Inhibitory assays

Inhibition of HCV NS3/4A protease activity in reaction mixtures containing MK-5172, vaniprevir, or the reference compounds danoprevir and TMC435 was determined in a time-resolved fluorescence assay. Cell-based HCV replicon assays were conducted in genotype 1b (con1) stable cell line HB1 (26) or a gt2a cell line (JFH) in the presence of either 10% fetal bovine serum (FBS) or 40% normal human serum (NHS). For in vitro resistance selections, 100,000 HB1 cells were seeded into a T162 Z-top flask and cultured in the presence of 0.5 mg/ml G418 and the desired concentration of MK-5172. Cells were cultured for approximately 3 weeks with regular exchanges of medium until sufficient cell death had occurred to enable distinct colonies to form. After expansion, total RNA was isolated, used as a template to generate NS3/4a cDNA, and sequenced using conventional molecular biology techniques.

Cell lines

A genotype/mutant panel of stable replicon cell lines

Preparation method

The solubility of this compound in DMSO is >38.4mg/mL. 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.

Reacting condition

2-10 nM, 3 weeks

Applications

MK-5172 demonstrated subnanomolar to low-nanomolar EC50s against genotypes 1a, 1b, and 2a. MK-5172 was efficacious across the genetically diverse range of genotype 1 infections encountered in clinical settings with EC50s ranged narrowly between 0.3 and 5.9 nM. In Genotype 1b replicon cells, pre- treatment with MK-5172 resulted in little apparent cell growth and limited recovery of replicon RNA levels.

Animal models

Chimpanzees, Dogs, Rats

Dosage form

Oral administration, 1 mg/kg, twice daily for 7 days

Application

MK-5172 demonstrated low to moderate clearance and a modest half-life in both rat and dog. Oral administration of MK-5172 (1 mg/kg) demonstrated modest bioavailability of 12 to 13%, with moderate plasma exposure in both species. The 24-h trough liver concentrations were 0.2 μM in rat and 1.4 μM in dog (1 mg/kg), yielding exposure multiples of 27- to 200-fold over the serum-adjusted replicon EC50. In chronic-HCV-infected chimpanzees harboring gt1a, gt1b, or gt1a NS3 R155K infections, treatment with MK-5172 (1 mg/kg, b.i.d.) demonstrated efficacy 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.

MK-5172 is a selective inhibitor of Hepatitis C Virus NS3/4a Protease [1].
Hepatitis C (HCV) virus is a member of the Flaviviridae family of viruses in the Hepacivirus genus and encoded by a 9.6-kb positive strand RNA genome [2].
In biochemical assays, MK-5172 inhibited a series of major genotypes and common mutants in a HCV NS3/4A protease enzymatic assay. In a cell-based replicon system, MK-5172 inhibited HCV with EC50 values of 2 nM against genotype 1a, 0.5 nM against genotype 1b, 8 nM against genotype 2a and 13 nM against genotype 3. Also, MK-5172 is effective against HCV genotypes 1a, 2a, 1b, 2b and 3a [2].
Treatment three chronically HCV-infected chimpanzees with a dose of 1 mg/kg twice daily for 7 days, Two of the chimpanzees had wild-type (WT) gt1a or gt1b infections with high viral titers (~106 IU/ml). A third chimpanzee had a modest viral titer (~104 IU/ml) that was gt1a NS3 R155K virus. MK-5172 (1 mg/kg) reduced viral titer of the gt1a (WT) infection to ~100 IU/ml within 2 days and the gt1b infection to 20 IU/ml. The gt1a NS3 R155K-infected chimp experienced a rapid ~2-log reduction in viral titer [2].
References:
[1]. Harper S, McCauley JA, Rudd MT, et al. Discovery of MK-5172, a Macrocyclic Hepatitis C Virus NS3/4a Protease Inhibitor. ACS Med Chem Lett, 2012, 3(4): 332-336.
[2]. Summa V, Ludmerer SW, McCauley JA, et al. MK-5172, a selective inhibitor of hepatitis C virus NS3/4a protease with broad activity across genotypes and resistant variants. Antimicrob Agents Chemother, 2012, 56(8): 4161-4167.