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.