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

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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.

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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    Zehui Liu and Qian Hu contributed equally to this work.

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