Hepatitis C virus (HCV) NS3/4A protease genotype assays

Published methods for the construction, expression, and purification of recombinant full-length NS3/4A complexes representing HCV genotypes 1a (H77c) and 1b (J4L6S) were used to generate homogeneous full-length NS3/4A protease complexes representing the six major HCV genotypes (HC-J6, HC-J8, S52, ED43, SA13 and HK-6A). The susceptibility of purified recombinant NS3/4A protease complexes was assessed using fluorescence resonance energy transfer (FRET) assays. The IC50 value was calculated.

Cell lines

HuH-7, MRC5, MT-2, HepG2, HeLa and HEK293 cells

Preparation method

Soluble in DMSO. General tips for obtaining a higher concentration: Please warm the tube at 37℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reaction Conditions

4 days

Applications

Asunaprevir inhibited HCV RNA replication in different cell lines, including liver, T lymphocytes, lung, cervix, and embryonic kidney. It showed no obvious activity against other RNA viruses.

Animal models

Rats

Dosage form

10 μM; p.o.; 60 mins

Applications

After oral dosing to the rat, Asunaprevir demonstrated modest oral bioavailability and a plasma AUC of 1.0 μM·h. However, at the 24th hrs after p.o. dosing, the liver levels of Asunaprevir were high at 15.2 μM, suggesting a hepatotropic distribution in vivo.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

Asunaprevir is an orally efficacious inhibitor of NS3 protease with IC50 value of 1nM [1].

Asunaprevir is an inhibitor of hepatitis C virus (HCV) NS3 protease. It can inhibit 6 major genotypes of HCV NS3/4A protease with IC50 values of 0.7nM, 0.3nM, 15nM, 78nM, 320nM, 1.6nM, 1.7nM and 0.9nM, respectively for genotype 1a, 1b, 2a, 2b, 3a, 4a, 5a and 6a, respectively. When using the purified recombinant full-length HCV NS3/4A protease complexes, asunaprevir shows the Ki values of 0.4nM and 0.2nM, respectively for genotype 1a and genotype 1b. The mechanism of the inhibition is that the acylsulfonamide of asunaprevir interacts with the catalytic site of NS3 protease in a noncovalent manner. Asunaprevir inhibits HCV RNA replication in different cell lines, including liver, T lymphocytes, lung, cervix, and embryonic kidney. It shows no obvious activity against other RNA viruses. The permeability of asunaprevir is similar to the compound with good absorption in humans. The tests of metabolism rate show that asunaprevir exhibits low to intermediate metabolic clearance. Plasma and tissue exposures in vivo indicate that asunaprevir displays a hepatotropic disposition [2].

References:
[1] Scola PM, Sun LQ, Wang AX, Chen J, Sin N, Venables BL, Sit SY, Chen Y, Cocuzza A, Bilder DM, D'Andrea SV, Zheng B, Hewawasam P, Tu Y, Friborg J, Falk P, Hernandez D, Levine S, Chen C, Yu F, Sheaffer AK, Zhai G, Barry D, Knipe JO, Han YH, Schartman R, Donoso M, Mosure K, Sinz MW, Zvyaga T, Good AC, Rajamani R, Kish K, Tredup J, Klei HE, Gao Q, Mueller L, Colonno RJ, Grasela DM, Adams SP, Loy J, Levesque PC, Sun H, Shi H, Sun L, Warner W, Li D, Zhu J, Meanwell NA, McPhee F. The discovery of asunaprevir (BMS-650032), an orally efficacious NS3 protease inhibitor for the treatment of hepatitis C virus infection. J Med Chem. 2014 Mar 13;57(5):1730-52.
[2] McPhee F, Sheaffer AK, Friborg J, Hernandez D, Falk P, Zhai G, Levine S, Chaniewski S, Yu F, Barry D, Chen C, Lee MS, Mosure K, Sun LQ, Sinz M, Meanwell NA, Colonno RJ, Knipe J, Scola P. Preclinical Profile and Characterization of the Hepatitis C Virus NS3 Protease Inhibitor Asunaprevir (BMS-650032). Antimicrob Agents Chemother. 2012 Oct;56(10):5387-96.