Design and synthesis of novel fluorene derivatives as inhibitors of pyruvate dehydrogenase kinase.

Activation of pyruvate dehydrogenase (PDH) by inhibition of pyruvate dehydrogenase kinase (PDHK) has the potential for the treatment of diabetes mellitus and its complications, caused by the malfunction of the glycolytic system and glucose oxidation. In this paper, we describe the identification of novel PDHK inhibitors with a fluorene structure. High-throughput screening using our in-house library provided compound 6 as a weak inhibitor that occupied the allosteric lipoyl group binding site in PDHK2. Structure-based drug design (SBDD) while addressing physicochemical properties succeeded in boosting inhibitory activity approximately 700-fold. Thus obtained compound 32 showed favorable pharmacokinetics profiles supported by high membrane permeability and metabolic stability, and exhibited activation of PDH in rat livers and a glucose lowering effect in Zucker fatty rats.

The discovery of 3,3-dimethyl-1,2,3,4-tetrahydroquinoxaline-1-carboxamides as AMPD2 inhibitors with a novel mechanism of action.

AMP deaminase 2 (AMPD2) has been thought to play an important role in energy homeostasis and immuno-oncology, while selective AMPD2 inhibitors are highly demanded to clarify the physiological function of AMPD2. In this report, we describe selective AMPD2 inhibitors inducing allosteric modulation. Based on hypothesis that compounds that exhibit increased inhibition by preincubation would cause conformational change of the enzyme, starting from HTS hit compound 4, we discovered compound 8 through the SAR study. From X-ray structural information of 8, this chemical series has a novel mechanism of action that changes the substrate pocket to prevent AMP from binding. Further elaboration of compound 8 led to the tool compound 21 which exhibited potent inhibitory activity of AMPD2 in ex vivo evaluation of mouse liver.

Effect of matcha consumption on gut microbiota in healthy Japanese individuals: study protocol for a double-blind crossover interventional study.

This study compares and clarifies the changes in intestinal flora resulting from the continuous consumption of two types of matcha. Healthy adults will consume two types of matcha tea for four weeks, and differences in the intestinal microflora before and after drinking will be compared. Gut microbiota will be identified using next-generation sequencing. Phylogenetic classification of the enterobacteria will be performed based on sequence similarities. The relative proportions of the classified enterobacteria to the total nucleotide sequences will be compared between the samples obtained from the two groups consuming different matcha. The continuous consumption of matcha may improve dysbiosis and prevent atherosclerosis. The effects may vary according to the type of matcha used. Trial registration: The study was registered with university hospital medical information network (UMIN) (UMIN000040303), and all participants gave their written informed consent. Registered 1 November 2020, https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000045982.

Identification of Potent and Selective Inhibitors of Fat Mass Obesity-Associated Protein Using a Fragment-Merging Approach.

Fat mass obesity-associated protein (FTO) is a DNA/RNA demethylase involved in the epigenetic regulation of various genes and is considered a therapeutic target for obesity, cancer, and neurological disorders. Here, we aimed to design novel FTO-selective inhibitors by merging fragments of previously reported FTO inhibitors. Among the synthesized analogues, compound 11b, which merges key fragments of Hz (3) and MA (4), inhibited FTO selectively over alkylation repair homologue 5 (ALKBH5), another DNA/RNA demethylase. Treatment of acute monocytic leukemia NOMO-1 cells with a prodrug of 11b decreased the viability of acute monocytic leukemia cells, increased the level of the FTO substrate N6-methyladenosine in mRNA, and induced upregulation of MYC and downregulation of RARA, which are FTO target genes. Thus, Hz (3)/MA (4) hybrid analogues represent an entry into a new class of FTO-selective inhibitors.

Discovery of Selective Transforming Growth Factor ß Type II Receptor Inhibitors as Antifibrosis Agents.

Historically, modulation of transforming growth factor ß (TGF-ß) signaling has been deemed a rational strategy to treat many disorders, though few successful examples have been reported to date. This difficulty could be partially attributed to the challenges of achieving good specificity over many closely related enzymes that are implicated in distinct phenotypes in organ development and in tissue homeostasis. Recently, fresolimumab and disitertide, two peptidic TGF-ß blockers, demonstrated significant therapeutic effects toward human skin fibrosis. Therefore, the selective blockage of TGF-ß signaling assures a viable treatment option for fibrotic skin disorders such as systemic sclerosis (SSc). In this report, we disclose selective TGF-ß type II receptor (TGF-ßRII) inhibitors that exhibited high functional selectivity in cell-based assays. The representative compound 29 attenuated collagen type I alpha 1 chain (COL1A1) expression in a mouse fibrosis model, which suggests that selective inhibition of TGF-ßRII-dependent signaling could be a new treatment for fibrotic disorders.

Identification of Diketopiperazine-Containing 2-Anilinobenzamides as Potent Sirtuin 2 (SIRT2)-Selective Inhibitors Targeting the "Selectivity Pocket", Substrate-Binding Site, and NAD+-Binding Site.

The NAD+-dependent deacetylase SIRT2 represents an attractive target for drug development. Here, we designed and synthesized drug-like SIRT2-selective inhibitors based on an analysis of the putative binding modes of recently reported SIRT2-selective inhibitors and evaluated their SIRT2-inhibitory activity. This led us to develop a more drug-like diketopiperazine structure as a "hydrogen bond (H-bond) hunter" to target the substrate-binding site of SIRT2. Thioamide 53, a conjugate of diketopiperazine and 2-anilinobenzamide which is expected to occupy the "selectivity pocket" of SIRT2, exhibited potent SIRT2-selective inhibition. Inhibition of SIRT2 by 53 was mediated by the formation of a 53-ADP-ribose conjugate, suggesting that 53 is a mechanism-based inhibitor targeting the "selectivity pocket", substrate-binding site, and NAD+-binding site. Furthermore, 53 showed potent antiproliferative activity toward breast cancer cells and promoted neurite outgrowth of Neuro-2a cells. These findings should pave the way for the discovery of novel therapeutic agents for cancer and neurological disorders.

High-Throughput Screening of Potential Skin Penetration-Enhancers Using Stratum Corneum Lipid Liposomes: Preliminary Evaluation for Different Concentrations of Ethanol.

In this study, we developed a technique for high-throughput screening (HTS) of skin penetration-enhancers using stratum corneum lipid liposomes (SCLLs). A fluorescent marker, sodium fluorescein (FL), entrapped in SCLLs was prepared to provide a preliminary evaluation of the effect of different concentrations of ethanol on the disruption effect of SCLLs, which is an alternative for skin penetration-enhancing effects. In addition, SCLLs containing a fluorescent probe (DPH, TMA-DPH, or ANS) were also prepared and utilized to investigate SCLL fluidity. The results using SCLL-based techniques were compared with conventional skin permeation and skin impedance test using hairless rat skin. The obtained correlations were validated between FL leakage, SCLL fluidity with various probes, or skin impedance and increases in the skin permeation enhancement ratio (ER) of caffeine as a model penetrant. As a result, FL leakage and SCLL fluidity using ANS were considered to be good indices for the skin penetration-enhancing effect, suggesting that the action of ethanol on the SC lipid and penetration-enhancing is mainly on the polar head group of intercellular lipids. In addition, this screening method using SCLL could be utilized as an alternative HTS technique for conventional animal tests. Simultaneously, the method was found to be time-saving and sensitive compared with a direct assay using human and animal skins.

Molecular Assembly of Wheat Gliadins into Nanostructures: A Small-Angle X-ray Scattering Study of Gliadins in Distilled Water over a Wide Concentration Range.

Gliadin, one of the major proteins together with glutenin composing gluten, affects the physical properties of wheat flour dough. In this study, nanoscale structures of hydrated gliadins extracted into distilled water were investigated primarily by small-angle X-ray scattering (SAXS) over a wide range of concentrations. Gliadins are soluble in distilled water below 10 wt %. Guinier analyses of SAXS profiles indicate that gliadins are present as monomers together with small amounts of dimers and oligomers in a very dilute solution. The SAXS profiles also indicate that interparticle interference appears above 0.5 wt % because of electrostatic repulsion among gliadin assemblies. Above 15 wt %, gliadins form gel-like hydrated solids. At greater concentrations, a steep upturn appears in the low-q region owing to the formation of large aggregates, and a broad shoulder appears in the middle-q region showing density fluctuation inside. This study demonstrates that SAXS can effectively disclose the nanostructure of hydrated gliadin assemblies.

Expression and characterization of protein disulfide isomerase family proteins in bread wheat.

BACKGROUND: The major wheat seed proteins are storage proteins that are synthesized in the rough endoplasmic reticulum (ER) of starchy endosperm cells. Many of these proteins have intra- and intermolecular disulfide bonds. In eukaryotes, the formation of most intramolecular disulfide bonds in the ER is thought to be catalyzed by protein disulfide isomerase (PDI) family proteins. The cDNAs that encode eight groups of bread wheat (Triticum aestivum L.) PDI family proteins have been cloned, and their expression levels in developing wheat grains have been determined. The purpose of the present study was to characterize the enzymatic properties of the wheat PDI family proteins and clarify their expression patterns in wheat caryopses. RESULTS: PDI family cDNAs, which are categorized into group I (TaPDIL1Aα, TaPDIL1Aß, TaPDIL1Aγ, TaPDIL1Aδ, and TaPDIL1B), group II (TaPDIL2), group III (TaPDIL3A), group IV (TaPDIL4D), and group V (TaPDIL5A), were cloned. The expression levels of recombinant TaPDIL1Aα, TaPDIL1B, TaPDIL2, TaPDIL3A, TaPDIL4D, and TaPDIL5A in Escherichia coli were established from the cloned cDNAs. All recombinant proteins were expressed in soluble forms and purified. Aside from TaPDIL3A, the recombinant proteins exhibited oxidative refolding activity on reduced and denatured ribonuclease A. Five groups of PDI family proteins were distributed throughout wheat caryopses, and expression levels of these proteins were higher during grain filling than in the late stage of maturing. Localization of these proteins in the ER was confirmed by fluorescent immunostaining of the immature caryopses. In mature grains, the five groups of PDI family proteins remained in the aleurone cells and the protein matrix of the starchy endosperm. CONCLUSIONS: High expression of PDI family proteins during grain filling in the starchy endosperm suggest that these proteins play an important role in forming intramolecular disulfide bonds in seed storage proteins. In addition, these PDI family proteins that remain in the aleurone layers of mature grains likely assist in folding newly synthesized hydrolytic enzymes during germination.

Molecular mechanisms of action of different concentrations of ethanol in water on ordered structures of intercellular lipids and soft keratin in the stratum corneum.

Ethanol (EtOH) is one of the bases in topically applied medicines that promote the skin permeation of drugs. Although the effects of EtOH have been attributed to structural modifications in the stratum corneum, the underlying mechanisms, especially the influence of different concentrations of EtOH, have not been examined extensively. Structural modifications in the stratum corneum of hairless mouse due to the application of EtOH/water mixture were herein investigated at the molecular level using synchrotron X-ray diffraction. The results revealed that all EtOH concentrations examined greatly modified the short lamellar structures containing the aqueous layer in intercellular lipids and the structure of keratin fibrils in corneocytes, which can take up hydrophilic compounds. However, the long lamellar and the hydrocarbon-chain packing structures were unaffected by EtOH. Changes to the short lamellar structures were not proportional to the concentration of EtOH. However, the keratin fibril structures changed gradually with increasing EtOH concentration. The X-ray diffraction experiments enabled the effects of different EtOH concentrations on the morphology of the stratum corneum to be assessed by using a number of experimental samples to avoid variations due to individual differences. The results indicated that alterations to the short lamellar structures appeared to be related to the skin permeability of drugs with the application of EtOH/water mixture, and monotonous structural changes in the keratin fibrils with an increase in EtOH concentration may contribute to this permeation as supplement. These results will be useful for the development of new drug formulations containing EtOH.

Discovery of INT131: a selective PPARγ modulator that enhances insulin sensitivity.

PPARγ is a member of the nuclear hormone receptor family and plays a key role in the regulation of glucose homeostasis. This Letter describes the discovery of a novel chemical class of diarylsulfonamide partial agonists that act as selective PPARγ modulators (SPPARγMs) and display a unique pharmacological profile compared to the thiazolidinedione (TZD) class of PPARγ full agonists. Herein we report the initial discovery of partial agonist 4 and the structure-activity relationship studies that led to the selection of clinical compound INT131 (3), a potent PPARγ partial agonist that displays robust glucose-lowering activity in rodent models of diabetes while exhibiting a reduced side-effects profile compared to marketed TZDs.

Characterization of protein-like fluorophores released from lake phytoplankton on the basis of fractionation and electrophoresis.

Three kinds of lake plankton were cultivated, and the properties of protein-like fluorophores released from the plankton were characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results were compared with those by gel chromatography with a fluorescence detector and three-dimensional excitation-emission matrix (3-DEEM). The concentrated protein-like fluorophores of algal dissolved organic matter (DOM) were successfully separated from the fulvic-like fluorophores, and analyzed using SDS-PAGE. SDS-PAGE analysis revealed that the protein-like fluorescence DOM released from Microcystis aeruginosa consisted of proteins with molecular weights of 17, 37, 50, 75, 150 kDa, and greater than 250 kDa. The results of SDS-PAGE were consistent with those of gel chromatography. Those substances with molecular weights greater than 250 kDa may be a polysaccharide-peptide complex, called peptidoglycan, which is a component of bacterial cell walls. The molecular weights of protein-like fluorescence DOM from Staurastrum dorsidentiferum were determined to be 37 and 50 kDa. For Cryptomonas ovata, its DOM was found to be composed of substances with molecular weights of between 10 and 150 kDa. The results by high-performance size exclusion chromatography with chemiluminescent nitrogen detection (HPSEC/CLND) analysis suggest that the protein-like fluorophores from the plankton might be composed of substances containing organic nitrogen.

Inhibitory effect of JTP-59557, a new triazole derivative, on intestinal phosphate transport in vitro and in vivo.

JTP-59557 [(-)-4-(2-tert-Butyl-4,5-dichlorophenyl)-5-(5-trifluoromethylpyridin-2-ylsulfanyl)-4H-[1,2,4]triazol-3-ol] showed an inhibitory effect on Na(+)-dependent inorganic phosphate (Pi) transport in intestinal brush border membrane vesicles with an IC(50) value of 0.40 microM in rabbit and with an IC(50) of 0.19 microM in rat, without affecting Na(+)-independent Pi and Na(+)-dependent d-glucose transport activities. In Chinese hamster ovary (CHO) cells expressing human type IIb Na/Pi cotransporter (type IIb), JTP-59557 decreased human type IIb-mediated Pi uptake with an IC(50) of 0.12 microM. In rabbit intestinal brush border membrane vesicles, JTP-59557 behaved as a noncompetitive inhibitor with respect to Pi. In an in vivo study, single administration of JTP-59557 significantly decreased the intestinal Pi absorption rate, when either Pi solution or laboratory chow was given to rats. In this report, we show that JTP-59557 is a potent, selective, stereospecific, noncompetitive inhibitor of intestinal Na/Pi cotransporters including type IIb, and it may represent a new class of intestinal Pi absorption inhibitor.