Piperine is a novel and potent DHODH inhibitor
DHODH activity was determined using a chromogen 2,6-dichloroindophenol (DCIP) based reduction assay, which is stoichiometrically equivalent to the oxidation of dihydroorotate (DHO) to orotate. As shown in Figure 1A-B, piperine inhibits DHODH enzymatic activity in a dose-dependent manner and has a half maximal inhibitory concentration (IC50) value of 0.88 ± 0.04 μM, which is comparable with that of A771726, a Food and Drug Administration approved DHODH inhibitor. We then determined the inhibition mode of piperine using the Lineweaver-Burk plot. The results indicated that piperine is uncompetitive inhibitor versus the substrate DHO (Figure 1C) and a noncompetitive for CoQ (Figure 1D). In addition, using enzymatic kinetic experiments assays, we obtained affinity (Ki) and kinetic parameters Kon, Koff, and residence time (RT). As shown in Figure 1E-F, piperine exhibits long residence time (Kon =1.28x102M-1s-1, Koff=9.11x10-5 s-1, RT=182.95 min) and high affinity (Ki= Koff/Kon=1.18 μM) for DHODH.