsRNA) by the helicase (28), and recently, Aydin et al (19) proposed a model in which the protease domain of NS3 binds to dsRNA as a clamp while the helicase unwinds the double-stranded template to allow access by NS5B to the negative strand for RNA synthesis. It is tempting to speculate that binding of telaprevir to the protease domain may inhibit RNA synthesis by reducing helicase activity, but a previous study has reported that telaprevir does not inhibit the helicase activity of full-length NS3 in vitro (29). In vivo, however, NS3 functions as part of a multiprotein replicase complex where binding of telaprevir may result in disruption of proteinprotein and protein-RNA interactions to block a function of NS3 in RNA synthesis.…”

Simultaneously Targeting the NS3 Protease and Helicase Activities for More Effective Hepatitis C Virus Therapy

“…In an earlier report, Ndjomou et al (2012) 10 studied the effects of a series of benzothiazole compounds derived from the yellow dye primuline on the various functions of the HCV NS3 protein. Many of these compounds, like NIH molecular probe ML283, 16 specifically inhibit NS3-catalyzed RNA and DNA unwinding, but they do not potently inhibit the ability of NS3 to cleave ATP and peptides.…”

“…1) inhibit both the NS3 helicase and protease functions in vitro , but not the ability of NS3 to cleave ATP, the fuel for helicase movement. 10 …”