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  • br Acknowledgement br Introduction Human dihydroorotate

    2019-08-20


    Acknowledgement
    Introduction Human dihydroorotate dehydrogenase (hDHODH), a flavin-dependent mitochondrial enzyme involved in de novo xpo 1 biosynthesis, is a validated therapeutic target for the treatment of autoimmune diseases such as rheumatoid arthritis and cancer [1], [2], [3]. Leflunomide (Fig. 1) is a disease-modifying anti-rheumatic drug that was approved more than 15 years ago for the treatment of rheumatoid arthritis and other autoimmune diseases [4]. Although associated with severe side effects such as diarrhea, abnormal liver tests, nausea, and hair loss [6], leflunomide acts as a prodrug and is rapidly converted into its active metabolite teriflunomide (also called A77 1726, Fig. 1), which is able to inhibit hDHODH in the low μM range [7], [8]. Since the introduction of leflunomide, the search for new potent hDHODH inhibitors that would display similar clinical benefits as leflunomide but without associated side effects, has been on going. One of the promising compounds was brequinar [9], which was discarded as a therapeutic agent due to a narrow therapeutic window and inconsistent pharmacokinetics [2]. Another compound, 4SC-101 (vidofludimus, Fig. 1) [10], is currently undergoing phase II clinical trials for inflammatory bowel disease [6], [11]. However, despite recent efforts [1], [6], [12], [13], [14], [15], the quest to add new hDHODH inhibitors to the human pharmacopoeia remains an urgent area of research. Earlier, we reported a series of innovative hDHODH inhibitors [5] designed by merging some structural features of leflunomide and brequinar and based on the acidic 4-hydroxy-1,2,5-oxadiazol-3-yl (hydroxyfurazan) moiety (Fig. 1, compounds 1 and 2). The acidic hydroxyfurazan, connected through an amide bridge to a substituted biphenyl lipophilic moiety, was suggested to play the role of brequinar\'s carboxylic group by interacting with Arg136 in the hDHODH subsite 2 [16]. Compounds 1 and 2 were able to potently inhibit DHODH on murine liver mitochondrial membranes (50 and 66 nM respectively) [5]. The degree of fluorine substitution at the phenyl ring adjacent to the oxadiazole moiety was strongly correlated with activity. In addition, the correlation between activity and stabilization of the compounds\' bioactive conformations was extensively studied [17]. Using a similar approach, in this work we describe new potent hDHODH inhibitors designed by selecting other acidic hydroxylated azoles, ideally substituting hydroxyfurazan in 1 and 2. The selection of hydroxylated azole systems (specifically hydroxythiadiazole, pyrazole and triazole) was run by the possibility of establishing interactions with the small lipophilic pocket created by Val143 and Val134 (subsite 4) and by their different acidic properties [18]. Supported by promising docking scores, nine candidate structures based on three acidic heterocycles were designed and synthetized (compounds 3–9, Fig. 2, Table S1). Synthetic strategies and detailed enzymatic and cell-based studies of the designed series are presented and discussed. The suggested binding modes of the most representative molecules were confirmed by high-resolution crystal structures of hDHODH in complex with the compounds 4–6.
    Result and discussion
    Conclusions In this paper we introduced a new generation of hDHODH inhibitors designed by scaffold hopping replacement of the acidic moiety of brequinar with different hydroxylated azoles. All the designed compounds can potently inhibit hDHODH in vitro, reaching an IC50 value of 16 nM in the best example, the thiadiazole 4. Moreover, when tested for antiproliferative activity, compounds 3–9 (but not 6a and 6b) were found to be effective in the same range of concentration as brequinar. In addition, compounds 4, 5 and 6 have lower cellular cytotoxicity than the leads, showing cytotoxic effects at 70-fold higher concentrations than those required to inhibit cell proliferation. These three promising hDHODH inhibitors, for which evidence suggests that antiproliferative activity depends on blocking the de novo pyrimidine biosynthesis, were tested for their immunosuppression activity showing promising effects on PBMC, similarly to brequinar. Compounds 4, 5 and 6 therefore represent original chemical scaffolds explored in the field of hDHODH inhibition and might lead to the discovery of new immunosuppressant and antiproliferative agents targeting hDHODH. The crystal structures of the most interesting hDHODH inhibitors from series 4, 5 and 6 will facilitate subsequent optimization both in terms of drug-like properties and pharmacokinetic characteristics. In addition, optimized analogues could undergo in vivo tests (e.g. collagen-induced arthritis, CIA) to evaluate their anti-arthritic activity. These studies are under development and will be the subjects of forthcoming publications.