Highly Sensitive Long Wavelength HCV Assay Kits and Substrates

Hepatitis C virus (HCV), discovered in 1989, infects approximately 170 million people worldwide.^{1, 2} It belongs to the Flaviviridae family of positive, single stranded RNA, and its polyprotein consists of structural proteins (C, E1, E2 and p7), and the non-structural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B), resulting from proteolytical cleavages of host signal peptidases; metalloprotease and serine proteases, respectively.^3-6 The protease responsible for the cleavage of HCV non-structural polyprotein at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B sites is the NS3/4A protease. Since these cleavages are essential for the maturation of the viral proteins, this protease has become one of the key targets for developing anti-HCV drugs.^7-11

Applying both peptide and fluorescent dye technologies, AnaSpec has developed an ultra-sensitive HCV NS3/4A protease FRET substrate that can detect the presence of HCV NS3/4A protease at levels of < 0.1 pmol. Based on an EDANS/DABCYL substrate described by Taliani, M. et al. (12), the donor and the quencher were substituted with 5-FAM and our trademark quencher, QXL^™ 520. This substrate found in SensoLyte^™ 520 HCV Protease Assay Kit (Cat# 71145), shows a 22-fold lower Km compared to the EDANS/DABCYL peptide (see Table 1), the substrate found in SensoLyte^™ 490 HCV Protease Assay Kit (Cat# 71126 and 72087). These kits and the SensoLyte^™ 620 kit (Cat# 71146) have been developed by AnaSpec for HTS screening of inhibitors and for measuring HCV NS3/4A activity (Table 2).

Beside being the producer of the world’s most sensitive HCV NS3/4A FRET substrate, AnaSpec also carries a series of mutated HCV NS3 serine proteases to complement our popular wild-type protease. These mutants provide researchers with additional tools with which to assess the implications and explore a response to the emergence of protease inhibitor (PI) resistant NS3 proteases

Effective HCV protease inhibitors (PIs) such as VX-95¹² and BILN 2061^13,14 have been found to reduce viral load; however, due to poor fidelity of the viral reverse transcriptase and RNA-dependent RNA polymerase, drug resistant mutations, consisting of single or multiple amino acid substitutions, have been identified in several labs and found to confer resistance to PIs.

Both wild-type and mutated proteases are recombinant fusion proteins with an NS3 protease domain and a fragment of the NS4A protein fused to its N-terminus. As a result of this fusion, these proteins are already in the active form, which makes pre-activation by pep4A or pep4AK unnecessary. Only a minimal amount of protease is needed (50-100 ng) to perform AnaSpec’s FRET-based activity assays - SensoLyte™ HCV Protease Assay Kits.

Figure 1 shows the initial hydrolysis velocity (Vo) of 5-FAM/QXL^™520 FRET substrate (contained in the SensoLyte^™ 520 HCV Protease Assay Kit (Cat# 71145), catalyzed by wild-type HCV NS3/4A protease at different substrate concentrations. Figure 2 shows that the two protease mutants consisting of single substitution of the alanine residue, A156S or A156T, can effectively cleave the 5-FAM/ QXL^™520 FRET peptide substrate.

On-line listings of recombinant NS3/4A proteases:
- Wild-type
- Mutated
- NS3 Protease Inhibitors

References:
1. Choo, QL. et al. Science 244, 359 (1989).
2. CDC and Prevention. Morb. Mortal. Wkly. Rep. 47, 1 (1998).
3. Choo, QL. et al. Proc. Natl. Acad. Sci. USA 88, 2451 (1991).
4. Hijikata, M. et al. Proc. Natl. Acad. Sci. USA 88, 5547 (1991).
5. Okamoto, H. et al. J. Gen. Virol. 72, 2697 (1991).
6. Takamizawa, A. et al. J. Virol. 65, 1105 (1991).
7. Sali, DL. et al. Biochem. 37, 3392 (1998).
8. Steinkuhler, C. et al. Biochem. 37, 8899 (1998).
9. Gallinari, P. et al. J. Virol. 72, 6758 (1998).
10. Hardy, RW. et al. J. Virol. 77, 2029 (2003).
11. Hamill, P. and F. Jean, Biochem. 44, 6586 (2005).
12. Taliani, M. et al. Anal. Biochem. 240, 60 (1996).
13. Lin, K. et al. Antimicrob. Agents Chemother. 50, 1813 (2006).
14. Faucher, AM. et al. Org.Lett. 6, 2901 (2004).
15. Hinrichsen, H. et al. Gastroenterology 127, 1347 (2004).

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