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).