Natural product piperine alleviates experimental allergic encephalomyelitis in mice by targeting dihydroorotate dehydrogenase

Article abstract of DOI:10.1016/j.bcp.2020.114000

Multiple sclerosis (MS) is the most popular chronic and debilitating inflammatory disease of the central nervous system (CNS) that remains incurable. Dihydroorotate dehydrogenase (DHODH) is critical to the activity of T lymphocytes and represents a potential therapeutic target for MS. Here we identify piperine, a bioactive constituent of black pepper, as a potent inhibitor of DHODH with an IC₅₀ value of 0.88 μM. Isothermal titration calorimetry and thermofluor assay demonstrate the directly interaction between piperine and DHODH. The co-complex crystal structure of DHODH and piperine at 1.98 ? resolution further reveal that Tyr356 residue of DHODH is crucial for piperine binding. Importantly, we show that piperine can inhibit T cell overactivation in a DHODH-dependent manner in concanavalin A-triggered T-cell assay and mixed lymphocyte reaction assay. Finally, piperine exhibits strong preventive and therapeutic effect in the MOG-induced experimental allergic encephalomyelitis (EAE), a useful model for studying potential treatments for MS, by restricting inflammatory cells infiltration into the CNS and preventing myelin destruction and blood–brain barrier (BBB) disruption. Taken together, these findings highlight DHODH as a therapeutic target for autoimmune disease of the nervous system, and demonstrate a novel role for piperine in the treatment of MS.

Full text of DOI:10.1016/j.bcp.2020.114000

Journal Pre-proofs
Natural Product Piperine Alleviates Experimental Allergic Encephalomyelitis
in Mice by Targeting Dihydroorotate Dehydrogenase
Zehui Liu, Qian Hu, Wanyan Wang, Sisi Lu, Dang Wu, Shuyin Ze, Jiacheng
He, Ying Huang, Wuyan Chen, Yechun Xu, Weiqiang Lu, Jin Huang
PII:
DOI:
Reference:
S0006-2952(20)30228-8
https://doi.org/10.1016/j.bcp.2020.114000
BCP 114000
To appear in:
Biochemical Pharmacology
Received Date:
Accepted Date:
20 February 2020
24 April 2020
Please cite this article as: Z. Liu, Q. Hu, W. Wang, S. Lu, D. Wu, S. Ze, J. He, Y. Huang, W. Chen, Y. Xu, W.
Lu, J. Huang, Natural Product Piperine Alleviates Experimental Allergic Encephalomyelitis in Mice by Targeting
Dihydroorotate Dehydrogenase, Biochemical Pharmacology (2020), doi: https://doi.org/10.1016/j.bcp.
2020.114000
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Natural Product Piperine Alleviates Experimental Allergic
Encephalomyelitis in Mice by Targeting Dihydroorotate
Dehydrogenase
Zehui Liu1,#, Qian Hu1,#, Wanyan Wang1, Sisi Lu1, Dang Wu1, Shuyin Ze2,
Jiacheng He2, Ying Huang3, Wuyan Chen4, Yechun Xu4, Weiqiang Lu2,*, Jin
Huang1,*
1Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East
China University of Science and Technology, Shanghai, China; 2Shanghai Key
Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School
of Life Sciences, East China Normal University, Shanghai, China; 3Guangdong
4
Institute for Drug Control, Guangdong, China; CAS Key Laboratory of
Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy
of Sciences (CAS), Shanghai, China.
#Zehui Liu and Qian Hu contributed equally to this work.
Correspondence: Jin Huang, Email: huangjin@ecust.edu.cn; Weiqiang Lu,
Email: wqlu@bio.ecnu.edu.cn.
1

Abstract
Multiple sclerosis (MS) is the most popular chronic and debilitating
inflammatory disease of the central nervous system (CNS) that remains
incurable. Dihydroorotate dehydrogenase (DHODH) is critical to the activity of
T lymphocytes and represents a potential therapeutic target for MS. Here we
identify piperine, a bioactive constituent of black pepper, as a potent inhibitor
of DHODH with an IC50 value of 0.88 μM. Isothermal titration calorimetry and
thermofluor assay demonstrate the directly interaction between piperine and
DHODH. The co-complex crystal structure of DHODH and piperine at 1.98 Å
resolution further reveal that Tyr356 residue of DHODH is crucial for piperine
binding. Importantly, we show that piperine can inhibit T cell overactivation in a
DHODH-dependent manner in concanavalin A-triggered T-cell assay and
mixed lymphocyte reaction assay. Finally, piperine exhibits strong preventive
and therapeutic effect in the MOG-induced experimental allergic
encephalomyelitis (EAE), a useful model for studying potential treatments for
MS, by restricting inflammatory cells infiltration into the CNS and preventing
myelin destruction and blood–brain barrier (BBB) disruption. Taken together,
these findings highlight DHODH as a therapeutic target for autoimmune
disease of the nervous system, and demonstrate a novel role for piperine in
the treatment of MS.
Key words: Multiple sclerosis; DHODH; piperine; Natural product
2

Abbreviations
BBB, blood–brain barrier; CD, circular dichroism; CNS: central nervous system;
CoQ0, Coenzyme Q0; DCIP: 2, 6-dichloroindophenol; DHO: dihydroorotate;
DHODH, dihydroorotate dehydrogenase; FMN, flavin mononucleotide; ITC,
isothermal titration calorimetry; MS, multiple sclerosis;
3

1. Introduction
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the
central nervous system (CNS) that affects approximately 2.5 million people
worldwide [1, 2]. MS lesions can emerge in the whole CNS and are most easily
observed in the white matter as focal regions of inflammation and
demyelination [3]. The cause of MS is still not well-known, although genetic
susceptibility and environmental factors have been associated with the MS
occurrence [4, 5]. MS is the leading cause of disability in young adults,
resulting in serious socioeconomic impact for patients and for society
(approximately $10 billion in United States) [6-8]. Therefore, development of
new therapeutics is important to improve the clinical benefit and life quality for
MS patients.
MS is widely considered to be an inflammatory T-cell-mediated
autoimmune disease of CNS [9]. In clinical MS patients and Experimental
Allergic Encephalomyelitis (EAE) preclinical model, immune system T
leukocytes transmigrate from the bloodstream into the CNS to assault the
myelin sheath surrounding nerve fibers [10]. Autoreactive T cells that can
recognize CNS antigens play a critical pathogenic role in the induction of CNS
inflammation and demyelination, which then promotes the progression of
neurological function deficit and physical disability [11]. In clinical, increased
population of activated T cells against the myelin were observed in MS
patients compared to healthy subjects. Autoreactive CD4+ T cells were
4

considered as the key driver events in EAE models, indicating important
immunopathologic features of MS. Currently, increasing animal experiment
results and clinical trials of MS demonstrate that CD8+ T cells may also
contribute to inflammation sustaining and tissue destruction in MS [12, 13].
Collectively, T lymphocytes have emerged as important players in the
pathogenic mechanism of CNS inflammation, representing a promising
therapeutic target of MS.
Human Dihydroorotate dehydrogenase (DHODH) is a rate-limiting
enzyme in the de novo pyrimidine biosynthesis pathway. DHODH binds to
flavin mononucleotide (FMN) cofactor in conjunction with ubiquinone to
catalyze the oxidation of dihydroorotate to orotate [14-16]. Human DHODH is
located on the intracellular mitochondrial inner membrane and thereby can be
categorized into class 2 DHODH family. As an essential protein that catalyzes
the conversion of DHO to orotate, DHODH is a critical enzyme in the
pyrimidine de novo biosynthesis of DNA and RNA. In highly proliferative cells,
such as activated T lymphocytes, increased de novo pyrimidine biosynthesis
can enable their superior growth capacity [17]. Blockade of de novo pyrimidine
biosynthesis by pharmacological inhibiting DHODH has been considered as a
promising therapeutic strategy for autoimmune diseases through inhibition of T
lymphocytes activation [18]. DHODH inhibitor teriflunomide (A771726) have
been approved for the treatment of MS [19, 20]. However, its observed
hepatotoxicity in clinical and potential teratogenicity in preclinical model should
5

be taken into consideration in clinical treatment.
In the present study, we identified piperine, a main bioactive constituent of
black pepper, as a natural inhibitor of human DHODH by using a panel of
biochemical and biophysical methods, including enzymatic assay, isothermal
titration calorimetry and X-ray crystal diffraction. We further demonstrated that
DHODH inhibition by piperine ameliorated MOG-induced EAE mouse model
accompanied with reduced inflammation as well as lessened myelin and
blood–brain barrier (BBB) destruction. Taken together, our data revealed that
piperine targets T cells in EAE by inhibition of DHODH and provided a potential
treatment strategy for MS patients.
2. Materials and Methods
2.1 Reagents
Piperine and A771726 were obtained from Sigma-Aldrich (Shanghai, China).
Fluorescent probe BSA-Cy5.5 and DBT were purchased from Biolead Biology
Sci&Tech (Beijing, China). MOG35-55 peptide was obtained from GL Biochem
(Shanghai, China). Antibodies against mouse CD4 (Product # 17-0041-81)
and CD8 (Product # 11-0081-81) were purchased from ebioscience (CA, USA).
All other reagents were purchased from Sigma-Aldrich unless otherwise
indicated.
2.2 Protein expression and purification
Human DHODH protein expression and purification were performed as
6

described in our previous studies [21]. Briefly, the cDNA of human DHODH
was cloned into a vector plasmid pET-19b (Novagen, Madison, WI, USA),
which contains an N-terminal 6xHis-tag. DHODH protein was expressed in
Escherichia coli strain BL21 (DE3) (Novagen) at 16 °C for 15 h in the presence
of 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG). The cells were
collected by centrifugation at 3,500 rpm, and re-suspended in binding buffer
(50 mM HEPES, 300 mM NaCl, 10% Glycerol, 1% Triton X-100, pH 7.7). The
cells were then lysed by sonication and centrifuged at 12,000 rpm for 30 min.
The supernatant was gathered and loaded in Ni-NTA column pre-equilibrated
with binding buffer. The binding buffer supplemented with step gradients of
imidazole (0, 100 mM, 150 mM, 300 mM, 500 mM) were used to elute DHODH
protein. The eluted target protein was collected for the further experiments.
2.3 DHODH inhibition Assay
DHODH enzymatic activity was determined using 2, 6-dichloroindophenol
(DCIP) method as previously described [22]. In brief, recombinant human
DHODH (15 nM) was incubated with varied concentrations of indicated
compounds for 10 min at room temperature. The assay buffer was composed
of 50 mM Tris (pH 8.0), 120 μM DCIP, 100 μM Coenzyme Q0 (CoQ0), 0.1%
Triton X-100. The enzymatic reaction was initiated by adding dihydroorotate
(DHO) with a final concentration of 500 μM and absorption at 600 nm was
continuously recorded for 6 min with EnVision Multimode Plate Reader (Perkin
Elmer, Waltham, MA). The IC50 values were calculated by GraphPad Prism 8
(GraphPad software, CA).
7

2.4 Fluorescence-based thermal shift assay
Briefly, SYPRO orange stock solution (Invitrogen, Carlsbad NY) was diluted
1:1000 into the assay buffer (25 mM Hepes, 150 mM NaCl, pH 7.5), and
purified DHODH was added with a final concentration of 5 μM [23]. Then, the
mixture (40 µL) was loaded into the 96-well iCycler iQ PCR plate (Bio-Rad, CA,
USA) with various concentrations of compounds. The plate was heated from
25 to 80 °C with a rate of 0.50 °C /min and the fluorescence intensity was
measured at Ex/Em=490/530 nm using a CF×96TM Real Time System
(Bio-Rad, Hercules, CA) according to the manufacturer’s instructions.
2.5 Enzymatic kinetic analysis
Lineweaver-Burk analysis was carried out as described previously [24]. Briefly,
a variety of concentrations of CoQ0 (25-200 µM), DHO (25-200 µM) and
compound (1-3 µM) were incubated with DHODH (20 nM) in reaction buffer
(50 mM Tris, pH 8.0, and 150 mM KCl). The enzymatic reaction was detected
at 287 nm using a Hitachi U-2000 spectrophotometer (Tokyo, Japan). In
addition, kinetic curves of DHODH activity in the presence of varied
concentrations of piperine was generated to calculate the Kon and Koff values.
All experiments were performed at 25 °C. The experiments were performed at
least three times.
2.6 Isothermal titration calorimetry (ITC)
Thermodynamic characterization of DHODH-piperine interactions was
performed using an iTC200 instrument (Microcal, GE Healthcare, PA, USA) as
described before [25]. Before titrations, the stock solutions of PIP and DHODH
8

protein were diluted with ITC buffer (50 mM HEPES, 300 mM NaCl, 10%
Glycerol, 0.1% Triton) at the concentrations of 100 µM and 20 µM, respectively.
All titrations of piperine into DHODH were performed by 19 identical injections
of 2 µl with a duration of 4 seconds per injection spaced at interval of 120
seconds between injections following an initial injection of 0.4 µl at 25 °C. The
reaction heat of injecting piperine into the DHODH were obtained and the data
was processed by the supplied MicroCal Origin software package (PA, USA).
The final concentration of DMSO in the assay buffer is less than 3%. All the
calorimetric data were calculated with the formula ΔG = ΔH − TΔS = −RT lnK,
where T is experimental temperature, R is gas constant, K is binding constant,
ΔG represents changes of free energy, ΔH represents changes of enthalpy
and ΔS represents changes of entropy of, respectively.
2.7 Fluorescence quenching assay
Fluorescence quenching analysis was performed as previously described [26].
The purified DHODH protein was incubated with various concentrations of
piperine at 4ꢀ°C for 1ꢀh and fluorescence spectra were detected using a Cary
Eclipse fluorescence spectrophotometer (Agilent Technologies, CA, USA).
Excitation wavelength was set at 280ꢀnm and emission spectra ranging from
285 to 600ꢀnm was recorded.
2.8 CD spectroscopy
Briefly, DHODH protein were dialyzed against 10 mM phosphate buffer (pH 7.4)
and incubated with different concentrations of piperine for 2 min at room
temperature. The circular dichroism (CD) spectra were measured by chirascan
9

circular dichroism spectrometer (Applied PhotoPhysics, United Kingdom) in a
quartz cell with a 0.5-cm path length.
2.9 Crystallization of DHODH-piperine complexes
Optimal crystals were grown by hanging drop vapor diffusion method at 20 °C
as previously reported [27]. Drops were prepared by mixing 1.0 µL of 20 mg/ml
DHODH protein solution which contained 2 mM piperine, 50 mM HEPES pH
7.7, 400 mM NaCl, 30% Glycerol, 1 mM EDTA, 10 mM (N,
N-dimethylundecylamine-N-oxide)
UDAO,
and
20.8
mM
N,
N-dimethyldecylamine- N-oxide (DDAO) with equal-volume precipitant solution
of 0.1 M acetate, pH 4.8, and 2.4-2.6 M ammonium sulfate. The drops were
incubated against 0.5 mL of 50 mM HEPES pH 7.7, 300 mM NaCl, 20%
Glycerol, 1 mM EDTA, 10 mM UDAO and 20.8 mM DDAO. Diffraction-quality
crystals were appeared usually in three weeks and reached the full size of
0.15× 0.15× 0.153 mm. Crystals were flash frozen in liquid nitrogen for data
collection.
2.10 Data collection and structure determination
X-ray diffraction data were collected at the synchrotron beamline BL19U1 in
SSRF, Shanghai, China. The crystals were maintained at 100 K during data
collection. Data were indexed, integrated and scaled using HKL3000 (HKL
Research, Charlottesville, VA). The structure of the complex was determined
10

by molecular replacement using molrep (CCP4) and PDB entry 4JTU as an
initial model. The resulting model was re-builded and refined in Coot. The
images were generated using the software PyMOL (Molecular Graphics
System, Version 2.2.3; Schrödinger, LLC). A summary of the data collection
and processing statistics was given in Table 1. The coordinates and structure
factors of the final model of DHODH-piprine were deposited in the Protein Data
Bank (PDB, www.rcsb.org, PDB ID: 6LZL).
2.11 DHODH activity in Jurkat cells
Intracellular DHODH enzymatic activities was analyzed as described before
[28]. Jurkat T was purchased from National Infrastructure of Cell Line
Resource (Shanghai, China) and used less than 8 passages. Jurkat T cells
were cultured in RPMI-1640 with 10% FBS and treated with different
concentrations of piperine. After 24 h incubation, cells were harvested by
centrifugation and lysed in NP-40 lysis buffer at 4°C for 15 min. Lysates were
clarified at 12000 rpm for 10 min and the subsequent supernatant was used in
DHODH reaction. Lysate (200 μL) was incubated in buffer A (the final
volume,1.0 mL), containing 500 mM DHO, 200 mM K2CO3-HCl (pH 8.0), 0.2
Triton X-100, and 100 μM CoQ0 at 37 °C for 1 h. Mixture of enzyme reaction
(200 μL) was mixed with buffer B, containing 1 mM 4-TFMBAO, 2 mM
K3[Fe(CN)6], and 20 mM K2CO3 and then heated at 80 °C for 5 min. The
reaction was stopped by cooling in ice and the fluorescence signal was
recorded using a PT1-QM4 steady-stead fluorimeter (PTI, USA) at excitation
and emission wavelengths of 340 and 460 nm, respectively.
11

2.12 Mouse spleen cell proliferation
Single-cell suspensions of spleens from individual C57BL/6 mice were
harvested as described previously [29]. All animal care and experimental
procedures in this study were conducted in compliance with the protocol
approved by the Institutional Animal Care and Use Committee at East China
University of Science and Technology. Briefly, the spleen was aseptically
taken from mice, cut into small pieces and crush gently through the cell
strainers
in
5
ml
1×HBSS.
Cells
were
suspended
in
Ammonium-Chloride-Potassium (ACK) lysing buffer to remove the
erythrocytes and then collected by centrifugation at 2000 rpm for 10 min. After
washing twice with 1×HBSS, cells were re-suspended in RPMI-1640 with 10%
FBS for culture. Spleen cells were seeded into 96-wells cell culture plates at a
density of 5×105 cells/well in RPMI-1640 with 10% FBS and subsequently
incubated with various concentrations of piperine in the presence or absence
of uridine (100 μM). Cell proliferation was stimulated by adding 10 μg/ml of
Con A. After 72 h treatment, cells growth was measured by MTT assay with
EnVision Multimode Plate Reader (Perkin Elmer, Waltham, MA).
2.13 Mixed lymphocyte reaction (MLR) assay
The two-way mouse mixed lymphocyte reaction was conducted as described
in our previous study [22]. All animal care and experimental procedures in this
study were conducted in compliance with the protocol approved by the
Institutional Animal Care and Use Committee at East China University of
Science and Technology. In brief, single-cell suspensions of spleens from
12

C57BL/6 and BALB/C were prepared as foregoing mentioned. Each set of
2×105 cells were seed into 96-wells plates in RPMI-1640 with 10% FBS and 50
μM β-mercaptoethanol. Cells were incubated for 4 days with indicated
concentrations of piperine in the presence or absence of uridine (100 μM). Cell
proliferation was evaluated by MTT as described above and the proliferation of
BALB/C spleen cells alone was served as background.
2.14 MOG35–55 induced chronic progressive EAE model in C57BL/6 mice
10-12 weeks old female C57BL/6 mice were purchased from Shanghai
Laboratory Animal Center. All animal care and experimental procedures in this
study were conducted in compliance with the protocol approved by the
Institutional Animal Care and Use Committee at East China University of
Science and Technology. Mice were housed for 7 days in a flow cabinet with a
12 h light/dark cycle and fed with standard rodent chow and tap water ad
libitum. For EAE induction, mice were immunized in the flank with 100 µg
MOG35-55 peptide dissolved in Complete Freund’s Adjuvant (CFA, including
extract 4 mg/ml M. tuberculosis H37Ra; Beijing Biolead Biology Sci &
Tech)[30]. The 200 ng/mouse pertussis toxin (PTX) was injected on day 0 and
day 2. Treatment with piperine or vehicle started after immunization after 12
days and kept 17 days. Clinical symptoms scoring for MOG35-55 induced EAE
was evaluated as follows: score 0, no obvious changes in motor functions;
score 0.5, distal paralysis of the tail; score 1, complete tail paralysis; score 1.5,
mild paresis of one or both hind legs; score 2, severe paresis of hind legs;
score 2.5, complete paralysis of one hind leg; score 3, complete paralysis of
both hind legs. It is noteworthy that outcome assessors were blinded to
13

treatment allocation.
2.15 Flow Cytometry Analysis
Preparation of individual cell suspensions from EAE mouse was performed as
described before [29] All animal care and experimental procedures in this
study were conducted in compliance with the protocol approved by the
Institutional Animal Care and Use Committee at East China University of
Science and Technology. Splenocytes were resuspended in FACS buffer
(PBS, pH7.4, supplemented with 1 mM EDTA and 2 % FBS) and then
incubated with APC-CD4+ (mouse) and FITC-CD8+ (mouse) antibodies for 2 h
at 4 °C in the dark, respectively. Flow cytometry analysis was performed on a
BD FACS Caliber equipped with Cell Quest software (BD Biosciences, NJ,
USA).
2.16 In vivo imaging
The permeability of blood-brain barrier (BBB) was assessed with bovine serum
albumin coupled with Cy5.5 (BSA-Cy5.5) as described before [31]. The
BSA-Cy5.5 (50 mg/kg) was injected by tail vein, and imaging was performed 6
h after injection. All animal care and experimental procedures in this study
were conducted in compliance with the protocol approved by the Institutional
Animal Care and Use Committee at East China University of Science and
Technology. The images were acquired using an IVIS Spectrum CT Imaging
System (PerkinElmer, Inc. USA) with Ex/Em=680/720 nm. Myelin imaging was
performed with a near-infrared dye, 3,3-diethylthiatricarbocyanine iodide (DBT),
that binds to myelin and can be used to evaluated demyelination [32]. DBT dye
14

(0.3 mg/kg) were administered to mice by intravenous injections in the tail vein.
The fluorescence signals of the mouse myelin were acquired using a IVIS
Spectrum CT Imaging System (PerkinElmer, Inc. USA) 5 min post-injection of
DBT with Ex/Em=745/800 nm.
2.17 Histochemical analysis
The spinal cords dissected from the spinal canal were fixed in 4% buffered
paraformaldehyde for 24 h and then embedded in paraffin. All animal care and
experimental procedures in this study were conducted in compliance with the
protocol approved by the institutional Animal Care and Use Committee at East
China University of Science and Technology. In the following step, the
standard tissue sections (5 μm) were subjected to hematoxylin and eosin
(H&E) staining and luxol fast blue (LFB) staining. Images were acquired using
a digital color charge-coupled device camera and NIS-Elements imaging
software. The organ sections (heart, liver, spleen, lung, and kidney) from
vehicle- or piperine- treated EAE mice was subjected to H&E analysis.
2.18 Data and Statistical analysis
Data were shown as means +/- standard deviation of at least three
independent experiments. Statistical differences were analyzed using
two-tailed Student t-test or one-way ANOVA followed by Tukey test as
indicated in the figure legend for each experiment. Statistical analysis was
performed using GraphPad Prism 8 software (GraphPad software, CA, USA)
and differences were considered statistically significant at P≤0.05.
15

3. Results
3.1 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 inhibited DHODH enzymatic activity in a dose-dependent manner with
a half maximal inhibitory concentration (IC50) value of 0.88 ± 0.04 μM, which
was comparable with that of A771726 (IC50=0.35±0.02 μM), a Food and Drug
Administration approved DHODH inhibitor. We then determined the inhibition
mode of piperine by using the Lineweaver-Burk plot. The results indicated that
piperine is an uncompetitive inhibitor versus the substrate DHO (Figure 1C)
and a noncompetitive for CoQ0 (Figure 1D), which is the same as A771726
[24]. In addition, we measured affinity (Ki) and kinetic parameters Kon, Koff, and
residence time (RT) by enzymatic kinetic assays. As shown in Figure 1E-F,
piperine exhibited long residence time (Kon =1.28x102 M-1s-1, Koff=9.11x10-5 s-1,
RT=182.95 min) and high affinity (Ki= Koff/Kon=0.71 μM) for DHODH. The
inhibitory constant (Ki) of A771726 for DHODH is 0.18 ±0.02 μM as reported
by a previously study [33].
3.2 Piperine directly interacts with DHODH protein
Next, we examined the physical interaction between piperine and DHODH
using circular dichroism (CD) spectroscopy. This technique is widely used to
probe the binding of ligand to protein through analyzing the induced CD (ICD)
spectrum [34]. Purified DHODH protein was incubated with different
concentrations of piperine in phosphate buffer, and detected by CD. As
16

presented in Figure 2A, piperine induced a significant alteration of CD
spectrum, indicating a changed secondary structure of DHODH protein.
Considering the existing of tryptophan residues (Trp 362) in human DHODH
protein, we performed an intrinsic fluorescence-quenching assay to further
confirm the binding of piperine to DHODH (24). In this assay, DHODH (20 μM)
was incubated with varied concentrations of piperine, and the fluorescence
signals were measured. As shown in Figure 2B, DHODH had a maximal
fluorescence intensity at 337 nm, while piperine induced a fluorescence
quenching in a dose-dependent manner, which suggested the binding of
piperine to DHODH.
To investigate DHODH protein thermal stabilization upon compound
binding, we used thermal shift assay (TSA) [35]. DHODH protein (5 μM) was
incubated with various concentrations of piperine and the mixture was
subjected to thermal scanning. The Melting temperature (Tm) was calculated
from the Melt curve. Figure 2C showed that piperine dramatically improved the
stability of DHODH protein as reflected by a more than 5.12 °C increase of Tm
value, which was comparable with A771726 (approximately 7.5 °C) [36].
Furthermore, the thermodynamic parameters of the interaction between
piperine and DHODH was examined by isothermal titration calorimetry (ITC).
The purified recombinant human DHODH protein was titrated with piperine
and the reaction heat was recorded. As shown in Figure 2D, piperine bound to
DHODH with a KD value of 1.97±0.20 μM according to the ITC titration curves,
which was comparable with the result of DHODH enzymatic kinetic analysis
(Ki=0.71 ± 0.01 μM). Taken together, these results showed the potent binding
potency of piperine to DHODH.
17

3.3 Piperine co-crystallizes with human DHODH
Then, we solved the X-ray cocrystal structure of piperine bound to DHODH at
a resolution of 1.98 Å (PDB code: 6LZL). The high-resolution structure showed
that piperine was located in the hydrophobic channel formed between helix α1
and α2 (Figure 3A). This channel could be roughly divided into an exclusive
hydrophobic entrance and a relatively polar narrow end, and capped by a
small hydrophobic pocket formed by side chains of Val134 and Val143 (Figure
3B). Thus, the amphipathic character of binding channel made it a genuine
pocket for potential ligands binding. Obviously, the electron density
unequivocally revealed that piperine matches this amphipathic requirement
well: the 1,3-benzodioxole ring located in the hydrophobic part of the channel
and the n-piperidinecarbonyl of the piperine deeply buried in the polar
environment. The 1,3-benzodioxole ring of piperine contacting with the
surrounding residues, including Met 43, Ala 59, Leu 68, Phe 98 Pro 364,
formed
a
protruding
hydrophobic
patch
(Figure
3C).
The
N-piperidinecarboxaldehyde moiety of the piperine bound to the phenolic
hydroxyl group of Tyr 356 through a hydrogen bond and overlays well on the
polar subsite (Figure 3D). The short hydrogen bond distance (2.7Å) between
the oxygen atom of piperine and the hydroxyl of Tyr 356 in the crystallographic
structure indicated a reliable hydrogen bond interaction. DHODH have two
important redox sites, one is where FMN oxidizes dihydroorotate to orotate
18

and the other is where ubiquinone oxidizes FMNH2 to FMN [37]. Piperine can
occupy the narrow region in the FMNH2-ubiquinone redox site, which can
impact the corresponding DHODH activity. Polar and hydrophobic interactions
were illustrated clearly by the two-dimensional view of piperine contacting with
DHODH in Figure 3E, and there is no water in the binding pocket.
Compared with apo-DHODH (PDB: 5K9D) [38], the binding of piperine
induced a dislocation of the loop region between Leu 68 and Arg 72. This
region following Helix α2 connects the N- and C-terminal domains of DHODH
and is considered as a gatekeeping function by controlling interaction and
dissociation of ubiquinone [39]. Morever, after bound with piperine, the side
chain of Leu 68 and Pro 69 moved by approximately 0.3-1.0 Å to avoid a steric
clash with the 1,3-benzodioxole ring of the inhibitor (Figure 4A). The side
chains of Met 43, Gln 47, Pro 52, His 56, Phe 62, Tyr 147 worked as the
primary interaction partners with piperine, also underwent a conformational
change.
We also compared the binding mode of piperine with A771726. After
superposed the coordinates of cocrystallized DHODH-piperine and
co-crystallized DHODH-A771726 (PDB: 1D3H) [37], it could be observed that
the binding motif of piperine in DHODH was seemingly the same as A771726
by placing the carbonyl oriented toward the polar pocket of binding site (Figure
4B). Besides maintaining the interaction with Tyr 356, piperine was found can
be able to reach the small hydrophobic pocket created by Val143 and Val134.
19

This small hydrophobic pocket has often been used to achieve additional
ligand interactions and to increase the binding affinity [40]. Compared with
A771726, the longer carbon chain of piperine caused the 1,3-benzodioxole
ring contacting with the surrounding residues formed a protruding hydrophobic
patch (Figures 3E and 4B).
3.4 Piperine reduces spleen lymphocytes proliferation by inhibiting
DHODH
Human DHODH is a critical enzyme for de novo pyrimidine synthesis, and it is
crucial to the activity of T-cells [41]. Multiple sclerosis is an immune-mediated
disease and blocking autoreactive T cells is considered to be a promising
strategy [42, 43]. To test the immunosuppressive effects of piperine, we
investigated the anti-proliferative potential of piperine on concanavalin A (Con
A)-stimulated mouse spleen lymphocytes expansion (Figure 5A). The
FDA-approved DHODH inhibitor A771726 was used as a positive control. As
shown in Figure 5B, piperine markedly impaired ConA-induced T lymphocytes
proliferation in a dose-dependent manner. Considering that DHODH is the
rate-limiting enzyme in the endogenous biosynthesis of uridine
monophosphate, we asked whether uridine treatment could rescue
piperine-induced anti-proliferation on T lymphocytes. As speculated, uridine
treatment completely eliminated piperine’s inhibition capacity on
ConA-induced lymphocytes proliferation (Figure 5B). Mixed lymphocyte
reaction (MLR) is a classical model for T-cell reactivity. Similarly, we found that
piperine exhibited strong suppressive activities on MLR in a dose-dependent
20

manner, while uridine supplement significantly recovered lymphocytes
proliferation (Figure 5C-D).
Then, we turned to investigate whether cellular DHODH activity is affected
by piperine. Cultured Jurkat T cells were treated with piperine and cellular
orotic
acid
was
quantified
by
a
fluorogenic
probe
4-trifluoromethyl-benzamidoxime [28] (Figure 5E). As shown in Figure 5F,
piperine treatment significantly reduced DHODH activity in Jurkat T cells in a
dose-dependent manner with an IC50 value of 0.75 μM. Taken together, these
results suggested that piperine can reduce lymphocytes proliferation via
directly inhibiting DHODH.
3.5 Piperine reduces clinical score and severity of EAE
MS is an inflammatory and autoimmune disease. We next sought to determine
whether pharmacological inhibition of DHODH by piperine would attenuate
experimental autoimmune encephalomyelitis (EAE), a valuable model for
human MS [44]. For EAE induction, C57BL/6 mice were immunized with
MOG35-55 followed by pertussis toxin (PTX) administration (Figure 6A). EAE
mice were administered daily with vehicle, A771726, or piperine (10 or 30
mg/kg) by i.p. injections from day 12 after MOG’s induction and clinical sign
was blinded evaluated using a 5-point scoring system. As shown in Figure 6B,
therapy with piperine suppressed the manifestation of clinical symptoms over
the course of the study. Meanwhile, the lower sum of scores for each individual
mouse indicated a reduced disease burden for most mice in the
piperine-treated group (Figure 6C). In the EAE disease progression, body
weight loss is considered as a quantitative surrogate marker for disease
21

severity [45]. Figure 6D showed a significant recovery in animal body weight
following piperine treatment during the EAE period. This schedule was referred
to as preventive treatment.
We then sought to investigate whether piperine treatment introduced at
the peak of the EAE clinical symptoms could reduce the severity of disease
(Figure 6E). Remarkably, piperine treatment starting at the peak of EAE (day
20) lead to an accelerated recovery from the EAE clinical signs when
compared with the vehicle group (Figure 6F). Furthermore, the sum of scores
for individual mouse and average body weight loss was also largely improved
(Figure 6G-H). H&E analysis of organs sections from vehicle- or piperine-
treated EAE mice suggested that piperine is well tolerant and safe following
oral dosage of 30 mg/kg every day in EAE mice (Figure 8). Altogether, these
results strongly suggested that piperine alleviates the disease severity of EAE
in mice as administrated in both preventive and therapeutic regimens.
3.6 Piperine alleviates inflammation, blood-brain-barrier and myelin
destruction
MS has numerous pathologic hallmarks as an inflammatory and autoimmune
CNS disease, such as leukocytes infiltration, blood-brain-barrier (BBB) and
myelin destruction. Spinal cords tissues of the mice under preventive
treatment were subjected to histological analysis on day 22 after MOG’s
immunization. As illustrated in Figure 7A, piperine treated-group showed
reduced leukocyte infiltration by hematoxylin and eosin (H&E) staining and
less demyelination in white matter by Luxol fast blue (LFB) staining as
compared with the vehicle-treated mice. The intensity of LFB staining of myelin
22

was measured by Image J and then scored visually in a semi-quantitative
manner [46]. The spinal cord section of the mice with piperine treatment
showed an increasing density compared with EAE mice, and there was a 50%
increase in total optical density.
We then aimed to investigate the effect of piperine on BBB permeability
using a fluorescent BSA conjugate probe BSA-Cy5.5. Figure 7B showed that
piperine significantly reduced the accumulation of BSA-Cy5.5 in the brain as
indicated by in vivo optical imaging when compared to the vehicle treated mice,
suggesting improved BBB integrity against EAE challenge. During the
pathological process of MS, an increased BBB permeability allows
lymphocytes activated in the periphery to infiltrate the CNS, which evoke a
local immune reaction resulting in axons and myelin damages. We next
examined whether piperine treatment alleviates myelin destruction in EAE
model using a myelin-binding near-infrared dye 3,3-diethylthiatricarbocyanine
iodide (DBT). DBT dye (0.3 mg/kg) was intravenously injected to mice and the
fluorescence signals were imaged. Figure 7C showed a higher fluorescent
intensity in piperine-treated group as compared to the vehicle group, indicating
reduced myelin destruction following piperine treatment.
We also investigated the effect of piperine on EAE-related T cell activation.
Flow cytometry analysis demonstrates that EAE challenge induced a
significantly increase of population of CD8+ and CD4+ T cell in spleen
(increased from 9.35±0.17% to 13.6±0.32% for CD8+ T cell and from
16.17±0.18% to 24.32±0.39% for CD4+ T cell) (Figure 7D-E). Remarkably,
piperine treatment group (30 mg/kg) showed a significantly reduction of CD8+
population, when compared with the vehicle-treated mice. In addition, reduced
23

CD4+ proportion was also observed following piperine administration. Taken
together, these data indicated that piperine can effectively ameliorate
inflammation and protect blood-brain-barrier and myelin in EAE mice.
4. Discussion
MS is a chronic disease of the CNS characterized by autoimmune
inflammation, demyelination, and axonal damage [3, 47]. The underlying
immunological disorders in MS result in a variety of neurological symptoms. A
number of studies have revealed a critical role of T cells in the pathogenesis of
EAE and MS, which makes them a promising target for drug discovery [48].
Pyrimidine nucleotides are critical for DNA and RNA synthesis and are
indispensable for the development and survival of mature T lymphocytes [17].
Human DHODH catalyzes the fourth committed step in the de novo pyrimidine
nucleotide biosynthetic pathway [37]. Pharmacological intervention of
pyrimidine biosynthesis by selectively inhibition of DHODH has been explored
for the development of new therapeutic agents against MS. Synthetic small
molecule A771726 have been approved for the treatment of MS as an
immunomodulatory drug [46]. However, the long elimination half-life of
A771726 (approximately 2 weeks) [49, 50] is associated with its observed
hepatotoxicity in clinical and potential teratogenicity in animal study [51]. More
safe and effective therapeutic agents targeting DHODH are needed to benefit
patient with MS.
24

Black pepper was widely used in traditional medicine as a natural herb for
the therapy of a variety of diseases including rheumatism, pains, and fevers
[52]. Piperine is the predominant active constituent of black pepper and also is
the most abundant alkaloid in pepper (approximately 98%). Previous research
has revealed that piperine has widely pharmacological activities, including
anti-inflammatory, antiarthritic effects, antioxidant and chemopreventive
activities [53, 54]. To the best of our knowledge, this is the first study of
revealing piperine’s potent protective activity in autoimmune demyelinating
disease.
Piperine was widely used as a flavoring ingredient in foods and beverages
manufacturers with well-documented safety since it is first isolated from
pepper in 1819 [52]. Using modern drug safety evaluation system, Bastaki M
et al organized a 90-day GLP compliant toxicity study in sprague-dawley rats
with multiple doses of piperine (5-50ꢀmg/kgꢀbw/day) to evaluate the safety of
daily uptake of piperine in foods. Notably, no adverse effects were observed
following piperine administration even at the highest dosage (50ꢀmg/kgꢀbw/day)
[55]. Herein, we present data that piperine has the ability to interact with
DHODH and reduce T cell proliferation via a DHODH dependent manner. We
further show that piperine effectively alleviates autoimmune response in
MOG-induced EAE mouse model without obvious toxicity following the oral
administration of piperine (30 mg/kg). As piperine exerts its pharmacological
effects in preclinical MS model through DHODH inhibition, and as piperine is a
25

clinical stage compound with excellent safety [56], drug repurposing of
piperine may be a valuable manipulation to benefit patients with MS.
According to the MS immunopathogenesis hypothesis, activated T cells
can cross the BBB and reactive with CNS proteins, including myelin basic
protein [MBP] and myelin oligodendrocyte glycoprotein [MOG] [57]. The BBB
penetration of a small molecule may facilitate targeting activated T cells in the
brain lesion. Intriguingly, piperine is capable of crossing the BBB as detected
in an in vitro parallel artificial membrane permeability assay [58]. Furthermore,
in vivo tissue distribution assay indicated that piperine can penetrate BBB in
rat [59]. So, the BBB penetration of piperine may lay a foundation for its
superior pharmacological activity in nervous system.
Multiple sclerosis (MS) has long been considered a CD4 T-cell disease
and emerging reports demonstrated that CD8 T cells also play a significant
role in disease process [60]. Current treatment strategies mainly focus on
eliminating or reducing these pathogenic T cells. In recent years, the clinical
success of B cell-targeting therapies raised the attention on B cells. [61, 62].
On the other hand, several studies have showed that other immune cells are
also related to MS pathogenesis. For instance, microglia/macrophages play a
dual role in the pathogenesis of MS. They can exert myelin regeneration and
repair effects but also contribute to neuronal damage by production of
inflammatory mediators [63, 64]. While, the therapeutic relevance of
macrophages/microglia are not well defined. In this study, we mainly focus on
26

T cells in EAE model based on these observations, while further studies are
needed to verify whether other immune cells contributed to the therapeutic
effect of piperine in EAE.
Piperine was identified as a less potent agonist of TRPV1 (EC50=37.9 μM)
[65]. Through a bioassay-guided isolation of the ethanol extract from the fruits
of Piper longum, Ro’s group have identified piperine as a moderate dual
MAO-A and MAO-B inhibitor with IC50 values of 20.9 μM and 7.0 μM,
respectively [16]. Piperine (10-100μM) was reported to reduce PMA-simulated
inflammation response by targeting NF-κB, C/EBP and AP-1 signaling [66].
Herein, we demonstrate that piperine is a potent, direct DHODH inhibitor with
an IC50 value of 0.88 ± 0.04 μM. Meanwhile, piperine effectively reduced T cell
proliferation through DHODH inhibition in vitro, suggesting a potential role of
piperine in immune function modulating. Furthermore, piperine treatment
significantly reduced immune cell infiltration in central nervous tissue and
resultant remission of EAE symptoms. The 13.2-15.8 h terminal elimination
half-life (t1/2) of piperine [67] is largely shorter than A771726. Since DHODH is
a main functional target of EAE, we corroborated the efficacy of piperine in MS
model by inhibiting DHODH activity, although it remains to be investigated in
the future whether other proteins were involved in the therapeutic effect of
piperine in EAE.
In summary, we demonstrate that piperine regulates the activation of T
lymphocytes through pharmacological inhibiting DHODH and blocking de novo
27

pyrimidine biosynthesis. Importantly, EAE disease progression was observably
alleviated following oral administration of piperine in both preventive and
therapeutic regimens. These compelling preclinical data presented here
suggest that piperine is a potential therapeutic agent for MS with a novel
mechanism-of-action, and further demonstrate that DHODH is a promising
therapeutic target for the treatment of MS.
Acknowledgements
This study was supported by the National Natural Science Foundation of China
(81773775, 81973362, 81830083), Shanghai Committee of Science and
Technology (18431900500, 19ZR1473500), Open Research Project of Key
Laboratory of High-Incidence-Tumor Prevention & Treatment (Guangxi
Medical University), Ministry of Education. We thank Dr. Wenming Qin at
SSRF beamline BL19U for assistance with data collection. We also thank
Research Center of Analysis and Test of East China University of Science and
Technology for technical assistance.
Conflict of Interest
Zehui Liu, Jin Huang, Weiqiang Lu and Lei Ma have applied for a china patent
on piperine.
28

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