The component E of PDHc PDHc E catalyzes the
The component E1 of PDHc (PDHc-E1) catalyzes the first step of multistep processes, using thiamine diphosphate (ThDP) and Mg2+ as cofactors.15, 16 Especially, this PDHc-E1 catalyzed process is a rate limiting step among multistep processes. Accordingly, the PDHc should be inactive by inhibiting the activity of PDHc-E1. In our work, E. coli PDHc-E1 was selected as target to design novel bactericides. Optimal binding model for inhibitors targeting the active site of E. coli PDHc-E1 has been built based on reported crystal structure of E. coli PDHc using molecular simulation. As human PDHc-E1 and porcine PDHc-E1 have a sequence similarity of 98.5%, the structure of porcine PDHc-E1 was built by homology modeling method use human PDHc-E1 (PDB code: 3EXE) as template. Therefore, the binding mode for inhibitors targeting the active site of porcine PDHc-E1 was also built using molecular simulation. Considering the importance of ThDP in the metabolic process of pyruvate, we can design novel ThDP analogs as E. coli PDHc-E1 inhibitors. According to literatures, some ThDP analogs (Fig. 1) with high binding affinities against E. coli PDHc-E1 have been prepared and studied greatly,1, 2, 19, 20 but no report about their application in agriculture. In fact, these reported ThDP analogs are unsuitable for the usage as microbicides due to their complex structure with highly charged pyrophosphate and poor bioavailability. In particular, these ThDP analogs had poor enzyme-selectivity between microorganisms and mammals, such as ThTTDP (Fig. 1), which acted as PDHc-E1 inhibitor displayed effective inhibition ( = 64 nM) of E. coli PDHc-E1, however, it kasugamycin also showed powerful binding affinity ( = 74 nM) to human PDHc-E1. According to the data of sequence alignment, E. coli PDHc-E1 and porcine PDHc-E1 have a lower sequence similarity of 29.5%.21, 22, 23 It is possible to obtain highly selective inhibitors targeting E. coli PDHc-E1. These reported ThDP analogs showed poor enzyme-selectivity between microorganisms and mammals due to their high structural similarity to ThDP. Crystal structure revealed that the binding mode of 4-aminopyrimidine part of ThDP with human PDHc-E1 (PDB code: 3EXE) or with E. coli PDHc-E1 (PDB code: 1L8A) was the same, while other structural part of ThDP had different binding modes toward the two enzymes. Therefore, on the basis of the structure of ThDP, 2-methylpyrimidine-4-ylamine moiety was remained, thiazolium ring (A part) in ThDP was modified, and further modification was focused on the pyrophosphate moiety (B part). In this work, thiazolium ring (A part) in ThDP was replaced by an acylhydrazone moiety to improve the flexibility of the molecule, which may be beneficial for producing selective. Moreover, the acylhydrazone moiety in new ThDP analogs could act as hydrogen donor (NH) or hydrogen receptor (CO and CN), which would beneficially interacted with the active site of E. coli PDHc-E1 by forming hydrogen bond. Meanwhile, the pyrophosphate moiety in ThDP (B part) was further modified to avoid the high charge of the pyrophosphate moiety. Therefore, the pyrophosphate moiety in ThDP was respectively replaced by diphenyl ether, N-substituted-benzamide or diphenylurea moiety. Thus, two novel series of (E)-N′-((4-amino-2-methylpyrimidin-5-yl)methylene)-4-substituted-phenoxybenzohydrazide 7 and (E)-N-(3-(2-((4-amino-2-methylpyrimidin-5-yl)methylene)hydrazinecarbonyl)phenyl)-substituted-benzamide 12 were first designed and synthesized, respectively (Fig. 2). In order to explore the effect of structure B part on PDHc-E1 inhibition. The structure B of 12 was modified to give a novel series of 15 by changing the position of amide group from meta to para-position. Finally, the amide group of 15 was further replaced by urea group to give novel compounds 19. Above the structural modification of ThDP was hoped to produce a good selective by forming especial potent inhibition binding the active site of E. coli PDHc-E1. Herein, we reported the synthesis of 4-aminopyrimidine derivatives 7, 12, 15, and 19. In order to find safe inhibitors for mammals, their enzyme-selectivity inhibition between E. coli PDHc-E1 and porcine PDHc-E1 were examined. The interaction between inhibitors and E. coli PDHc-E1 or porcine PDHc-E1 was studied by molecular docking, site-directed mutagenesis and enzymatic assays. The antibacterial activity of these title compounds with potent inhibition against E. coli PDHc-E1 was further examined.