Figure legends
Figure 1. Piperine potently inhibit DHODH enzymatic
activity
(A) Chemical structure of piperine.
(B) Dose-response curves of piperine and A771726 against human DHODH.
(C) Piperine is an uncompetitive DHODH inhibitor versus substrate DHO.
Assays
were performed using varied substrate concentrations (25 to 200 μM) with
either DMSO (●), 1.0 μM (■) or 3.0 μM (▲) piperine as mentioned in
methods. Lineweaver–Burk plot was used to reveal the inhibition
characteristics of piperine against DHODH.
(D) piperine is noncompetitive inhibitor for CoQ0. The enzymatic
reactions mixture contains the varied CoQ0 (25 to 200 μM) with either
DMSO (●), 1.0 μM (■) or 3.0 μM (▲) (▲) piperine. Lineweaver–Burk plots
were used to reveal the inhibition mode of piperine against DHODH.
(E) Enzymatic kinetic curves of DHODH with a series of concentrations of
piperine from 0.025 to 50 μM.
(F) Analysis of the enzymatic kinetic curves shown in (E) generated the
following kinetic parameters and affinity for DHODH:
Kon=1.28x102M-1s-1,
Koff=9.11x10-5 s-1and residence time (RT) =182.95 min, as well as Ki=
Koff/Kon=0.71
± 0.01 μM, respectively. Results are the mean ± SEM of three independent
experiments.
Figure 2. Piperine directly
bind to DHODH protein
(A) Representative data of piperine binding to DHODH detected by CD.
Purified DHODH protein (20 μM) was incubated with 10 μM PIP and
subjected to CD spectroscopy analysis.
(B) Binding of piperine to DHODH was determined by intrinsic
fluorescence quenching assay. DHODH protein (20 μM) was treated with
increasing concentrations of piperine (10 μM to 50 μM) for 30 min. The
fluorescence emission (290-500 nm) was recorded with an excitation
wavelength of 280 nm.
The
dashed line indicates the fluorescence spectrum of piperine (10 μM).
(C) Thermal shift assay reveasl that piperine significantly stabilizes
DHODH and results in a thermal shift over 5.12°C (molar ratio 1:20).
(D) Thermodynamic characterization of DHODH-piperine interactions by
ITC. DHODH protein was titrated with piperine in ITC buffer as mentioned
in methods. Binding curves were fitted as a single binding event using
MicroCal origin software package. Data are presented as mean ± SEM of
three independent experiments
Figure 3. Piperine co-crystallize with DHODH
(A-B) The overall structure of DHODH in complex with piperine. Structure
of DHODH-piperine complex, with protein backbones shown as a gray
surface. Superposition of the PIP molecule (pink) is shown in stick
representation. The residues of α1 and α2 (orange), involved in
hydrophobic contacts with PIP, is exhibited as carton.
(C-D) Ubiquinone-binding patch of DHODH with PIP. The 2Fo-Fc electron
density maps of PIP in DHODH is contoured at 1.5σ. Dashed lines indicate
potential hydrogen bonds. The residues involved in ligand binding are
labelled with yellow sticks.
(E) Two-dimensional view of PIP interactions with DHODH. Residues
accounting for polar and hydrophobic interactions are shown in blue and
green boxes.
Figure
4. Piperine inhibits mouse spleen cell proliferation via inhibition of
DHDOH.
(A) Schematic diagram of concanavalin-A (Con A) induced spleen cell
activation.
(B) Spleen cells isolated from naive C57BL/6 mice were cultured with 10
μg/mL of Con A in the presence of various concentration of piperine.
After culture for 3 days, cell viability was determined using MTT assay.
Each value indicates the mean ± SD of three independently experiments.
(C) Schematic diagram of mixed lymphocyte reaction (MLR) assay.
(D) Single-cell suspensions of spleens from C57BL/6 and BALB/C were
prepared as described in methods section. Each set of
2×105 cells were cultured in 96-well cell culture
plates with a serial dilution of piperine. Cell proliferation was
evaluated after 4 days by MTT as described above. Each value indicates
the mean ± SD of three independently experiments.
(E) Principle of the fluorescence-based assay for cellular DHODH
activity. DHO was firstly enzymatic catalyzed to orotic acid by cellular
DHODH and orotic acid was further detected by a fluorescence probe
4-trifluoromethyl-benzamidoxime (4-TFMBAO).
(F) The enzymatic activities of DHODH in Jurkat T cells treated with a
series of concentrations of piperine were determined by fluorescence
assay as described in (E).