A time-resolved, internally quenched fluorescence assay to characterize inhibition of hepatitis C virus nonstructural protein 3–4A protease at low enzyme concentrations

Mao, Shi‐Shan; DiMuzio, Jillian; McHale, Carolyn; Burlein, Christine; Olsen, David B.; Carroll, Steven S.

doi:10.1016/j.ab.2007.10.041

Cited by 32 publications

(31 citation statements)

References 34 publications

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“…This finding implies that helicase and protease activities might be mutually exclusive, or that their viral functions are completely unrelated. Importantly, it had been reported that NS3-4A protease activity is very sensitive to salt concentrations when the protein is studied at low concentrations (Ͻ1 nM) and less sensitive to salt concentrations at higher protein concentrations (Ն1 nM) (22). We therefore conducted protease assays with relatively high NS3-4A concentrations (40 -120 nm) that fall within the range typically employed for RNA helicase assays (12,13,25,31).…”

Section: Discussionmentioning

confidence: 99%

“…Protein Purification-Because there are a multitude of reports describing the purification of various autocleaved and reconstituted forms of the NS3-4A complex (17,22,23,26,27) and because all of these factors greatly influence NS3 enzymatic function (13), we believe it is necessary to describe our constructs, purification strategies, and reconstitutions in detail. All proteins were purified according to the methods described in Beran et al, (13,25).…”

Section: Methodsmentioning

confidence: 99%

“…Moreover, these screens should utilize constructs in which NS3-4A is produced through autoproteolysis rather than reconstitution. Indeed, a recent study comparing the effects of NS3-4A serine protease inhibitors on full-length NS3-4A produced through autocleavage, NS3 ϩ NS4A peptide, and NS3 protease domain ϩ NS4A peptide demonstrated that BILN 2061 and VX-950 are much better inhibitors of autocleaved NS3-4A serine protease activity than of reconstituted NS3 ϩ NS4A peptide or NS3 protease domain ϩ NS4A (22). The greater protease activity of autocleaved NS3-4A creates the potential for increased sensitivity when measuring protease activity in the presence of new drugs (i.e.…”

Section: Proteinmentioning

confidence: 99%

“…Nonetheless, the reconstituted complexes retain signif- icant levels of activity and provide valuable tools for structure/ function studies (5,22).…”

Section: Ns3-4a Exhibits Robust Serine Protease Activity Under a Varimentioning

confidence: 99%

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Hepatitis C Viral NS3-4A Protease Activity Is Enhanced by the NS3 Helicase

Beran¹,

Pyle

2008

Journal of Biological Chemistry

100

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Non-structural protein 3 (NS3) is a multifunctional enzyme possessing serine protease, NTPase, and RNA unwinding activities that are required for hepatitis C viral (HCV) replication. HCV non-structural protein 4A (NS4A) binds to the N-terminal NS3 protease domain to stimulate NS3 serine protease activity. In addition, the NS3 protease domain enhances the RNA binding, ATPase, and RNA unwinding activities of the C-terminal NS3 helicase domain (NS3hel). To determine whether NS3hel enhances the NS3 serine protease activity, we purified truncated and full-length NS3-4A complexes and examined their serine protease activities under a variety of salt and pH conditions. Our results indicate that the helicase domain enhances serine protease activity, just as the protease domain enhances helicase activity. Thus, the two enzymatic domains of NS3-4A are highly interdependent. This is the first time that such a complete interdependence has been demonstrated for a multifunctional, single chain enzyme. NS3-4A domain interdependence has important implications for function during the viral lifecycle as well as for the design of inhibitor screens that target the NS3-4A protease.NS3-4A is a multifunctional enzyme with serine protease and helicase activities that are essential for hepatitis C viral (HCV) 3 replication (1-3). The NS3 N-terminal domain adopts a chymotrypsin-like fold, and it displays serine protease activity in the presence of the NS4A co-factor protein (4, 5) (Fig. 1A). The genomic HCV RNA is initially translated into a long polyprotein that must be cleaved into the core, envelope, and nonstructural (NS) proteins, which are required for viral replication and packaging. The NS3-4A protease is essential for this process, as it cleaves several sites within the HCV polyprotein (4). It may also enhance HCV replication by cleaving host proteins such as Cardif (also known as IPS-1, MAVS, or VISA), which are involved in innate immunity (6 -9). In this way, NS3-4A may down-regulate the cellular antiviral response.The NS3 C-terminal domain (NS3hel) belongs to the DEXH/ D-box subgroup of helicase superfamily 2 (10). It exhibits NTPase activity and, in the presence of the protease domain, it is a reactive RNA helicase (11-13). The NS3 helicase, along with the HCV NS5A phosphoprotein and the HCV NS5B RNAdependent RNA polymerase, is required for replication of the viral RNA genome (14) and is possibly required for viral packaging as well (15). Thus, NS3-4A must be able to function as a serine protease, an RNA helicase, and a packaging enzyme in an HCV-infected cell in order for the virus to propagate.Recently, we observed that RNA binding and unwinding by NS3hel are greatly enhanced by the covalently attached NS3 protease domain (13). This suggests that, despite their seemingly unrelated enzymatic functions, the helicase and protease domains have evolved to become interdependent and functionally coupled. It is well established that the NS3 protease domain requires binding of NS4A for reactivity (4, 16 -18), and that the protease d...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Proteinmentioning

confidence: 99%

“…Nonetheless, the reconstituted complexes retain signif- icant levels of activity and provide valuable tools for structure/ function studies (5,22).…”

Section: Ns3-4a Exhibits Robust Serine Protease Activity Under a Varimentioning

confidence: 99%

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Hepatitis C Viral NS3-4A Protease Activity Is Enhanced by the NS3 Helicase

Beran¹,

Pyle

2008

Journal of Biological Chemistry

100

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“…Recombinant HCV NS3/4A protease was expressed and purified from Escherichia coli. Inhibition of HCV NS3/4A protease activity in reactions containing MK-7009 or reference compounds VX-950 and SCH503034 was determined in a time-resolved fluorescence assay, as described previously (29). Cell-based HCV replicon assays were conducted with the previously described genotype 1b (con1) stable cell line HB1 (2,24) in the presence of either 10% fetal bovine serum (FBS) or 50% normal human serum (NHS), as described previously (32).…”

Section: Compoundmentioning

confidence: 99%

MK-7009, a Potent and Selective Inhibitor of Hepatitis C Virus NS3/4A Protease

Liverton¹,

Carroll

DiMuzio

et al. 2010

Antimicrob Agents Chemother

Self Cite

144

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The administration of hepatitis C virus (HCV) NS3/4A protease inhibitors to patients with chronic HCV infections has demonstrated that they have dramatic antiviral effects and that compounds acting via this mechanism are likely to form a key component of future anti-HCV therapy. We report here on the preclinical profile of MK-7009, an inhibitor of genotype 1a and 1b proteases at subnanomolar concentrations with modestly shifted potency against genotype 2a and 2b proteases at low nanomolar concentrations. Potent activity was also observed in a cell-based HCV replicon assay in the presence of added human serum (50%). In multiple species evaluated in preclinical studies, the MK-7009 concentrations in the liver were maintained at a significant multiple of the cell-based replicon 50% effective concentration over 12 to 24 h following the administration of moderate oral doses (5 to 10 mg per kg of body weight). MK-7009 also had excellent selectivity against both a range of human proteases and a broad panel of pharmacologically relevant ion channels, receptors, and enzymes. On the basis of this favorable profile, MK-7009 was selected for clinical development and is currently being evaluated in controlled clinical trials with both healthy volunteers and HCV-infected patients.

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Generation and Quantitation of Infectious Hepatitis C Virus Derived from Cell Culture (HCVcc)

Paulson

2010

CP Pharmacology

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The development of robust genotype 1b and genotype 1a hepatitis C virus (HCV) replicon systems has enabled the convenient in vitro study of part of the virus life cycle. This unit describes detailed protocols for generating and measuring infectious HCV, or cell-culture-derived infectious HCV (HCVcc). The HCVcc infectious system has two essential components: (1) cells that are permissive to de novo infection and allow effective replication of the full virus life cycle; and (2) a virus genome that has robust and efficient replication in tissue culture. The assays in this unit are based on protocols designed for Huh-7-derived cell lines that allow robust replication of HCV and are permissive to infection. These protocols are important for the implementation of drug discovery efforts relative to the entire infectious virus life cycle.

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A time-resolved, internally quenched fluorescence assay to characterize inhibition of hepatitis C virus nonstructural protein 3–4A protease at low enzyme concentrations

Cited by 32 publications

References 34 publications

Hepatitis C Viral NS3-4A Protease Activity Is Enhanced by the NS3 Helicase

Hepatitis C Viral NS3-4A Protease Activity Is Enhanced by the NS3 Helicase

MK-7009, a Potent and Selective Inhibitor of Hepatitis C Virus NS3/4A Protease

Generation and Quantitation of Infectious Hepatitis C Virus Derived from Cell Culture (HCVcc)

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