In this study, a series of novel amide derivatives and sulfamide derivatives as potential E. coli PDHc E1 inhibitors were designed and synthesized by optimizing the linker between triazole and benzene ring moieties based on the structure of lead compound I as thiamin diphosphate (ThDP) analogs. Their inhibitory activity against E. coli PDHc E1 were examined in vitro and their inhibitory activity against microbial diseases were further evaluated. Most of these compounds exhibit good inhibitory activity against E. coli PHDc E1 (IC50 1.99 to 25.66 μM) and obvious antibacterial activity. 5a, 5c and 9i showed 90–100% antibacterial activity against Xanthomonas oryzae pv. oryzae (Xoo), Acidovorax avenae subsp. avenae (Aaa) and cyanobacteria. Sulfamide derivatives 9 showed more potent inhibitory activity against E. coli PDHc E1 (IC50 < 14 μM) than that of amide derivatives 5 or lead compound I. Especially 9d (IC50 = 2.95 μM) and 9k (IC50 = 1.99 μM) exhibited not only the most powerful inhibitory potency against E. coli PDHc E1, but also 9k showed 99% antibacterial activity against Aaa at 500 μg mL−1 and almost the best inhibition of 97% against cyanobacteria at 20 μg mL−1. Furthermore, the binding mode of 5d and 9d to E. coli PDHc E1 was analyzed by a molecular docking method. The possible interactions of 9d with the important residues of E. coli PDHc E1 were further verified via site-directed mutagenesis enzymatic assays, and fluorescence spectral analysis. Both theoretical and experimental results revealed that 9d could display a more powerful interaction than that of 5d or I by forming a hydrogen bond between a sulfamide linkage and residues Lsy392, Tyr599 and His106 at active site of E. coli PDHc E1. 9k, 9d and 9i with both potent enzyme inhibition and significant antibacterial activity, could be used as novel lead compounds for further optimization. These results proved that a series of compounds with potential antibacterial activity could be obtained by the biorational design of E. coli PDHc 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-E1.1,3,4-Oxadiazole pyrimidine derivative 11d exhibited most powerful inhibitory potency against Escherichia coli PDHc-E1 (IC50 = 0.97 μM) and cyanobacteria (EC50 = 0.83 μM).

•The binding modes of TNP-AMP into the human liver FBPase has been re-examined.•Not only AMP but also FBP can competitively inhibit the binding of TNP-AMP to FBPase.•TNP-AMP can bind to both the active site and the allosteric site.•K274 is important for TNP-AMP binding to the active site of FBPase.•K274L is good candidate for exploring the allosteric site with TNP-AMP as fluorescent probe.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.

Cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphoshatase (cy-FBP/SBPase) is a potential enzymatic target for screening of novel inhibitors that can combat harmful algal blooms. In the present study, we targeted the substrate binding pocket of cy-FBP/SBPase. A series of novel hit compounds from the SPECs database were selected by using a pharmacophore-based virtual screening strategy. Most of the compounds tested exhibited moderate inhibitory activities (IC50 = 20.7–176.9 μM) against cy-FBP/SBPase. Compound 2 and its analogues 10 and 11 exhibited strong inhibitory activities, with IC50 values of 20.7, 13.4, and 19.0 μM against cy-FBP/SBPase in vitro and EC50 values of 12.3, 10.9, and 2.9 ppm against cyanobacteria Synechocystis PCC6803 in vivo, respectively. The compound 10 was selected in order to perform a refined docking study to investigate the rational binding mode of inhibitors with cy-FBP/SBPase. Furthermore, possible interactions of the residues with inhibitors were examined by site-directed mutagenesis, enzymatic assays, and fluorescence spectral analyses. The results provide insight into the binding mode between the inhibitors and the substrate binding pocket. The observed theoretical and experimental results are in concert, indicating that the modeling strategies and screening methods employed are appropriate to search for novel lead compounds having both structural diversity and high inhibitory activity against cy-FBP/SBPase.

Cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphoshatase (Cy-FBP/SBPase) is an important target enzyme for finding inhibitors to solve harmful algal bloom (HAB). In this study, as potential inhibitors of Cy-FBP/SBPase, a series of novel chromone-connecting benzohydrazone compounds (Novel N′-((4-oxo-4H-chromen-3-yl)methylene)benzohydrazide) were designed and synthesized. Their inhibitory activities against Cy-FBP/SBPase were further examined in vitro. Some of these compounds, such as f6–f8, f11, f12 and f16, exhibit higher inhibitory activities (IC50 = 11.2–16.1 μM), especially, the compound f7 was identified as the most potent inhibitor with IC50 value of 11.2 μM. The probable binding-mode of compound f7 was further analyzed carefully by molecular docking methods. These results indicate that compound f7 could be used as a lead compound for further optimization and might have potential to be developed as a new algicide.As potential inhibitors of Cy-FBP/SBPase, a series of novel type chromone-benzohydrazone derivatives were designed and synthesized. Amongst these compounds, the compound f7 exhibits the most potent inhibitory activity (IC50 = 11.2 μM). The probable binding mode of compound f7 was analyzed by molecular docking, as shown in Figure 1.

Fructose-1,6-/sedoheptulose-1,7-bisphosphatase (FBP/SBPase) is a potential important target enzyme for finding inhibitors to solve harmful algal bloom. In this paper, the interactions between FBP/SBPase and metal ions were studied by enzyme activity analysis, fluorescence and molecular modeling method. The enzyme activity analysis showed that FBP/SBPase can be activated by Mg2+ or Mn2+ but cannot be activated by Ca2+ or Zn2+. Spectroscopic analysis of emission quenching showed that quenching mechanism of FBP/SBPase with Mg2+ or Mn2+ was static quenching mechanism while that of Ca2+ or Zn2+ was dynamic quenching process. Hydrogen bonds and van der Waals interaction might be the predominant intermolecular forces in stabilizing FBP/SBPase-Mg2+ while hydrophobic forces were the predominant intermolecular forces in stabilizing FBP/SBPase-Mn2+. Microenvironment and conformation of FBP/SBPase were changed in binding reaction. The effect of metal ions and important amino acid residues on FBP/SBPase–metal ion complex was also discussed by molecular modeling study.Graphical abstractHighlights► We firstly present a detailed exploration of interaction between metal ions with cyanobacteria FBP/SBPase. ► FBP/SBPase can be activated by Mg2+ or Mn2+ by static quenching mechanism. ► Hydrogen bonds and van der Waals might be the predominant forces in stabilizing FBP/SBPase-ion2+ complexes. ► The effects of Mg2+ and Mn2+ on FBP/SBPase were due to the change of different α-helix or loop area.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (class II HMGR) could serve as a potential target to discover drugs fighting against the invasive diseases originated from Streptococcus pneumoniae, one of the major causes of bacterial disease in human. However, no strongly effective inhibitors of class II HMGR have been found so far. In the present study, for the first time, four annonaceous acetogenins (ACGs) were explored for the inhibition on S. pneumoniae HMGR. The results showed that the ACGs had higher inhibitory activities against S. pneumoniae HMGR with Ki values in the range of 6.45–20.49 μM than the statin drug lovastatin (Ki = 116.25 μM), a classical inhibitor of class I HMGR. Then, three-dimensional modeling and docking simulations analyzed the possible binding mode of ACGs to S. pneumoniae HMGR and suggested a kind of novel structural and binding mode for designing promising inhibitor candidates of the targeted enzyme S. pneumoniae II HMGR.

Cytochrome P450 sterol 14α-demethylases (CYP51s) are essential enzymes in sterol biosynthesis and well-known as the target of antifungal drugs. All fungal CYP51s are integral membrane proteins, making structural and biophysical characterization more challenging. The X-ray crystallographic structure of CYP51 isolated from Mycobacterium tuberculosis (MT-CYP51) is the unique reported one hitherto. In the present study, a homology modeling three-dimensional structure of CYP51 from Penicillium digitatum (PD-CYP51) was generated by CPHmodels, in which the accuracy of sequence alignment could be improved by taking into account further structural conservation information, using MT-CYP51 as the template. Interaction mechanism between the active site of PD-CYP51 and its inhibitors were further investigated by molecular dynamics simulating and molecular docking. With the effective docking process and interaction analysis information, structure-based virtual screening was performed to pick out the thirty new potential inhibiting compounds with structural diversity by using a new virtual screening strategy including Flex-Pharm/PMF/GOLD//FlexX/PMF/GOLD molecular docking procedures, and finally, seven new hit compounds out of SPECs database with potent inhibitory ability were validated by bioaffinity assays at enzyme level and on P. digitatum in vitro. The positive results indicated that all modeling strategies and screening processes presented in the current study most like to be an encouraging way in search of novel lead compounds with structural diversity for the specifically individual fungal CYP51s of both plants and human pathogens in the future.

Rice cultivar zhonghua11 (Oryza sativa L. ssp. japonica) plants with decreased sedoheptulose-1, 7-bisphosphatase (SBPase) were obtained by transformation with the rice SBPase antisense gene under the control of the maize ubiquitin promoter. The transgenic and wild-type plants were grown at different nitrogen levels (0.1, 1, or 10 mM NH4NO3). Growth rates of the seedlings were measured by the changes in dry weight, and the photosynthetic carbon reduction activities and the potential efficiency of photosystem II were measured by CO2 assimilation and Fv/Fm, respectively. At low N, there are strong effects on growth and photosynthesis when SBPase was reduced by genetic manipulation. Decreased SBPase activity led to a decrease in the amount of starch accumulated in the leaves at all N levels and the decrease was much more prominent in low N than that in high N, but the starch allocation between shoot and root was unaltered. The analysis of chlorophyll fluorescence and SBPase activity indicated that the decrease of growth and photosynthesis at different N levels were not related to the function of PSII but to the activity of SBPase. Western blot analysis showed the content of SBPase in thylakoid membranes was much more than in the stroma fractions in transgenic plants at low N. Results suggested that low N in addition to a 34% decrease in SBPase activity is sufficient to diminish photosynthesis and limit biomass production. Decreased SBPase activity may reduce the N use efficiency of photosynthesis and growth and alter biomass allocation.