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  • Moreover maintaining the pyrimidine pool is not the only imp


    Moreover, maintaining the pyrimidine pool is not the only important role played by DHODHs. In Toxoplasma gondii, for instance, DHODH plays a second essential function [38] possibly coupled to the mitochondrial respiratory activity, where it replenishes ubiquinol levels and prevents reactive oxygen species formation [39]. As a first step towards evaluating the potential of DHODH as a drug target against schistosomiasis, we cloned gene fragments encoding SmDHODH and its human homologue, HsDHODH, using identical constructs. Both enzymes were overexpressed in E. coli and purified using metal ion affinity chromatography. Tag free SmDHODH and HsDHODH were kinetically characterized and an inhibition assay was optimized to allow for the screening of ligands and cross-validation studies. Well known class 2 DHODH inhibitors, atovaquone, A77126, brequinar [5] and DSM265 [29,40,41] were tested against both enzymes and a multimethod approach combining homology modeling and comparative analysis of molecular dynamics trajectories were used to provide the molecular basis of inhibitor selectivity. Our results indicate that selective inhibition of SmDHODH is feasible and can be used towards the development of novel chemical entities to be tested for the treatment of schistosomiasis.
    Experimental procedures
    Results and discussion
    Conclusions The discovery and development of novel antischistosomal drugs is of utmost importance. It is our interested to evaluate DHODH as a drug target against schistosomiasis. As a first step towards this goal, DHODH from Schistosoma mansoni was expressed in E. coli and characterized by combining biochemical, biophysical and theoretical methods. SmDHODH showed to catalyze the conversion of dihydroorotate into orotate using quinone as LDN193189 Hydrochloride 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.
    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.