Natural product piperine alleviates experimental allergic encephalomyelitis in mice by targeting dihydroorotate dehydrogenase
Graphical abstract
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], [7], [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 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], [15], [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 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.
Section snippets
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.
Protein expression and purification
Human DHODH protein expression and purification were
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 Fig. 1A and 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
Discussion
MS is a chronic disease of the CNS characterized by autoimmune inflammation, demyelination, and axonal damage [3], [45]. 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 [46]. Pyrimidine nucleotides are critical for DNA and RNA synthesis and are indispensable for the development and survival of mature T
CRediT authorship contribution statement
Zehui Liu: Writing - original draft, Methodology, Data curation. Qian Hu: Formal analysis, Visualization. Wanyan Wang: Software. Sisi Lu: Investigation. Dang Wu: Formal analysis. Shuyin Ze: Software. Jiacheng He: . Ying Huang: Resources. Wuyan Chen: Validation. Yechun Xu: Resources. Weiqiang Lu: Conceptualization, Writing - review & editing. Jin Huang: Supervision.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This study was supported by the National Natural Science Foundation of China (81773775, 81973362, 81972828), 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
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Drug repurposing of ilepcimide that ameliorates experimental autoimmune encephalomyelitis via restricting inflammatory response and oxidative stress
2023, Toxicology and Applied PharmacologyCitation Excerpt :Ilepcimide is a piperidine derivative which was first synthesized by Peking University Pharmaceutical Company as a structural analog of piperine, a major bioactive phytochemical of black pepper (Piper nigrum Linn) (Xiao et al., 2015). In our previous study, we have showed that piperine alleviates experimental allergic encephalomyelitis (EAE) in mice through inhibiting T-lymphocyte driven abnormal inflammatory responses (Liu et al., 2020). Considering the structural similarly of ilepcimide and piperine, and the well-documented safety of ilepcimide, we are inspired to repurpose ilepcimide for MS therapy.
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2022, European Journal of Medicinal ChemistryCitation Excerpt :The hydrophilic heads of hDHODH inhibitors are mainly responsible for their key hydrogen-bond binding with protein. We analyzed seventeen crystal structures of hDHODH complexed with inhibitors, suggesting that most of hDHODH inhibitors formed direct or water-mediated hydrogen bonds with residues Gln47, Arg136 and Thr360 with the frequency values of 14, 12 and 9 (Fig. 3A, Table S1) [11,24–37]. Interestingly, the hydrophilic head of teriflunomide formed a hydrogen-bond network with Gln47, Arg136 and Thr360 via two water molecules (Fig. 3B).
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2022, European Journal of Medicinal ChemistryCitation Excerpt :It is also used as antioxidant, antitumor, antimicrobial, anti-depressive and anti-inflammatory agent [128]. A recent study shows that piperine, as a natural hDHODH inhibitor [129], can inhibit T cell overactivation and exhibits strong preventive and therapeutic effect in the MOG-induced experimental allergic encephalomyelitis (EAE). Thermal shift assay (TSA) studies show that piperine has a strong binding ability with hDHODH (Ki = 0.71 μM).
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Zehui Liu and Qian Hu contributed equally to this work.