Introduction
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS) that afflicts approximately 2.5 million people worldwide(Baecher-Allan, Kaskow & Weiner, 2018; Goverman, 2009). MS lesions can emerge in the whole CNS and are most easily observed in the white matter as focal regions of inflammation and demyelination(Reich, Lucchinetti & Calabresi, 2018). The well-defined cause of MS still remains elusive now, although genetic susceptibility and environmental factors have been associated with the MS occurrence(Ascherio, 2013; Dendrou, Fugger & Friese, 2015a). MS is the leading cause of disability in young adults, resulting in serious socioeconomic consequences for the individual patient and for society (approximately $10 billion in United States)(Adelman, Rane & Villa, 2013; Holland, Schneider, Rapp & Kalb, 2011; Jennum, Wanscher, Frederiksen & Kjellberg, 2012). Therefore, development of innovative therapeutic is an urgent need 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(Ridderstad Wollberg et al., 2014). In clinical MS and EAE model, immune system T leukocytes transmigrate from the bloodstream into the CNS to assault the myelin sheath surrounding nerve fibers(Zozulya & Wiendl, 2008). Autoreactive T cells indiscriminately recognize CNS antigens play a critical pathogenic role in the induction of CNS inflammation and demyelination, and thereby in the progression of neurological function deficit and physical disability(T Hart & Massacesi, 2009). In clinical, increased population of activated T cells against the myelin were observed in MS patients compared to healthy human. Autoreactive CD4+ T cells were considered as the key driver events in EAE models, indicating important immunopathologic features of MS(Dendrou, Fugger & Friese, 2015a). Currently, increasing animal experiment results and clinical trials data of MS demonstrate that CD8+ T-cells may also contribute to inflammation sustaining and tissue destruction in MS(Friese & Fugger, 2005; Lassmann & Bradl, 2017). Collectively, T lymphocytes have emerged as important players in the pathogenic mechanism of CNS autoimmune 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(Löffler, Jöckel & Schuster, 1997; Munier-Lehmann, Vidalain, Tangy & Janin, 2013; Sykes et al., 2016). Human DHODH is located in 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 player in the pyrimidine de novo biosynthesis of DNA and RNA. In high proliferative cells, such as activated T lymphocytes, increasedde novo pyrimidine biosynthesis can confer their superior growth capacity(Quemeneur, Gerland, Flacher, Ffrench, Revillard & Genestier, 2003). Blockade of de novo pyrimidine biosynthesis by pharmacological targeting of DHODH has been considered as a promising therapeutic strategy for autoimmune diseases through inhibition of T lymphocytes activation. Teriflunomide (A77 1726) have been approved for the treatment of MS through blocking pyrimidine de novobiosynthesis at the level of DHODH(Dimitrova, Skapenko, Herrmann, Schleyerbach, Kalden & Schulze-Koops, 2002; Ruckemann et al., 1998). While, its observed hepatotoxicity in clinical and potential teratogenicity in preclinical model limit its benefit.
In the present study, we identified piperine, a main bioactive constituent of traditional medicine black pepper, as a natural inhibitor of human DHODH 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 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.