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  • br Acknowledgments We thank Jon Clardy

    2019-09-06


    Acknowledgments We thank Jon Clardy, Juan Manuel Domínguez, José Francisco García Bustos, and the GlaxoSmithKline (GSK) Plasmodium falciparum dihydroorotate dehydrogenase project team for their contributions, helpful advice, and discussions.
    Introduction Dihydroorotate dehydrogenase (DHODH) is a flavin-dependent enzyme that involves in the rate-limiting step in de novo pyrimidine biosynthesis (Munier-Lehmann et al., 2013). DHODHs could be divided into two classes according to amino SecinH3 sequence, cellular localization, and cofactor dependence (Bjornberg et al., 1997). Class 1 DHODHs, which exist chiefly in prokaryotes, are cytosolic enzymes using fumarate or NAD+ as electron acceptor (Nagy et al., 1992, Nielsen et al., 1996). Human dihydroorotate dehydrogenase (hDHODH) belongs to the class 2 enzymes, and catalyzes oxidative conversion of dihydroorotate (DHO) to orotate with ubiquinone as electron acceptor on the inner mitochondrial membrane (Jockel et al., 1998). Pyrimidines are required for the biosynthesis of DNA, RNA and phospholipid, which significantly affect cell proliferation and metabolism (Cherwinski et al., 1995, Nara et al., 2000). In humans, there are two routes for the synthesis of pyrimidines: salvage pathway and de novo synthesis. For rapidly proliferating cells, the salvage pathway could not supply sufficient pyrimidines for survival. Therefore, hDHODH is essential to activated T cells, as they depend heavily on de novo nucleotide synthesis. Inhibition of hDHODH activity decreases cellular levels of uridine monophosphate and arrests T-cells proliferation (Gummert et al., 1999, Loffler et al., 2005), which has been proved to be beneficial for the treatment against autoimmune diseases such as multiple sclerosis (O’Connor et al., 2006), lupus erythematosus and rheumatoid arthritis (RA) (O’Doherty et al., 2012). As the inhibitor of hDHODH, leflunomide was approved by FDA in 1999 for the treatment of RA and psoriatic arthritis (Kaltwasser et al., 2004, Smolen et al., 1999). Teriflunomide (A771726) is the active metabolite of leflunomide (Kieseier et al., 2009) and is indicated for the treatment of multiple sclerosis (Straus Farber et al., 2016). However, they have severe side effects that limit their application, such as liver damage (Redaelli et al., 2015), respiratory tract infections, and diarrhea (Chan et al., 2016). Development of new small molecular inhibitors specially targeted hDHODH still remains a compelling therapeutic goal. Ascochlorin (ASC) is a prenyl-phenol compound that was originally isolated from the phytophathogenic fungus Ascochyta visiae as an antiviral agent (Nawata et al., 1969, Tamura et al., 1968). In addition to its antimicrobial activity, ASC and its derivatives exhibit various physiological activities, including hypolipidemic activity (Hosokawa et al., 1981b), suppression of hypertension (Hosokawa et al., 1981a), anti-tumor activity (Hong et al., 2005), and activation of nuclear receptors (Togashi et al., 2003). Moreover, an ASC derivative has been reported to suppress the immune responses (Tsuruga et al., 2007), but its exact mechanism remains unknown. When we screened the hDHODH inhibitors from the microbial metabolites library through enzyme-based high-throughput screening (HTS), ASC and its derivatives (Fig. 1) were discovered as a new structural class of hDHODH inhibitors with strong inhibitory activity. In this study, we reported for the first time the inhibition of ASC against hDHODH, and evaluated its in vitro and in vivo anti-inflammatory and immunosuppressive effects.
    Materials and methods
    Results
    Discussion As the critical enzyme for de novo pyrimidine biosynthesis, hDHODH is considered as a promising target for the development of anti-proliferative, anti-inflammatory and immunosuppressive drugs since rapid cell proliferation often relays on de novo synthesis of pyrimidine nucleotides (Davidson and Diamond, 2001). Although leflunomide has been approved as treatment for RA, it couldnot meet the clinical demand on SecinH3 the safety and efficacy. Thus, research institutes and pharmaceutical companies still try to discover more potent hDHODH inhibitors such as LAS-186323 (Norman, 2013), vidofludimus (Kulkarni et al., 2010), FK-778 (Wlodarczyk et al., 2012), and some inhibitors are currently developed in clinical trials. The aims of this study were to find novel hDHODH inhibitors through HTS and to assess their pharmacological activity in vitro and in vivo for development of new immunosuppressive agents. In search for hDHODH inhibitors, ASC and its derivatives were discovered as a new class of high potent hDHODH inhibitors from a microbial metabolites library of NCPC.