Optimizing drug-like properties of selective butyrylcholinesterase inhibitors for cognitive improvement: Enhancing aqueous solubility by disrupting molecular plane.

Most recently, worldwide interest in butyrylcholinesterase (BChE) as a potential target for treating Alzheimer's disease (AD) has increased. In this study, the previously obtained selective BChE inhibitors with benzimidazole-oxadiazole scaffold were further structurally modified to increase their aqueous solubility and pharmacokinetic (PK) characteristics. S16-1029 showed improved solubility (3280 µM, upgraded by 14 times) and PK parameters, including plasma exposure (AUC0-inf = 1729.95 ng/mL*h, upgraded by 2.6 times) and oral bioavailability (Fpo = 48.18%, upgraded by 2 times). S16-1029 also displayed weak or no inhibition against Cytochrome P450 (CYP450) and human ether a-go-go related gene (hERG) potassium channel. In vivo experiments on tissue distribution revealed that S16-1029 could cross the blood-brain barrier (BBB) and reach the central nervous system (CNS). In vivo cognitive improvement efficacy and good in vitro target inhibitory activity (eqBChE IC50 = 11.35 ± 4.84 nM, hBChE IC50 = 48.1 ± 11.4 nM) were also assured. The neuroprotective effects against several AD pathology characteristics allowed S16-1029 to successfully protect the CNS of progressed AD patients. According to the findings of this study, altering molecular planarity might be a viable strategy for improving the drug-like property of CNS-treating drugs.

Genome-wide identification of CBL gene family in Salvia miltiorrhiza and the characterization of SmCBL3 under salt stress.

In plants, CBL mediated calcium signaling is widely involved in the response to plant stresses of adversity. However, to date, no comprehensive studies have been conducted on CBL family members in Salvia miltiorrhiza. Herein, we identified 8 SmCBLs in S. miltiorrhiza, and phylogenetic analysis classified SmCBLs into four groups. Analysis of cis-acting elements revealed that SmCBLs mostly have light-responsive and hormone-responsive elements. Tissue expression analysis indicated that almost all of SmCBLs were expressed in roots than in leaves and flowers. SmCBL3 responded to Abscisic Acid (ABA), polyethylene glycol (PEG), and NaCl treatments. Transgenic Arabidopsis thaliana that overexpressed SmCBL3 had higher germination rates and longer roots than the wild type (WT) when exposed to salt stress. Additionally, the transgenic lines exhibited higher levels of chlorophyll, proline, superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activity and SOS1, NHX1 and P5CS1 expression than WT, and lower levels of malondialdehyde (MDA). Furthermore, SmCBL3 interacts with SmCIPK9. In conclusion, we analyzed the protein physicochemical properties, evolutionary relationships, gene structures, and expression profiles of the SmCBL gene families in S. miltiorrhiza. Overexpression of SmCBL3 improves the salt tolerance of transgenic Arabidopsis. This study demonstrated that SmCBL3 is a positive regulator of plant salt tolerance, so the use of overexpressed SmCBL3 may serve as a potential strategy to enhance plant salt tolerance.

Cloning and functional characterization of FtWRKY29, a low phosphorus stress-inducible transcription factor in Tartary buckwheat.

The regulation of the expression of genes related to abiotic stress in plants is significantly influenced by the binding of the transcription factor (TF) WRKY to the W-box elements in their promoters. The findings of this study have confirmed that the ability of Tartary buckwheat (Fagopyrum tataricum Gaertn.) to tolerate phosphorus (P) deficiency is regulated by FtWRKY29, which is classified as a member of group II of the WRKY family. The roots predominantly exhibited an enhanced expression of FtWRKY29, which was significantly upregulated in response to low-P-induced stress. The W-box motif was bound to by FtWRKY29 which enhanced the transcription of genes and was localized to the nucleus. The overexpression of FtWRKY29 in Arabidopsis thaliana produced transgenic lines that exhibited phenotypes typical of diminished sensitivity to low-P-induced stress by promoting root growth, increasing P-uptake, and regulating the accumulation of anthocyanin. The low-P-responsive genes, PHT1;1, PHT1;4, and PHO1 were significantly up-regulated in these lines. In addition, the overexpression of FtWRKY29 restored the P-absorption ability of the wrky75 mutant to a certain extent. Moreover, the binding of FtWRKY29 to the promoter of PHT1;1 activated its expression in tobacco. It was also observed that FtWRKY29 interacts with AtMPK3, AtMPK6, FtMPK3, and FtMPK7. This study provides preliminary evidence that FtWRKY29 improved the tolerance of transgenic A. thaliana plants to low-P-induced stress and deepened the understanding of the regulatory mechanism behind the same in Tartary buckwheat.

Functional pleiotropism, diversity, and redundancy of Salvia miltiorrhiza Bunge JAZ family proteins in jasmonate-induced tanshinone and phenolic acid biosynthesis.

Jasmonate (JA) signaling regulates plant growth and development, biotic and abiotic stress tolerance, and primary and secondary metabolism biosynthesis. It is extensively modulated by JA-ZIM-domain (JAZ) family genes. In previous work, we obtained nine SmJAZ genes of Salvia miltiorrhiza and proved that SmJAZ8 was the core repressor of JA-induced tanshinone and phenolic acid biosynthesis. Here, we demonstrate that SmJAZ3 and SmJAZ4 act as repressors of JA-induced biosynthesis of tanshinones and salvianolic acid B (Sal B). This suggests that SmJAZ3/4 are functionally redundant in tanshinone and Sal B biosynthesis. SmJAZ1/2/5/6/9 are activators of JA-induced tanshinone biosynthesis and repressors of JA-induced Sal B biosynthesis. This demonstrates the redundancy and diversity of SmJAZ1/2/5/6/9 functions. Besides, SmJAZ10 inhibited JA-induced Sal B synthesis, but had no effect on the synthesis of tanshinone. Two-hybrid screening (Y2H) showed that SmJAZs formed homologous or heterogeneous dimers. Y2H and firefly luciferase complementation imaging (LCI) assays revealed that SmJAZs also formed a complex regulatory network with SmMYC2a, SmMYC2b, SmMYB39, and SmPAP1. Quantitative reverse transcription-PCR (qRT-PCR) indicated that SmJAZs regulated each other at the transcriptional level. Herein, we prove that SmJAZs have functional pleiotropism, diversity, and redundancy in JA-induced tanshinone and phenolic acid biosynthesis. This study provides an important clue for further understanding the inherent biological significance and molecular mechanisms of the JAZ family as the gene number increases during plant evolution.

The Original Form of C4-Photosynthetic Phosphoenolpyruvate Carboxylase Is Retained in Pooids but Lost in Rice.

Poaceae is the most prominent monocot family that contains the primary cereal crops wheat, rice, and maize. These cereal species exhibit physiological diversity, such as different photosynthetic systems and environmental stress tolerance. Phosphoenolpyruvate carboxylase (PEPC) in Poaceae is encoded by a small multigene family and plays a central role in C4-photosynthesis and dicarboxylic acid metabolism. Here, to better understand the molecular basis of the cereal species diversity, we analyzed the PEPC gene family in wheat together with other grass species. We could designate seven plant-type and one bacterial-type grass PEPC groups, ppc1a, ppc1b, ppc2a, ppc2b, ppc3, ppc4, ppcC4, and ppc-b, respectively, among which ppc1b is an uncharacterized type of PEPC. Evolutionary inference revealed that these PEPCs were derived from five types of ancient PEPCs (ppc1, ppc2, ppc3, ppc4, and ppc-b) in three chromosomal blocks of the ancestral Poaceae genome. C4-photosynthetic PEPC (ppcC4 ) had evolved from ppc1b, which seemed to be arisen by a chromosomal duplication event. We observed that ppc1b was lost in many Oryza species but preserved in Pooideae after natural selection. In silico analysis of cereal RNA-Seq data highlighted the preferential expression of ppc1b in upper ground organs, selective up-regulation of ppc1b under osmotic stress conditions, and nitrogen response of ppc1b. Characterization of wheat ppc1b showed high levels of gene expression in young leaves, transcriptional responses under nitrogen and abiotic stress, and the presence of a Dof1 binding site, similar to ppcC4 in maize. Our results indicate the evolving status of Poaceae PEPCs and suggest the functional association of ppc1-derivatives with adaptation to environmental changes.

Discovery of novel VEGFR-2 inhibitors embedding 6,7-dimethoxyquinazoline and diarylamide fragments.

VEGF/VEGFR-2 signaling plays a critical part in tumor angiogenesis. Inhibition of this pathway has been considered as a promising approach for cancer treatment. In this work, a series of 6,7-dimethoxy-4-anilinoquinazoline derivatives bearing diarylamide moiety were designed, synthesized and evaluated as potent inhibitors of VEGFR-2 kinase. Their in vitro antiproliferation activities against two human cancer cell lines Hep-G2 and MCF-7 have also been determined. Among them, compound 14b exhibited the most potent inhibitory activity against VEGFR-2 with IC50 value of 0.016 ± 0.002 µM and it showed the most potent antiproliferative effect against Hep-G2 and MCF-7 with IC50 values at low-micromolar range. Molecular docking studies revealed that these compounds represented by the most potent compound 14b could bind well to the ATP-binding site of VEGFR-2, which suggested that compound 14b could be a potential anticancer agent targeting VEGFR-2.

Tartary Buckwheat Transcription Factor FtbZIP5, Regulated by FtSnRK2.6, Can Improve Salt/Drought Resistance in Transgenic Arabidopsis.

bZIP transcription factors have been reported to be involved in many different biological processes in plants. The ABA (abscisic acid)-dependent AREB/ABF-SnRK2 pathway has been shown to play a key role in the response to osmotic stress in model plants. In this study, a novel bZIP gene, FtbZIP5, was isolated from tartary buckwheat, and its role in the response to drought and salt stress was characterized by transgenic Arabidopsis. We found that FtbZIP5 has transcriptional activation activity, which is located in the nucleus and specifically binds to ABRE elements. It can be induced by exposure to PEG6000, salt and ABA in tartary buckwheat. The ectopic expression of FtbZIP5 reduced the sensitivity of transgenic plants to drought and high salt levels and reduced the oxidative damage in plants by regulating the antioxidant system at a physiological level. In addition, we found that, under drought and salt stress, the expression levels of several ABA-dependent stress response genes (RD29A, RD29B, RAB18, RD26, RD20 and COR15) in the transgenic plants increased significantly compared with their expression levels in the wild type plants. Ectopic expression of FtbZIP5 in Arabidopsis can partially complement the function of the ABA-insensitive mutant abi5-1 (abscisic acid-insensitive 5-1). Moreover, we screened FtSnRK2.6, which might phosphorylate FtbZIP5, in a yeast two-hybrid experiment. Taken together, these results suggest that FtbZIP5, as a positive regulator, mediates plant tolerance to salt and drought through ABA-dependent signaling pathways.

A WRKY transcription factor, FtWRKY46, from Tartary buckwheat improves salt tolerance in transgenic Arabidopsis thaliana.

The WRKY transcription factor family includes plant-specific transcription factors that are widely involved in plant biotic and abiotic stress responses, growth and development. Tartary buckwheat is a type of small grain with strong resistance to adverse growing conditions. No systematic exploration of the WRKY family in Tartary buckwheat has yet been reported. In this paper, we report the FtWRKY46 gene from Tartary buckwheat and study its role in salt tolerance. FtWRKY46 has transcriptional activation activity in yeast, and FtWRKY46 fused to yellow fluorescent protein localizes to the nucleus. Further studies have found that its transcriptional activation region is located at the N-terminus. A yeast one-hybrid assay indicated that FtWRKY46 could bind to a W-box and activate reporter gene expression. Similarly, transient cotransfection showed that FtWRKY46 could specifically bind to W-box regions and activate reporter gene expression in plants. Furthermore, ectopic expression of FtWRKY46 could enhance Arabidopsis tolerance to salt stress. More specifically, the seed germination rate, root length, chlorophyll content and proline content were significantly higher in transgenic plants ectopically expressing FtWRKY46 than in WT plants after salt stress (P < 0.05), while MDA levels were significantly lower than in WT plants (P < 0.05). Additionally, salt treatment increased the expression of stress-related genes. To summarize, our results suggest that ectopic expression of FtWRKY46 enhance the stress tolerance of transgenic plants by modulating ROS clearance and stress-related gene expression.

Tartary buckwheat transcription factor FtbZIP83 improves the drought/salt tolerance of Arabidopsis via an ABA-mediated pathway.

Plants are subjected to a variety of abiotic stresses during their lifetime, and drought and salt stress are some of the main causes of reduced crop yields. Previous studies have shown that AREB/ABFs within bZIP transcription factors are involved in plant drought and salt stress responses in an ABA-dependent manner. However, the properties and functions of AREB/ABFs in Fagopyrum tataricum, a cereal with good resistance to abiotic stresses, are poorly understood. In this study, a gene encoding an AREB/ABF, designated FtbZIP83, was first isolated from Tartary buckwheat. Expression analysis in Tartary buckwheat indicated that FtbZIP83 was significantly induced by abscisic acid (ABA), NaCl and polyethylene glycol (PEG). The overexpression of FtbZIP83 in Arabidopsis resulted in increased drought/salt tolerance, which was attributed not only to higher proline (Pro) contents and antioxidant enzyme activity in transgenic lines compared with controls but also to the lower reactive oxygen species (ROS) accumulation and malondialdehyde (MDA) content. In addition, we found that FtbZIP83 was able to respond to drought and salt stress by upregulating the transcript abundance of downstream ABA-inducible gene. Furthermore, promoter sequence analysis showed that ABREs were present, and the activity of the FtbZIP83 promoter in transgenic Arabidopsis after drought stress was significantly higher than that under normal conditions. Based on the potential signalling pathways involved in AREB/ABFs, we also screened for the interaction protein FtSnRK2.6/2.3, which may phosphorylate FtbZIP83. Collectively, these results provide evidence that FtbZIP83, as a positive regulator, responds to drought/salt stress via an ABA-dependent signalling pathway composed of SnRK2-AREB/ABF.

Diverse biological effects of glycosyltransferase genes from Tartary buckwheat.

BACKGROUND: Tartary buckwheat (Fagopyrum tataricum) is an edible cereal crop whose sprouts have been marketed and commercialized for their higher levels of anti-oxidants, including rutin and anthocyanin. UDP-glucose flavonoid glycosyltransferases (UFGTs) play an important role in the biosynthesis of flavonoids in plants. So far, few studies are available on UFGT genes that may play a role in tartary buckwheat flavonoids biosynthesis. Here, we report on the identification and functional characterization of seven UFGTs from tartary buckwheat that are potentially involved in flavonoid biosynthesis (and have varying effects on plant growth and development when overexpressed in Arabidopsis thaliana.) RESULTS: Phylogenetic analysis indicated that the potential function of the seven FtUFGT proteins, FtUFGT6, FtUFGT7, FtUFGT8, FtUFGT9, FtUFGT15, FtUFGT40, and FtUFGT41, could be divided into three Arabidopsis thaliana functional subgroups that are involved in flavonoid biosynthesis of and anthocyanin accumulation. A significant positive correlation between FtUFGT8 and FtUFGT15 expression and anthocyanin accumulation capacity was observed in the tartary buckwheat seedlings after cold stress. Overexpression in Arabidopsis thaliana showed that FtUFGT8, FtUFGT15, and FtUFGT41 significantly increased the anthocyanin content in transgenic plants. Unexpectedly, overexpression of FtUFGT6, while not leading to enhanced anthocyanin accumulation, significantly enhanced the growth yield of transgenic plants. When wild-type plants have only cotyledons, most of the transgenic plants of FtUFGT6 had grown true leaves. Moreover, the growth speed of the oxFtUFGT6 transgenic plant root was also significantly faster than that of the wild type. At later growth, FtUFGT6 transgenic plants showed larger leaves, earlier twitching times and more tillers than wild type, whereas FtUFGT15 showed opposite results. CONCLUSIONS: Seven FtUFGTs were isolated from tartary buckwheat. FtUFGT8, FtUFGT15, and FtUFGT41 can significantly increase the accumulation of total anthocyanins in transgenic plants. Furthermore, overexpression of FtUFGT6 increased the overall yield of Arabidopsis transgenic plants at all growth stages. However, FtUFGT15 shows the opposite trend at later growth stage and delays the growth speed of plants. These results suggested that the biological function of FtUFGT genes in tartary buckwheat is diverse.