SmDHODH showed to catalyze the conversion of dihydroorotate

SmDHODH showed to catalyze the conversion of dihydroorotate into orotate using quinone as 2-Palmitoylglycerol acceptor following a ping-pong mechanism. Despite the overall resemblance to the human homologue enzyme, substitutions and differences in the conformational states adopted by HsDHODH and SmDHODH structures may allow the identification of selective inhibitors against the parasite enzyme. In addition, comparison between SmDHODH and PfDHODH structures suggest that inhibitors of PfDHODH could be exploited as potential inhibitors of SmDHODH. In fact, we believe that the search for SmDHODH inhibitors as well as the evaluation of SmDHODH as a drug target against schistosomiasis can be strongly benefited by the extraordinary work already performed in the development of class 2 DHODH inhibitors. Based on the structural and biochemical similarities shared among class 2 DHODHs, it is reasonable to consider that the strategy of testing chemical scaffolds already developed to inhibit class 2 DHODHs, including chemotherapeutic agents already in the market or undergoing clinical trials, can shortcut the identification of pharmacophore groups as well as potent SmDHODH inhibitors. Thus, considering the biological relevance of nucleotide biosynthesis, and the limited investment on the development of new treatments against neglected diseases, it is reasonable to consider that the possibility of repurposing existing drugs based on the selective inhibition of class 2 DHODHs can be a useful strategy to accelerate the drug development for schistosomiasis due to lower costs, reduced risk and the possibility of decreased time to market due to availability of preclinical data.
Introduction
Chemistry The synthetic pathway used to obtain the final products is very straightforward (Scheme 1). The benzyloxy-substituted 1,2,5-oxadiazolecarboxylic acid 8, a product we have described elsewhere (see Experimental), was transformed into the corresponding carbonyl chloride 9 by action of thionyl chloride. This intermediate was treated with the appropriately substituted anilines 10a–n in the presence of pyridine, to afford the related amides 11a–n. The same products were also obtained by direct coupling of the acid 8 with the corresponding anilines using HBTU (2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) in DMF. The amides 11a–n, upon hydrogenation on Pd/C in THF, yielded the expected final compounds 7a–n.
Inhibition of DHODH The final products 7a–n were assessed for their DHODH inhibitory activity on rat liver mitochondrial/microsomal membranes. A procedure adapted from the literature was employed (see Experimental), in which oxidation of DHO to ORO is monitored by following the concomitant reduction of the chromophore 2,6-dichlorophenolindophenol (DCIP) [24]; A771726 and BQN were taken as references. The reduction of DCIP was detected by the decrease in absorbance at 650 nm. The initial rate of the enzymatic reaction in the presence (v) and in the absence (V) of inhibitor was measured, and the IC50 value was calculated from Eq. (1):where [I] is the concentration of the inhibitor. The results are collected in Table 1.