Protease Assay: The HCV protease assay herein was applied to investigate the HCV-protease inhibitory activity of the prepared compounds as described above. The method of the HCV protease assay was described in D. T. Phuong, C. M. Ma, M. Hattori and J. S. J in: Inhibitory Effects of Antrodins A-E from Antrodia cinnamomea and Their Metabolites on Hepatitis C Virus Protease. Phytotherapy Research, 23, 582-584, 2009. Two micro liters of a compound solution (using DMSO as solvent) was placed in 384 well micro plate, then 8 ul of HCV NS3/4A protease (0.5 g/mL) was added to the well containing a sample and the plate was agitated. Finally, 10 uL of freshly prepared substrate (Ac-Asp-Glu-Dap(QXL 520)-Glu-Glu-Abu-COO-Ala-Ser-Cys(5-FAMsp)-NH2) (100 dilution of a DMSO stock solution) was added with sequential rotational shaking. The reaction mixture was incubated for 30 min at 37 C. The fluorimetric analyses were performed on an automated TECAN GENios plate reader with excitation wavelength at 485 nm.
Inhibition Assay: In addition, the dipeptidyl peptidase-IV (DPPIV) assay herein was applied to investigate the DPPIV inhibitory activity of the prepared compounds as described above. The method of the DPPIV assay was described in Lin et al., 1998. Inhibition of dipeptidyl peptidase IV by fluoroolefin-containing N-peptidyl-O-hydroxylamine peptidomimetics. Proc. Natl. Acad. Sci. USA Vol. 95, pp. 14020-14024.