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  • br Results and discussion br Conclusion


    Results and discussion
    Conclusion Based on the structure of ThDP, in this study, a series of novel ThDP analogs 6a-6g and 8a-8g were designed by optimizing triazole-benzene linker and modifying the substituent group of triazole ring. Then they were synthesized as potential inhibitors of Cy-PDHc E1 by using click chemistry. All the compounds 6a-6g (IC50 3.37–4.48 µM) had much better inhibitory activity against Cy-PDHc E1 than that of lead compounds Ia-Id. Compounds 8a-8g with iodine (I) in the 5-position of 1, 2, 3-triazole showed higher inhibitory activity (IC50 = 1.48–2.35 µM) against Cy-PDHc E1 than that of compounds 6a-6g. Especially ack1 inhibitor 8d exhibited highest inhibitory activity (IC50 = 1.48 µM). These results showed that the ester group of compounds 6a-6g and 8a-8g was the most important factor for their inhibitory activities against Cy-PDHc E1, at the same time, the introduction of iodine group to the 5-position of 1, 2, ack1 inhibitor 3-triazole was to the benefit for increasing the inhibitory activity of compounds 8a-8g against Cy-PDHc E1. It is more interesting that all the compounds 6a-6g and 8a-8g had stronger inhibition against Cy-PDHc E1 (inhibitory rate >90%) than that of their inhibition against porcine PDHc E1 (inhibitory rate only 7.31–21.0%) in vitro at the same concentration (100 µM), indicating that they owned possibly lower toxicity and more eco-friendly characterizations as potential algicide. Furthermore, the corresponding compounds 6a-6g and 8a-8g exhibited dramatically high algicide activities (EC50 = 0.74–2.44 µM). Especially compounds 8a-8g showed better algicide activities even than that of CuSO4. The results of molecular docking together with site-directed mutagenesis and fluorescence spectral analysis showed that triazole-benzene linker and the substituent group of triazole ring are the possibly important reasons for the higher inhibition activity of compound 8d. These results indicated that compound 8d could be used as a hit compound for further optimization and might have potential to be developed as a new algicide.
    Experimental procedures
    Introduction The post-translational modifier ubiquitin influences virtually all aspects of eukaryotic biology (Hershko and Ciechanover, 1998). The single-chain ubiquitin-activating enzyme E1 (UBA1) adenylates the C terminus of ubiquitin (Ub), followed by Ub transfer onto the UBA1 catalytic cysteine (Haas et al., 1983). Thioesterified Ub is subsequently relayed to a ubiquitin-conjugating enzyme (E2) and, with the aid of ubiquitin ligases (E3), onto target proteins (Hershko and Ciechanover, 1998). Besides Ub, there are 16 additional ubiquitin-like proteins (Ubls) including SUMO and NEDD8, which are activated by cognate E1s (Schulman and Harper, 2009, Cappadocia and Lima, 2017). SUMO and NEDD8 E1s are heterodimers (SAE1/UBA2 and APPBP1/UBA3) with UBA2 and UBA3, respectively, representing the catalytic subunit (Lee and Schindelin, 2008, Schulman and Harper, 2009, Streich and Haas, 2010). Ever since the approval by the US Food and Drug Administration of the proteasome inhibitor bortezomib for the treatment of multiple myeloma and mantle cell lymphoma, extensive research has been carried out to target the components of the ubiquitin proteasome system for cancer therapy. UBA1, 2, and 3 were established as promising oncological drug targets (da Silva et al., 2013). Furthermore, targeting UBA1 may overcome clinical resistance to bortezomib and be more effective in treating solid tumors (An and Statsyuk, 2015a, Xu et al., 2010). Wilkinson et al. (1990) demonstrated that a non-hydrolyzable analog of the ubiquitin adenylate could potently inhibit the ubi-quitin-activating enzyme. Later, Ub-AMSN and SUMO-AMSN probes, which mimic the ubiquitin and SUMO adenylates, respectively, were shown to selectively inhibit Uba1 and the SUMO E1 (Lu et al., 2010); however, due to their large size these semisynthetic protein inhibitors could not be used for therapeutic purposes. Adenosyl sulfamates were described as cell membrane-permeable E1 inhibitors with the NEDD8 E1-selective inhibitor MLN4924 as first-in-class representative (Soucy et al., 2009). These compounds form N-acylsulfamates with Ub/Ubls (Nawrocki et al., 2012) in the presence of ATP and the E1 (Brownell et al., 2010, Chen et al., 2011) via a mechanism whereby the sulfamate NH2 group attacks the E1∼Ub/Ubl thioester to produce a covalent Ub/Ubl-inhibitor adduct. The resulting adduct mimics the Ub/Ubl adenylate and binds tightly (with picomolar affinity) to the E1, thus causing its inhibition (Chen et al., 2011). Although adenosyl sulfamates specifically inhibit E1 enzymes, their specificities differ vastly as reflected in low-nanomolar to high-micromolar half-maximal inhibitory concentration (IC50) values reported for these compounds toward various E1 enzymes (Figure S1). Recently, the adenosyl sulfamates MLN7243 (Traore et al., 2014), a UBA1-selective inhibitor, and ABPA3 (An and Statsyuk, 2015a), a dual inhibitor of the Ub and NEDD8 E1s, were reported. Despite the structure of MLN4924 bound to NEDD8 E1, knowledge of how chemically diverse adenosyl sulfamates occupy the ATP-binding pocket of UBA1 and display variable specificities toward different E1 enzymes has been missing.