Target Fishing Reveals a Novel Mechanism of N-Acylamino Saccharin Derivatives Targeting Glyceraldehyde-3-Phosphate Dehydrogenase toward Cyanobacterial Blooms Control.

Based on current challenges of poor targeting and limited choices in chemical control methods of cyanobacterial blooms (CBs), identifying new targets is an urgent and formidable task in the quest for target-based algaecides. This study discovered N-acylamino saccharin derivatives exhibiting potent algicidal activity. Thus, using N-acylamino saccharin as the probes, glyceraldehyde-3-phosphate dehydrogenase from cyanobacterial (CyGAPDH) was identified as a new target of algaecides through the activity-based protein profiling (ABPP) strategy for the first time. Building upon the structure of Probe2, a series of derivatives were designed and synthesized, with compound b6 demonstrating the most potent inhibitory activity against CyGAPDH and Synechocystis sp. PCC6803 (IC50 = 1.67 µM and EC50 = 1.15 µM). Furthermore, the potential covalent binding model of b6 to the cysteine residue C154 was explored through covalent possibility prediction, LC-MS experiments, substrate competitive inhibition experiments, and molecular docking. Especially, the results revealed C154 as a crucial covalent binding site, with residues T184 and R11 forming robust hydrophobic interactions and H181 establishing significant hydrogen-bonding interactions with b6, highlighting their potential as essential pharmacophores. In summary, this study not only identifies a novel target of algaecides for the control of CB but also lays the solid foundation for the development of targeted covalent algaecides.

Role of Argonaute proteins in RNAi pathway in Plutella xylostella: A review.

Argonaute (Ago) proteins act as key elements in RNA interference (RNAi) pathway, orchestrating the intricate machinery of gene regulation within eukaryotic cells. Within the RNAi pathway, small RNA molecules, including microRNA (miRNA), small interfering RNA (siRNA), and PIWI-interacting RNA (piRNA), collaborate with Ago family member proteins such as Ago1, Ago2, and Ago3 to form the RNA-induced silencing complex (RISC). This RISC complex, in turn, either cleaves the target mRNA or inhibits the process of protein translation. The precise contributions of Ago proteins have been well-established in numerous animals and plants, although they still remain unclear in some insect species. This review aims to shed light on the specific roles played by Ago proteins within the RNAi mechanism in a destructive lepidopteran pest, the diamondback moth (Plutella xylostella). Furthermore, we explore the potential of double-stranded RNA (dsRNA)-mediated RNAi as a robust genetic tool in pest management strategies. Through an in-depth examination of Ago proteins and dsRNA-mediated RNAi, this review seeks to contribute to our understanding of innovative approaches for controlling this pest and potentially other insect species of agricultural significance.

DOX_BDW: Incorporating Solvation and Desolvation Effects of Cavity Water into Nonfitting Protein-Ligand Binding Affinity Prediction.

Accurate prediction of the protein-ligand binding affinity (PLBA) with an affordable cost is one of the ultimate goals in the field of structure-based drug design (SBDD), as well as a great challenge in the computational and theoretical chemistry. Herein, we have systematically addressed the complicated solvation and desolvation effects on the PLBA brought by the difference of the explicit water in the protein cavity before and after ligands bind to the protein-binding site. Based on the new solvation model, a nonfitting method at the first-principles level for the PLBA prediction was developed by taking the bridging and displaced water (BDW) molecules into account simultaneously. The newly developed method, DOX_BDW, was validated against a total of 765 noncovalent and covalent protein-ligand binding pairs, including the CASF2016 core set, Cov_2022 covalent binding testing set, and six testing sets for the hit and lead compound optimization (HLO) simulation. In all of the testing sets, the DOX_BDW method was able to produce PLBA predictions that were strongly correlated with the corresponding experimental data (R = 0.66-0.85). The overall performance of DOX_BDW is better than the current empirical scoring functions that are heavily parameterized. DOX_BDW is particularly outstanding for the covalent binding situation, implying the need for considering an electronic structure in covalent drug design. Furthermore, the method is especially recommended to be used in the HLO scenario of SBDD, where hundreds of similar derivatives need to be screened and refined. The computational cost of DOX_BDW is affordable, and its accuracy is remarkable.

Identification and Analysis of Small Molecule Inhibitors of CRISPR-Cas9 in Human Cells.

Genome editing tools based on CRISPR-Cas systems can repair genetic mutations in situ; however, off-target effects and DNA damage lesions that result from genome editing remain major roadblocks to its full clinical implementation. Protein and chemical inhibitors of CRISPR-Cas systems may reduce off-target effects and DNA damage. Here we describe the identification of several lead chemical inhibitors that could specifically inhibit the activity of Streptococcus pyogenes Cas9 (SpCas9). In addition, we obtained derivatives of lead inhibitors that could penetrate the cell membrane and inhibit SpCas9 in cellulo. Two of these compounds, SP2 and SP24, were able to improve the specificity of SpCas9 in cellulo at low-micromolar concentration. Furthermore, microscale thermophoresis (MST) assays showed that SP24 might inhibit SpCas9 activity by interacting with both the SpCas9 protein and the SpCas9-gRNA ribonucleoprotein complex. Taken together, SP24 is a novel chemical inhibitor of SpCas9 which has the potential to enhance therapies that utilize SpCas9.

N-Acylamino Saccharin as an Emerging Cysteine-Directed Covalent Warhead and Its Application in the Identification of Novel FBPase Inhibitors toward Glucose Reduction.

With a resurgence of covalent drugs, there is an urgent need for the identification of new moieties capable of cysteine bond formation. Herein, we report on the N-acylamino saccharin moieties capable of novel covalent reactions with cysteine. Their utility as alternative electrophilic warheads was demonstrated through the covalent modification of fructose-1,6-bisphosphatase (FBPase), a promising target associated with cancer and type 2 diabetes. The cocrystal structure of title compound W8 bound with FBPase unexpectedly revealed that the N-acylamino saccharin moiety worked as an electrophile warhead that covalently modified the noncatalytic C128 site in FBPase while releasing saccharin, suggesting a previously undiscovered covalent reaction mechanism of saccharin derivatives with cysteine. Treatment of title compound W8 displayed potent inhibition of glucose production in vitro and in vivo. This newly discovered reactive warhead supplements the current repertoire of cysteine covalent modifiers while avoiding some of the limitations generally associated with established moieties.

Cov_DOX: A Method for Structure Prediction of Covalent Protein-Ligand Bindings.

A handful of molecular docking tools have been extended to enable a covalent docking. However, all of them face the challenge brought by the covalent bond between proteins and ligands. Many covalent drug design scenarios still heavily rely on demanding crystallographic experiments for accurate binding structures. Aiming at filling the gap between covalent dockings and crystallographic experiments, we develop and validate a hybrid method, dubbed as Cov_DOX, in this work. Cov_DOX achieves an overall success rate of 81% with RMSD < 2 Å for the Top 1 pose prediction in the validation against a test set including 405 crystal structures for covalent protein-ligand complexes, covering various types of the warhead chemistry and receptors. Such accuracy is not far from the much more demanding crystallographic experiments, in sharp contrast to the performance of the covalent docking front runners (success rate: 40-60%).

Structure-Guided Discovery of the Novel Covalent Allosteric Site and Covalent Inhibitors of Fructose-1,6-Bisphosphate Aldolase to Overcome the Azole Resistance of Candidiasis.

Fructose-1,6-bisphosphate aldolase (FBA) represents an attractive new antifungal target. Here, we employed a structure-based optimization strategy to discover a novel covalent binding site (C292 site) and the first-in-class covalent allosteric inhibitors of FBA from Candida albicans (CaFBA). Site-directed mutagenesis, liquid chromatography-mass spectrometry, and the crystallographic structures of APO-CaFBA, CaFBA-G3P, and C157S-2a4 revealed that S268 is an essential pharmacophore for the catalytic activity of CaFBA, and L288 is an allosteric regulation switch for CaFBA. Furthermore, most of the CaFBA covalent inhibitors exhibited good inhibitory activity against azole-resistant C. albicans, and compound 2a11 can inhibit the growth of azole-resistant strains 103 with the MIC80 of 1 µg/mL. Collectively, this work identifies a new covalent allosteric site of CaFBA and discovers the first generation of covalent inhibitors for fungal FBA with potent inhibitory activity against resistant fungi, establishing a structural foundation and providing a promising strategy for the design of potent antifungal drugs.

Biological evaluation and SAR analysis of novel covalent inhibitors against fructose-1,6-bisphosphatase.

Fructose-1,6-bisphosphatase (FBPase) is an attractive target for affecting the GNG pathway. In our previous study, the C128 site of FBPase has been identified as a new allosteric site, where several nitrovinyl compounds can bind to inhibit FBPase activity. Herein, a series of nitrostyrene derivatives were further synthesized, and their inhibitory activities against FBPase were investigated in vitro. Most of the prepared nitrostyrene compounds exhibit potent FBPase inhibition (IC50 < 10 µM). Specifically, when the substituents of F, Cl, OCH3, CF3, OH, COOH, or 2-nitrovinyl were installed at the R2 (meta-) position of the benzene ring, the FBPase inhibitory activities of the resulting compounds increased 4.5-55 folds compared to those compounds with the same groups at the R1 (para-) position. In addition, the preferred substituents at the R3 position were Cl or Br, thus compound HS36 exhibited the most potent inhibitory activity (IC50 = 0.15 µM). The molecular docking and site-directed mutation suggest that C128 and N125 are essential for the binding of HS36 and FBPase, which is consistent with the C128-N125-S123 allosteric inhibition mechanism. The reaction enthalpy calculations show that the order of the reactions of compounds with thiol groups at the R3 position is Cl > H > CH3. CoMSIA analysis is consistent with our proposed binding mode. The effect of compounds HS12 and HS36 on glucose production in primary mouse hepatocytes were further evaluated, showing that the inhibition was 71% and 41% at 100 µM, respectively.

Development of disulfide-derived fructose-1,6-bisphosphatase (FBPase) covalent inhibitors for the treatment of type 2 diabetes.

Fructose-1,6-bisphosphatase (FBPase), as a key rate-limiting enzyme in the gluconeogenesis (GNG) pathway, represents a practical therapeutic strategy for type 2 diabetes (T2D). Our previous work first identified cysteine residue 128 (C128) was an important allosteric site in the structure of FBPase, while pharmacologically targeting C128 attenuated the catalytic ability of FBPase. Herein, ten approved cysteine covalent drugs were selected for exploring FBPase inhibitory activities, and the alcohol deterrent disulfiram displayed superior inhibitory efficacy among those drugs. Based on the structure of lead compound disulfiram, 58 disulfide-derived compounds were designed and synthesized for investigating FBPase inhibitory activities. Optimal compound 3a exhibited significant FBPase inhibition and glucose-lowering efficacy in vitro and in vivo. Furthermore, 3a covalently modified the C128 site, and then regulated the N125-S124-S123 allosteric pathway of FBPase in mechanism. In summary, 3a has the potential to be a novel FBPase inhibitor for T2D therapy.

Identification of the New Covalent Allosteric Binding Site of Fructose-1,6-bisphosphatase with Disulfiram Derivatives toward Glucose Reduction.

Fructose 1,6-bisphosphatase (FBPase) has attracted substantial interest as a target associated with cancer and type 2 diabetes. Herein, we found that disulfiram and its derivatives can potently inhibit FBPase by covalently binding to a new C128 allosteric site distinct from the original C128 site in APO FBPase. Further identification of the allosteric inhibition mechanism reveals that the covalent binding of a fragment of 214 will result in the movement of C128 and the dissociation of helix H4 (123-128), which in turn allows S123 to more easily form new hydrogen bonds with K71 and D74 in helix H3 (69-72), thereby inhibiting FBPase activity. Notably, both disulfiram and 212 might moderately reduce blood glucose output in vivo. Therefore, our current findings not only identify a new covalent allosteric site of FBPase but also establish a structural foundation and provide a promising way for the design of covalent allosteric drugs for glucose reduction.

Cov_FB3D: A De Novo Covalent Drug Design Protocol Integrating the BA-SAMP Strategy and Machine-Learning-Based Synthetic Tractability Evaluation.

De novo drug design actively seeks to use sets of chemical rules for the fast and efficient identification of structurally new chemotypes with the desired set of biological properties. Fragment-based de novo design tools have been successfully applied in the discovery of noncovalent inhibitors. Nevertheless, these tools are rarely applied in the field of covalent inhibitor design. Herein, we present a new protocol, called Cov_FB3D, which involves the in silico assembly of potential novel covalent inhibitors by identifying the active fragments in the covalently binding site of the target protein. In this protocol, we propose a BA-SAMP strategy, which combines the noncovalent moiety score with the X-Score as the molecular mechanism (MM) level, and the covalent candidate score with the PM7 as the QM level. The synthetic accessibility of each suggested compound could be further evaluated with machine-learning-based synthetic complexity evaluation (SCScore). An in-depth test of this protocol against the crystal structures of 15 covalent complexes consisting of BTK inhibitors, KRAS inhibitors, EGFR inhibitors, EphB1 inhibitors, MAGL inhibitors, and MAPK inhibitors revealed that most of these inhibitors could be de novo reproduced from the fragments by Cov_FB3D. The binding modes of most generated reference poses could accurately reproduce the known binding mode of most of the reference covalent adduct in the binding site (RMSD ≤ 2 Å). In particular, most of these inhibitors were ranked in the top 2%, using the BA-SAMP strategy. Notably, the novel human ALDOA inhibitor (T1) with potent inhibitory activity (0.34 ± 0.03 µM) and greater synthetic accessibility was successfully de novo designed by this protocol. The positive results confirm the abilities of Cov_FB3D protocol.

Discovery of novel allosteric site and covalent inhibitors of FBPase with potent hypoglycemic effects.

Fructose-1,6-bisphosphatase (FBPase) is an essential enzyme of GNG pathway. Significant advances demonstrate the FBPase plays a critical role in treatment of diabetes. Numerous FBPase inhibitors were developed by targeting AMP site, nevertheless, none of these inhibitors has exhibited suitable potency and druggability. Herein, a new allosteric site (C128) on FBPase was discovered, and several nitrostyrene compounds exhibiting potent FBPase inhibitions were found covalently bind to C128 site on FBPase. Mutagenesis suggest that C128 is the only cysteine that can influence FBPase inhibition, the N125-S124-S123 pathway was most likely involved in allosteric signaling transmission between C128 and active site. However, these nitrostyrenes may bind with multiple cysteine besides C128 in FBPase. To improve pocket selectivity, a series of novel compounds (14a-14n) were re-designed rationally by integrating fragment-based covalent virtual screening and machine-learning-based synthetic complexity evaluation. As expected, the mass spectrometry validated that the proportion of title compounds binding to the C128 in FBPase was significantly higher than that of nitrostyrenes. Notably, under physiological and pathological conditions, the treatment of compounds 14b, 14c, 14i or 14n led to potent inhibition of glucose production, as well as decreased triglyceride and total cholesterol levels in mouse primary hepatocytes. We highlight a novel paradigm that molecular targeting C128 site on FBPase can have potent hypoglycemic effect.

Conformation Search Across Multiple-Level Potential-Energy Surfaces (CSAMP): A Strategy for Accurate Prediction of Protein-Ligand Binding Structures.

Accurate protein binding structure determination presents a great challenge to both experiment and theory. Here, in this work, we propose a new DOX protocol which combines the ensemble molecular Docking as the coarse-level, structure Optimization with the semiempirical quantum mechanics methods as the medium level, and the eXtended ONIOM ( XO) calculations as the fine level. The fundamental of the DOX protocol relies on the Conformation Search Across Multiple-level Potential-energy surfaces (CSAMP) strategy, where the conformation spaces of a funnel-like structure are searched from the coarse level with hundreds of candidates to the medium level with around 10 top candidates to the fine level with the final top 1 or 2 binding modes. An in-depth test for the protocol set up against 28 crystallographic data consisting of HMGR-statins, SDase-inhibitors, 3HNRase-inhibitors, and NA-inhibitors yielded a satisfactory result with ∼0.5 Šroot-mean-square deviations (RMSDs) on geometries and ∼0.8 kcal/mol absolute error of relative binding energies on average. A further larger scale validation on the Astex test set (including 85 diverse structures) revealed an impressive performance with a RMSD < 2 Šsuccess rate of 99%, suggesting DOX is a promising computational route toward accurate prediction of the protein-ligand binding structures.

Design, synthesis and algicides activities of thiourea derivatives as the novel scaffold aldolase inhibitors.

By using a new Fragment-Based Virtual Screen strategy, two series of novel FBA-II inhibitors (thiourea derivatives) were de novo discovered based on the active site of fructose-1, 6-bisphosphate aldolase from Cyanobacterial (CyFBA). In comparison, most of the N-(2-benzoylhydrazine-1-carbonothioyl) benzamide derivatives (L14∼L22) exhibit higher CyFBA-II inhibitory activities compared to N-(phenylcarbamothioyl) benzamide derivatives (L1∼L13). Especially, compound L14 not only shows higher CyFBA-II activity (Ki = 0.65 µM), but also exhibits most potent in vivo activity against Synechocystis sp. PCC 6803 (EC50 = 0.09 ppm), higher (7-fold) than that of our previous inhibitor (EC50 = 0.6 ppm). The binding modes of compound L14 and CyFBA-II were further elucidated by jointly using DOX computational protocol, MM-PBSA and site-directed mutagenesis assays. The positive results suggest that strategy adopted in this study was promising to rapidly discovery the potent inhibitors with novel scaffolds. The satisfactory algicide activities suggest that the thiourea derivatives is very likely to be a promising lead for the development of novel specific algicides to solve Cyanobacterial harmful algal blooms (CHABs).

In silico screening of a novel scaffold for fructose-1,6-bisphosatase (FBPase) inhibitors.

Fructose-1, 6-bisphosphatase (FBPase) has been regarded as an attractive drug target to control blood glucose against Type 2 diabetes (T2D). In this study, by using the strategy of pharmacophore-based virtual screening, a novel scaffold inhibitor targeted the AMP allosteric site of human liver FBPase were screened, their inhibitory activities were further tested. The experimental results showed that compound H27 exhibited high inhibitory activities with the IC50 value of 5.3 µM. Therefore, compound H27 was chosen as the probe molecule, it's possible binding conformation targeted into FBPase was identified by using DOX2.0 strategy. The importance of key residues (T27, T31, K112 and R140) in allosteric site of FBPase for the binding inhibitors were validated by mutation experiments. The agreement between theory and experiment suggest that the interactional information of FBPase and inhibitors (H27) were reliable. On basis of these rational interactional information, the compound H29 was further designed to exhibit more potential FBPase inhibition (IC50 = 2.5 µM).

Natural products as sources of new fungicides (IV): Synthesis and biological evaluation of isobutyrophenone analogs as potential inhibitors of class-II fructose-1,6-bisphosphate aldolase.

Several recently identified antifungal compounds share the backbone structure of acetophenones. The aim of the present study was to develop new isobutyrophenone analogs as new antifungal agents. A series of new 2,4-dihydroxy-5-methyl isobutyrophenone derivatives were prepared and characterized by 1H, 13C NMR and MS spectroscopic data. These products were evaluated for in vitro antifungal activities against seven plant fungal pathogens by the mycelial growth inhibitory rate assay. Compounds 3, 4a, 5a, 5b, 5e, 5f and 5g showed a broad-spectrum high antifungal activity. On the other hand, for the first time, these compounds were also assayed as potential inhibitors against Class II fructose-1,6-bisphosphate aldolase (Fba) from the rice blast fungus, Magnaporthe grisea. Compounds 5e and 5g were found to exhibit the inhibition constants (Ki) for 15.12 and 14.27 µM, respectively, as the strongest competitive inhibitors against Fba activity. The possible binding-modes of compounds 5e and 5g were further analyzed by molecular docking algorithms. The results strongly suggested that compound 5g could be a promising lead for the discovery of new fungicides via targeting Class II Fba.

Design and optimization of N-acylhydrazone pyrimidine derivatives as E. coli PDHc E1 inhibitors: Structure-activity relationship analysis, biological evaluation and molecular docking study.

By targeting the thiamin diphosphate (ThDP) binding site of Escherichia coli (E. coli) pyruvate dehydrogenase multienzyme complex E1 (PDHc E1), a series of novel 'open-chain' classes of ThDP analogs A, B, and C with N-acylhydrazone moieties was designed and synthesized to explore their activities against E. coli PHDc E1 in vitro and their inhibitory activity against microbial diseases were further evaluated in vivo. As a result, A1-23 exhibited moderate to potent inhibitory activities against E. coli PDHc E1 (IC50=0.15-23.55µM). The potent inhibitors A13, A14, A15, C2, had strong inhibitory activities with IC50 values of 0.60, 0.15, 0.39 and 0.34µM against E. coli PDHc E1 and with good enzyme-selective inhibition between microorganisms and mammals. Especially, the most powerful inhibitor A14 could 99.37% control Xanthimonas oryzae pv. Oryzae. Furthermore, the binding features of compound A14 within E. coli PDHc E1 were investigated to provide useful insights for the further construction of new inhibitor by molecular docking, site-directed mutagenesis, and enzymatic assays. The results indicated that A14 had most powerful inhibition against E. coli PDHc E1 due to the establishment of stronger interaction with Glu571, Met194, Glu522, Leu264 and Phe602 at active site of E.coli PDHc E1. It could be used as a lead compound for further optimization, and may have potential as a new microbicide.

Structure-Based Rational Design of Novel Inhibitors Against Fructose-1,6-Bisphosphate Aldolase from Candida albicans.

Class II fructose-1,6-bisphosphate aldolases (FBA-II) are attractive new targets for the discovery of drugs to combat invasive fungal infection, because they are absent in animals and higher plants. Although several FBA-II inhibitors have been reported, none of these inhibitors exhibit antifungal effect so far. In this study, several novel inhibitors of FBA-II from C. albicans (Ca-FBA-II) with potent antifungal effects were rationally designed by jointly using a specific protocols of molecular docking-based virtual screening, accurate binding-conformation evaluation strategy, synthesis and enzymatic assays. The enzymatic assays reveal that the compounds 3c, 3e-g, 3j and 3k exhibit high inhibitory activity against Ca-FBA-II (IC50 < 10 µM), and the most potential inhibitor is 3g, with IC50 value of 2.7 µM. Importantly, the compounds 3f, 3g, and 3l possess not only high inhibitions against Ca-FBA-II, but also moderate antifungal activities against C. glabrata (MIC80 = 4-64 µg/mL). The compounds 3g, 3l, and 3k in combination with fluconazole (8 µg/mL) displayed significantly synergistic antifungal activities (MIC80 < 0.0625 µg/mL) against resistant Candida strains, which are resistant to azoles drugs. The probable binding modes between 3g and the active site of Ca-FBA-II have been proposed by using the DOX (docking, ONIOM, and XO) strategy. To our knowledge, no FBA-II inhibitors with antifungal activities against wild type and resistant strains from Candida were reported previously. The positive results suggest that the strategy adopted in this study are a promising method for the discovery of novel drugs against azole-resistant fungal pathogens in the future.

New insight into the binding modes of TNP-AMP to human liver fructose-1,6-bisphosphatase.

Human liver fructose-1,6-bisphosphatase (FBPase) contains two binding sites, a substrate fructose-1,6-bisphosphate (FBP) active site and an adenosine monophosphate (AMP) allosteric site. The FBP active site works by stabilizing the FBPase, and the allosteric site impairs the activity of FBPase through its binding of a nonsubstrate molecule. The fluorescent AMP analogue, 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP) has been used as a fluorescent probe as it is able to competitively inhibit AMP binding to the AMP allosteric site and, therefore, could be used for exploring the binding modes of inhibitors targeted on the allosteric site. In this study, we have re-examined the binding modes of TNP-AMP to FBPase. However, our present enzyme kinetic assays show that AMP and FBP both can reduce the fluorescence from the bound TNP-AMP through competition for FBPase, suggesting that TNP-AMP binds not only to the AMP allosteric site but also to the FBP active site. Mutagenesis assays of K274L (located in the FBP active site) show that the residue K274 is very important for TNP-AMP to bind to the active site of FBPase. The results further prove that TNP-AMP is able to bind individually to the both sites. Our present study provides a new insight into the binding mechanism of TNP-AMP to the FBPase. The TNP-AMP fluorescent probe can be used to exam the binding site of an inhibitor (the active site or the allosteric site) using FBPase saturated by AMP and FBP, respectively, or the K247L mutant FBPase.

Rational design, synthesis and biological evaluation of 1,3,4-oxadiazole pyrimidine derivatives as novel pyruvate dehydrogenase complex E1 inhibitors.

On the basis of previous study on 2-methylpyrimidine-4-ylamine derivatives I, further synthetic optimization was done to find potent PDHc-E1 inhibitors with antibacterial activity. Three series of novel pyrimidine derivatives 6, 11 and 14 were designed and synthesized as potential Escherichia coli PDHc-E1 inhibitors by introducing 1,3,4-oxadiazole-thioether, 2,4-disubstituted-1,3-thiazole or 1,2,4-triazol-4-amine-thioether moiety into lead structure I, respectively. Most of 6, 11 and 14 exhibited good inhibitory activity against E. coli PHDc-E1 (IC50 0.97-19.21 µM) and obvious inhibitory activity against cyanobacteria (EC50 0.83-9.86 µM). Their inhibitory activities were much higher than that of lead structure I. 11 showed more potent inhibitory activity against both E. coli PDHc-E1 (IC50<6.62 µM) and cyanobacteria (EC50<1.63 µM) than that of 6, 14 or lead compound I. The most effective compound 11d with good enzyme-selectivity exhibited most powerful inhibitory potency against E. coli PDHc-E1 (IC50=0.97 µM) and cyanobacteria (EC50=0.83 µM). The possible interactions of the important residues of PDHc-E1 with title compounds were studied by molecular docking, site-directed mutagenesis, and enzymatic assays. The results indicated that 11d had more potent inhibitory activity than that of 14d or I due to its 1,3,4-oxadiazole moiety with more binding position and stronger interaction with Lsy392 and His106 at active site of E. coli PDHc-E1.