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In an effort to explore new-type high-efficiency antifungal agents, 25 novel L -carvone-based 1,3,4-thiadiazole-amide derivatives were designed, synthesized, and structurally characterized by IR, 1 H NMR, 13 C NMR, and high-resolution mass spectrometry (HRMS) analyses. The antifungal activity of the target compounds was preliminarily assayed at a concentration of 50 μg/mL, and boscalid, a commercialized fungicide identified as a succinate dehydrogenase inhibitor (SDHI), was employed as the positive control. It was found that all of the target compounds showed moderate to potent antifungal activity against the tested fungi compared to boscalid. Surprisingly, compound 4b exhibited broad-spectrum and significant inhibition activity against the growth of eight phytopathogenic strains with inhibitory rates of 67-89%. Further, the results of the EC 50 value test suggested that the EC 50 values of compound 4b against Physalospora piricola and Colletotrichum orbiculare were 16.33 and 18.06 μg/mL, respectively, and both of them were better than those of boscalid (16.64 and >50). Therefore, compound 4b deserves further study as a lead compound for novel fungicides. In addition, the inhibitory activity of compound 4b against succinate dehydrogenase (SDH) was evaluated as well to prove that compound 4b (IC 50 = 3.38 μM) displayed higher SDH-inhibition activity than boscalid (IC 50 = 7.02 μM). The binding mode of compound 4b and SDH was simulated by molecular docking and found to be similar to that of boscalid. The structure-activity relationships (SARs) of the target compounds were analyzed by establishing a 3D-QSAR model. Besides, a 4b -loaded complex 4b/CSTA on a reported L -carvone-based nanochitosan carrier CSTA containing the 1,3,4-thiadiazole-amide group was constructed, and its sustained release performance was investigated in the EtOH-H 2 O system (1:9, v/v). The complex 4b/CSTA exhibited preferred sustained release performance, indicating its potential for developing environmentally friendly nanofungicides.

The hyperactivity of urease enzyme leads to various complications including gastritis and peptic ulcer. A diverse variety of natural and synthetic inhibitors have shown a tremendous potential to inhibit the urease enzyme, thus decreasing the hyperactivity and reducing the risk for the development of urinary calculi and other similar problems. Therefore, we herein report a family of fused heterocycles such as triazolothiadiazoles ( 4a - h , 5a - f ) and triazolothiadiazines ( 6a - h ) as potential antiurease agents with IC 50 values in the range 10.41-41.20 µM. Several compounds were identified as potential lead candidates. Among them, compounds 4e and 4f from triazolothiadiazole series showed the highest inhibitory potential with IC 50 values of 11.62 ± 0.34 and 10.35 ± 0.14 µM), respectively, whereas 6e from triazolothiadiazine series emerged as the most potent inhibitor with an IC 50 value of 10.41 ± 0.13 µM. These compounds exhibited two-fold strong inhibitory efficacy against urease as compared to standard inhibitor, thiourea (IC 50 = 22.48 ± 0.67 µM). The mechanistic insights from kinetics experiments for compounds 4e , 4f , and 6e revealed the competitive mode of inhibition with K i values of 8.65 ± 0.004, 7.04 ± 0.012, and 8.31 ± 0.007 µM, respectively. The in vitro results were further explored through in silico computational docking analysis which reflects that binding of ligands with Ni ions and His492 play a crucial role in urease inhibition. In silico predicted physicochemical properties and ADME profile reflect drug-like nature of these molecules.Communicated by Ramaswamy H. Sarma.

Protoporphyrinogen oxidase (PPO, EC 1.3.3.4) has a high status in the development of new inhibitors. To develop novel and highly effective PPO inhibitors, active substructure linking and bioisosterism replacement strategies were used to design and synthesize novel tetrahydrophthalimide derivatives containing oxadiazole/thiadiazole moieties, and their inhibitory effects on Nicotiana tobacco PPO ( Nt PPO) and herbicidal activity were evaluated. Among them, compounds B11 ( K i = 9.05 nM) and B20 ( K i = 10.23 nM) showed significantly better inhibitory activity against Nt PPO than that against flumiclorac-pentyl ( K i = 46.02 nM). Meanwhile, compounds A20 and B20 were 100% effective against three weeds ( Abutilon theophrasti , Amaranthus retroflexus , and Portulaca oleracea ) at 37.5 g a.i./ha. It was worth observing that compound B11 was more than 90% effective against three weeds ( Abutilon theophrasti , Amaranthus retroflexus , and Portulaca oleracea ) at 18.75 and 9.375 g a.i./ha. It was also safer to rice, maize, and wheat than flumiclorac-pentyl at 150 g a.i./ha. In addition, the molecular docking results showed that compound B11 could stably bind to Nt PPO and it had a stronger hydrogen bond with Arg98 (2.9 Å) than that of flumiclorac-pentyl (3.2 Å). This research suggests that compound B11 could be used as a new PPO inhibitor, and it could help control weeds in agricultural production.

Cancer cells frequently exhibit uncoupling of the glycolytic pathway from the TCA cycle (i.e., the "Warburg effect") and as a result, often become dependent on their ability to increase glutamine catabolism. The mitochondrial enzyme Glutaminase C (GAC) helps to satisfy this 'glutamine addiction' of cancer cells by catalyzing the hydrolysis of glutamine to glutamate, which is then converted to the TCA-cycle intermediate α-ketoglutarate. This makes GAC an intriguing drug target and spurred the molecules derived from bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (the so-called BPTES class of allosteric GAC inhibitors), including CB-839, which is currently in clinical trials. However, none of the drugs targeting GAC are yet approved for cancer treatment and their mechanism of action is not well understood. Here, we shed new light on the underlying basis for the differential potencies exhibited by members of the BPTES/CB-839 family of compounds, which could not previously be explained with standard cryo-cooled X-ray crystal structures of GAC bound to CB-839 or its analogs. Using an emerging technique known as serial room temperature crystallography, we were able to observe clear differences between the binding conformations of inhibitors with significantly different potencies. We also developed a computational model to further elucidate the molecular basis of differential inhibitor potency. We then corroborated the results from our modeling efforts using recently established fluorescence assays that directly read out inhibitor binding to GAC. Together, these findings should aid in future design of more potent GAC inhibitors with better clinical outlook., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Src homology-2 domain-containing protein tyrosine phosphatase 1 (SHP1) is mainly restricted to hematopoietic and epithelial cells and widely accepted as a convergent node for oncogenic cell-signaling cascades. The development of efficient methods for rapidly tracing and inhibiting the SHP1 activity in complex biological systems is of considerable significance for advancing the integration of diagnosis and treatment of the related disease. With this aim, we designed and synthesized five 2-phenyl-1,3,4-thiadiazole derivatives (PT2, PT5, PT8, PT9 and PT10) here based on the reported SHP1 inhibitors (PT1, PT3, PT4, PT6 and PT7). The photophysical properties and inhibitory activities of these 2-phenyl-1,3,4-thiadiazole derivatives (PT1-PT10) against SHP1 were thoroughly studied from the theoretical simulation and experimental application aspects. The representative compound PT10 exhibited a larger quantum yield than the other molecules because of the smaller geometric relaxation and reorganization energy of the excited state, which was consistent with the results from the fluorescence experiments in organic solvents. In addition, PT10 showed a selective fluorescence response for SHP1 activity and low cytotoxicity in HeLa cells. Lastly, it indicated the potential application in two-photon cell fluorescence imaging in the future according to the calculated excellent two-photon absorption properties. In this contribution, firstly, we offered the fluorescent and activated molecule PT10 against SHP1, which achieved the integration of visualization and inhibitory activity of SHP1 preliminarily at the enzyme molecular level.

Importance of extracellular nucleotides is widely understood. These nucleotides act as ligand for P2X and P2Y receptors and modulate a variety of biological functions. However, their extracellular concentration is maintained by a chain of enzymes termed as ecto-nucleotidases. Amongst them, nucleoside triphosphate diphosphohydrolases (NTPDases) is an important enzyme family responsible for the dephosphorylation of these nucleotides. Overexpression of NTPDases leads to many pathological conditions such as cancer and thrombosis. So far, only a few NTPDase inhibitors have been reported. Considering this scarcity of (NTPDase) inhibitors, a number of thiadiazole amide derivatives were synthesized and screened against human (h)-NTPDases. Several compounds showed promising inhibitory activity; compound 5a (IC 50 (µM); 0.05 ± 0.008) and 5g (IC 50 (µM); 0.04 ± 0.006) appeared to be the most distinguished molecules corresponding to h-NTPDase1 and -2. However, h-NTPDase3 was the least susceptible isozyme and only three compounds (5d, 5e, 5j) strongly inhibited h-NTPDase3. Interestingly, compound 5e was recognized as the most active compound that showed dual inhibition against h-NTPDase3 as well as against h-NTPDase8. For better comprehension of binding mode of these inhibitors, most potent inhibitors were docked with their respective isozyme., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Renal ischemia/reperfusion (I/R) injury is a main cause of acute kidney injury (AKI). Aquaporin (AQP)-1 water channel in the kidney is critical for the maintenance of water homeostasis and the urinary concentrating ability. Increasing evidence supports an important role of autophagy in the pathogenesis of AKI induced by renal I/R. The purpose of the present study is to investigate whether activation of autophagy prevents downregulation of AQP1 protein induced by renal I/R and potential molecular mechanisms. Renal I/R induced consistently reduced protein expression of AQP1, 2, and 3, as well as sodium cotransporters Na + -K + -2Cl - cotransporter and α-Na,K-ATPase, which was associated with increased urine output and decreased creatinine clearance in rats. Renal I/R also suppressed autophagy and increased inflammatory responses in the kidney. 4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), the glycogen synthase kinase-3β inhibitor, ameliorated renal injury under I/R, activated autophagy and markedly increased expression of AQPs and sodium transporters in the kidney, which was associated with improved urine output and creatinine clearance in rats. Hypoxia/reoxygenation (H/R) induced suppression of autophagy and downregulation of AQP1 in murine inner medullary collecting duct 3 (IMCD3) cells, which was fully prevented by TDZD-8 treatment. Inhibition of autophagy by 3-methyladenine or Atg5 gene knockdown attenuated recovery of AQP1 protein expression induced by TDZD-8 in IMCD3 cells with H/R. Interleukin-1 beta (IL-1β) decreased the abundance of AQP1 protein in IMCD3 cells. H/R induced increases in protein expression of nod-like receptor pyrin domain-containing 3 and IL-1β, which was reversed by TDZD-8. In conclusion, TDZD-8 treatment prevented downregulation of AQP1 expression under renal I/R injury, likely via activating autophagy and decreasing IL-1β production., (© 2021 Federation of American Societies for Experimental Biology.)

The efficacy of ionizing radiation (IR) for head and neck cancer squamous cell carcinoma (HNSCC) is limited by poorly understood mechanisms of adaptive radioresistance. Elevated glutaminase gene expression is linked to significantly reduced survival (p < 0.03). The glutaminase inhibitor, telaglenastat (CB-839), has been tested in Phase I/II cancer trials and is well tolerated by patients. This study investigated if telaglenastat enhances the cellular response to IR in HNSCC models. Using three human HNSCC cell lines and two xenograft mouse models, we examined telaglenastat's effects on radiation sensitivity. IR and telaglenastat combinatorial treatment reduced cell survival (p ≤ 0.05), spheroid size (p ≤ 0.0001) and tumor growth in CAL-27 xenograft bearing mice relative to vehicle (p ≤ 0.01), telaglenastat (p ≤ 0.05) or IR (p ≤ 0.01) monotherapy. Telaglenastat significantly reduced the Oxygen Consumption Rate/Extracellular Acidification Rate ratio in CAL-27 and HN5 cells in the presence of glucose and glutamine (p ≤ 0.0001). Telaglenastat increased oxidative stress and DNA damage in irradiated CAL-27 cells. These data suggest that combination treatment with IR and telaglenastat leads to an enhanced anti-tumor response. This pre-clinical data, combined with the established safety of telaglenastat justifies further investigation for the combination in HNSCC patients., (Published by Elsevier B.V.)

In analogy to antiviral acyclic nucleoside phosphonates, a series of 5-amino-3-oxo-1,2,4-thiadiazol-3(2H)-ones bearing a 2-phosphonomethoxyethyl (PME) or 3-hydroxy-2-(phosphonomethoxy)propyl (HPMP) group at the position 2 of the heterocyclic moiety has been synthesized. Diisopropyl esters of PME- and HPMP-amines have been converted to the N-substituted ureas and then reacted with benzoyl, ethoxycarbonyl, and Fmoc isothiocyanates to give the corresponding thiobiurets, which were oxidatively cyclized to diisopropyl esters of 5-amino-3-oxo-2-PME- or 2-HPMP- 1,2,4-thiadiazol-3(2H)-ones. The phosphonate ester groups were cleaved with bromotrimethylsilane, yielding N 5 -protected phosphonic acids. The subsequent attempts to remove the protecting group from N 5 under alkaline conditions resulted in the cleavage of the 1,2,4-thiadiazole ring. Similarly, compounds with a previously unprotected 5-amino-1,2,4-thiadiazolone base moiety were stable only in the form of phosphonate esters. The series of twenty-one newly prepared 1,2,4-thiadiazol-3(2H)-ones were explored as potential inhibitors of cysteine-dependent enzymes - human cathepsin K (CatK) and glycogen synthase kinase 3β (GSK-3β). Several compounds exhibited an inhibitory activity toward both enzymes in the low micromolar range. The inhibitory potency of some of them toward GSK-3β was similar to that of the thiadiazole GSK-3β inhibitor tideglusib, whereas others exhibited more favorable toxicity profile while retaining good inhibitory activity., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: GSK3β is an intracellular regulatory kinase that is dysregulated in multiple tissues in type 1 myotonic dystrophy, a rare neuromuscular disorder that manifests at any age. AMO-02 (tideglusib) inhibits GSK3β activity in preclinical models of type 1 myotonic dystrophy and promotes cellular maturation as well as normalizes aberrant molecular and behavioral phenotypes. This phase 2 study assessed the pharmacokinetics, safety and tolerability, and preliminary efficacy of AMO-02 in adolescents and adults with congenital and childhood-onset type 1 myotonic dystrophy., Methods: Sixteen subjects (aged 13 to 34 years) with congenital and childhood-onset type 1 myotonic dystrophy received 12 weeks of single-blind fixed-dose oral treatment with either 400 mg (n = 8) or 1000 mg (n = 8) AMO-02 (NCT02858908). Blood samples were obtained for pharmacokinetic assessment. Safety assessments, such as laboratory tests and electrocardiograms, as well as efficacy assessments of syndromal, cognitive, and muscular functioning, were obtained., Results: AMO-02 plasma concentrations conformed to a two-compartment model with first-order absorption and elimination, and dose-dependent increases in exposure (area under the curve) were observed. AMO-02 was generally safe and well-tolerated. No early discontinuations due to adverse events or dose adjustments of AMO-02 occurred. The majority of subjects manifested clinical improvement in their central nervous system and neuromuscular symptoms after 12 weeks of treatment compared with the placebo baseline, with a larger response noted at the 1000 mg/day dose level. AMO-02 exposure (cumulative area under the curve) was significantly correlated (P < 0.01) with change from baseline on several key efficacy assessments., Conclusion: AMO-02 has favorable pharmacokinetic and clinical risk/benefit profiles meriting further study as a potential treatment for congenital and childhood-onset type 1 myotonic dystrophy., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Sortase A (SrtA) anchors surface proteins to the cell wall envelope, and it has attracted increasing interesting as a potential antivirulence target. Several small-molecule inhibitors for SrtA have been developed, but target validation remains largely underexplored. Herein, we report a new class of SrtA inhibitors that supports antivirulence therapy through small-molecule targeting of SrtA. Tideglusib ( TD ), a drug candidate for myotonic dystrophy, was outstanding in high-throughput screening. A concise synthetic route quickly provided TD analogues, and the structure-activity relationships for SrtA inhibition have been established from those analogues. Several compounds largely retained the in vitro potency and exhibited a better solubility than TD . Additionally, TD attenuated virulence-related phenotypes in vitro and protected mice against lethal S. aureus USA300 bacteremia. Our study indicates that TD and its analogues could be new candidates as SrtA inhibitors with potential in the development of new antivirulence agents.

Tuberculosis, caused by Mycobacterium tuberculosis, is a serious infectious disease and remains a global health problem. There is an increasing need for the discovery of novel therapeutic agents for its treatment due to the emerging multi-drug resistance. Herein, we present the rational design and the synthesis of eighteen new thiadiazolylhidrazones (TDHs) which were synthesized by intramolecular oxidative N-S bond formation reaction of 2-benzylidene-N-(phenylcarbamothioyl)hydrazine-1-carboximidamide derivatives by phenyliodine(III) bis(trifluoroacetate) (PIFA) under mild conditions. The compounds were characterized by various spectral techniques including FTIR, 1 H NMR, 13 C NMR and HRMS. Furthermore, the proposed structure of TDH12 was resolved by single-crystal X-ray analysis. The compounds were evaluated for their in vitro antitubercular activity against M. tuberculosis H37Rv. Among them, some compounds exhibited remarkable antimycobacterial activity, MIC = 0.78-6.25 μg/mL, with low cytotoxicity. Additionally, the most active compounds were screened for their biological activities against M. tuberculosis in the nutrient starvation model. Enzyme inhibition assays and molecular docking studies revealed enoyl acyl carrier protein reductase (InhA) as the possible target enzyme of the compounds to show their antitubercular activities., Competing Interests: Declaration of competing interest The authors declared no potential conflicts of interest., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

The discovery of antibiotics around the middle twentieth century led to a decrease in the interest in antimycobacterial fatty acids. In order to re-establish the importance of naturally abundant fatty acid, a series of fatty acid-thiadiazole derivatives were designed and synthesized based on molecular hybridization approach. In vitro antimycobacterial potential was established by a screening of synthesized compounds against Mycobacterium tuberculosis H37Rv strain. Among them, compounds 5a, 5d, 5h, and 5j were the most active, with compound 5j exhibiting minimum inhibitory concentration of 2.34 μg/ml against M.tb H37Rv. Additionally, the compounds were docked to determine the probable binding interactions and understand the mechanism of action of most active molecules on enoyl-acyl carrier protein reductases (InhA), which is involved in the mycobacterium fatty acid biosynthetic pathway., (© 2019 John Wiley & Sons A/S.)

Background: The ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) terminate nucleotide signaling via the hydrolysis of extracellular nucleoside-5'-triphosphate and nucleoside- 5'-diphosphate, to nucleoside-5'-monophosphate and composed of eight Ca2+/Mg2+ dependent ectonucleotidases (NTPDase1-8). Extracellular nucleotides are involved in a variety of physiological mechanisms. However, they are rapidly inactivated by ectonucleotidases that are involved in the sequential removal of phosphate group from nucleotides with the release of inorganic phosphate and their respective nucleoside. Ectonucleoside triphosphate diphosphohydrolases (NTPDases) represent the key enzymes responsible for nucleotides hydrolysis and their overexpression has been related to certain pathological conditions. Therefore, the inhibitors of NTPDases are of particular importance in order to investigate their potential to treat various diseases e.g., cancer, ischemia and other disorders of the cardiovascular and immune system., Methods: Keeping in view the importance of NTPDase inhibitors, a series of thiadiazolopyrimidones were evaluated for their potential inhibitory activity towards NTPDases by the malachite green assay., Results: The results suggested that some of the compounds were found as non-selective inhibitors of isozyme of NTPDases, however, most of the compounds act as potent and selective inhibitors. In case of substituted amino derivatives (4c-m), the compounds 4m (IC50 = 1.13 ± 0.09 μM) and 4g (IC50 = 1.72 ± 0.08 μM) were found to be the most potent inhibitors of h-NTPDase1 and 2, respectively. Whereas, compound 4d showed the best inhibitory potential for both h-NTPDase3 (IC50 = 1.25 ± 0.06 μM) and h-NTPDase8 (0.21 ± 0.02 μM). Among 5a-t derivatives, compounds 5e (IC50 = 2.52 ± 0.15 μM), 5p (IC50 = 3.17 ± 0.05 μM), 5n (IC50 = 1.22 ± 0.06 μM) and 5b (IC50 = 0.35 ± 0.001 μM) were found to be the most potent inhibitors of h-NTPDase1, 2, 3 and 8, respectively. Interestingly, the inhibitory concentration values of above-mentioned inhibitors were several folds greater than suramin, a reference control. In order to determine the binding interactions, molecular docking studies of the most potent inhibitors were conducted into the homology models of NTPDases and the putative binding analysis further confirmed that selective and potent compounds bind deep inside the active pocket of the respective enzymes., Conclusion: The docking analysis proposed that the inhibitory activity correlates with the hydrogen bonds inside the binding pocket. Thus, these derivatives are of interest and may further be investigated for their importance in medicinal chemistry., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

It has been demonstrated that glutamine metabolism has become the main energy and building blocks supply for the growth and viability of a potentially large subset of malignant tumors. The glutamine metabolism often depends upon mitochondrial glutaminase (GLS) activity, which converts glutamine to glutamate and serves as a significant role for bioenergetic processes. Thus, recently, the GLS has become a key target for small molecule therapeutic intervention. Numerous medicinal chemistry studies are currently aimed at the design of novel and potent inhibitors for GLS, however, to date, only one compound (named CB-839) have entered clinical trials for the treatment of advanced solid tumors and hematological malignancies. The perspective summarizes the progress in the discovery and development of GLS inhibitors, including the potential binding site, biochemical techniques for inhibitor identification, and approaches for identifying small-molecule inhibitors, as well as future therapeutic perspectives in glutamine metabolism are also put forward in order to provide reference and rational for the drug discovery of novel and potent glutamine metabolism modulators.

Kidney-type glutaminase [KGA/isoenzyme glutaminase C (GAC)] is becoming an important tumor metabolism target in cancer chemotherapy. Its allosteric inhibitor, CB839, showed early promise in cancer therapeutics but limited efficacy in in vivo cancer models. To improve the in vivo activity, we explored a bioisostere replacement of the sulfur atom in bis-2-(5-phenylacetamido-1,2,4-thiadiazol)ethyl sulfide and CB839 analogues with selenium using a novel synthesis of the selenadiazole moiety from carboxylic acids or nitriles. The resulting selenadiazole compounds showed enhanced KGA inhibition, more potent induction of reactive oxygen species, improved inhibition of cancer cells, and higher cellular and tumor accumulation than the corresponding sulfur-containing molecules. However, both CB839 and its selenium analogues show incomplete inhibition of the tested cancer cells, and a partial reduction in tumor size was observed in both the glutamine-dependent HCT116 and aggressive H22 liver cancer xenograft models. Despite this, tumor tissue damage and prolonged survival were observed in animals treated with the selenium analogue of CB839.

Target identification is an essential basis for novel-pesticide development in new molecular design and lead optimization. 3-(4-Methyl-1,2,3-thiadiazolyl)-6-trichloromethyl[1,2,4]triazolo[3,4- b][1,3,4]thiadizole (YZK-C22) is a novel fungicide candidate with specific antifungal activity. We investigated its mode of action, and our studies indicated that YZK-C22 showed no cross resistance against Saccharomyces cerevisiae mutants with classic fungicide targets. Mec1 and Rad53 are two kinases that respond to DNA-replication damage, and the efficacy test showed that YZK-C22 could not perform its fungicidal activity by inhibiting DNA repair. Target screening by drug-affinity-responsive target stability (DARTS) showed that pyruvate kinase (PK), a key enzyme in the glycolytic pathway, was the potent new fungicidal target of YZK-C22. Fifty-eight differentially expressed proteins (DEPs) primarily involved in the metabolic process were identified by isobaric tags for relative and absolute quantification analysis (iTRAQ) in S. cerevisiae, and protein expression in the citrate cycle decreased with treatment of 5 mg/L YZK-C22, which was consistent with the results of DARTS. Molecular-docking analysis further validated that YZK-C22 could dock into the active center of PK instead of phosphoenolpyruvate. The enzyme activity of PK from S. cerevisiae was competitively inhibited with a K i of 3.33 ± 0.28 μmol/L, and the cell-growth inhibition of S. cerevisiae was released by supplementation with pyruvic acid, whereas the growth of S. cerevisiae was not recovered by adding PK's substrate (phosphoenolpyruvate) or allosteric regulator (fructose-1,6-bisphosphate). The present studies uncovered and validated the primary target of the new, potent fungicidal candidate YZK-C22; our results provide a successful, valuable, and applicable case of target discovery and identification for novel-fungicide development.

Background: Sevoflurane has been shown to protect against myocardial ischemia/reperfusion (I/R) injury in animals, while its cardioprotection is lost if the ischemic insult is too long. In this study, we proposed a prevailing hypothesis that GSK-3β inhibitor-mediated activation of GSK-3β/β-catenin signaling pathway provides additional cardioprotection in sevoflurane preconditioned rats following I/R injury., Methods: Rats were subjected to treatment with TDZD-8, a GSK-3β inhibitor, 5 minutes prior to sevoflurane preconditioning and 30-minute ischemia and 120-minute reperfusion. Furthermore, in order to find out whether this cardioprotection is linked with mitochondrial function and endoplasmic reticulum stress (ERS), we isolated mitochondria from rat hearts perfused with TDZD-8 and determined the alternations of ERS markers., Results: Sevoflurane preconditioning or GSK-3β inhibitor treatment prevented cardiomyocyte apoptosis, phosphorylated GSK-3β and accelerated total β-catenin expression levels, reduced mitochondrial permeability transition pore (MPTP) activity, promoted the recovery of mitochondrial membrane potential and decreased the expression levels of GRP78, caspase-12 and C/EBP homology protein (CHOP) in rats under I/R condition, suggesting sevoflurane preconditioning or TDZD-8 activate the GSK-3β/β-catenin signaling pathway, improve mitochondria function and suppress ERS occurrence., Conclusions: Taken together, the findings obtained from the study support the concept that sevoflurane preconditioning confers cardioprotection against myocardial I/R injury and GSK-3β/β-catenin signaling activation mediated by TDZD-8 as a novel target to prolong cardioprotection by sevoflurane anaesthesia.

Cytosolic protein tyrosine phosphatase epsilon (cyt-PTPε) plays a central role in controlling differentiation and function of osteoclasts, whose overactivation causes osteoporosis. Based on our previous study reporting a number of cyt-PTPε inhibitory chemical compounds, we carried out a further and extended analysis of our compounds to examine their effects on cyt-PTPε-mediated dephosphorylation and on osteoclast organization and differentiation. Among five compounds showing target selectivity to cyt-PTPε over three other phosphatases in vitro, two compounds exhibited an inhibitory effect against the dephosphorylation of cellular Src protein, the cyt-PTPε substrate. Moreover, these two compounds caused destabilization of the podosome structure that is necessary for the bone-resorbing activity of osteoclasts, and also attenuated cellular differentiation of monocytes into osteoclasts, without affecting cell viability. Therefore, these findings not only verified anti-osteoclastic effects of our cyt-PTPε inhibitory compounds, but also showed that cyt-PTPε expressed in osteoclasts could be a putative therapeutic target worth considering., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

One lead 3,6-disubstituted 1,2,4-triazolo[3,4-b][1,3,4]thiadiazole was identified as an inhibitor of shikimate dehydrogenase with antitubercular activity. Following up this compound, we optimized the lead through systematic modification of the 3 and 6 positions. The antitubercular activities in vitro, shikimate dehydrogenase inhibitory activities and cytotoxicity of derivatives were determined. We found IMB-SD62 with lower cytotoxicity and better activity. Thus, we studied the in vivo efficacy of IMB-SD62 against Mycobacterium tuberculosis and pharmacokinetics of IMB-SD62. In vivo acute M. tuberculosis H37Rv infection assay, IMB-SD62 showed antitubercular activity with the mean lung CFU counts decreasing 1.7 lg. The plasma pharmacokinetics study in rats showed that the oral bioavailability of IMB-SD62 was 14% and the half time was 1.05 h. The results of tissue distribution indicated that IMB-SD62 was mainly absorbed by liver and lung. In vitro metabolism study suggested that the metabolic ways of IMB-SD62 were dealkylated, oxidized and demethylated. CYP enzyme inhibition of IMB-SD62 in human liver microsomes was also evaluated. IMB-SD62 showed barely inhibition on CYP3A4 and CYP2D6. The excretion study manifested that IMB-SD62 was mainly eliminated by fecal excretion in rats. We concluded that based on these pharmaceutical properties, IMB-SD62 has the potential to be developed into new TB drug., (Copyright © 2018. Published by Elsevier Ltd.)

The thiadiazole scaffold is an important core moiety in a variety of clinical drug candidates targeting a range of diseases. For example, the 2,4,5-substituted 1,3,4-thiadiazole scaffold is present in a lead compound and at least two clinical candidates targeting the human motor protein Eg5, against neoplastic diseases. An inhibitor named K858 has in vivo activity in various mouse xenografts whereas the clinical candidates (S)-ARRY-520 and (R)-Litronesib have entered clinical trials with the former one in phase III clinical trials either alone or in combination with a proteasome inhibitor against relapsed/refractory multiple myeloma. Astonishingly, structural data are lacking for all thiadiazole-containing Eg5 inhibitors. Here we report the structure determination of two crystal forms of the ternary Eg5-ADP-K858 complex, locking the motor in the so-called final inhibitor bound state, thus blocking ADP release, a crucial stage for Eg5 activity. K858 acts at the established allosteric inhibitor-binding pocket formed of helix α2, loop L5 and helix α3. The structure of the complex has far reaching consequences for thiadiazole containing Eg5 inhibitors. For example, we could rationalise the structure-activity relationship in the crucial 5-position of the thiadiazole scaffold and the complex will serve in the future as a basis for strucutre-based drug design., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

GSK-3 inhibitors are an emerging tool for clinical interventions in human diseases and represent a niche area in combinational therapy. They possess diverse facets in applications of nervous system disorders, Type 2 diabetes, regenerative medicine and cancer. However, conflicting reports suggest the controversial role of GSK-3 inhibitors in cancers. This review aims to highlight the rise of GSK-3 inhibitors as tools for molecular-targeted research and its shift to a promising drug candidate. The review also focuses on key GSK-3 inhibitors and their roles in cancer and regenerative medicine with special emphasis to tideglusib. In addition, the decisive roles of GSK-3 in various molecular pathways will be concisely reviewed. Finally, this review concludes the emergence of GSK-3 inhibitors as a 'double-edged sword' in the treatment against human diseases cautioning researchers about the potential ramifications of off-target pharmacological effects., Competing Interests: The authors declare no conflict of interest., (© Georg Thieme Verlag KG Stuttgart · New York.)

In cystic fibrosis (CF), deletion of phenylalanine 508 (F508del) in the CFTR channel is associated with misfolding and premature degradation of the mutant protein. Among the known proteins associated with F508del-CFTR processing, the ubiquitin ligase RNF5/RMA1 is particularly interesting. We previously demonstrated that genetic suppression of RNF5 in vivo leads to an attenuation of intestinal pathological phenotypes in CF mice, validating the relevance of RNF5 as a drug target for CF. Here, we used a computational approach, based on ligand docking and virtual screening, to discover inh-02, a drug-like small molecule that inhibits RNF5. In in vitro experiments, treatment with inh-02 modulated ATG4B and paxillin, both known RNF5 targets. In immortalized and primary bronchial epithelial cells derived from CF patients homozygous for the F508del mutation, long-term incubation with inh-02 caused significant F508del-CFTR rescue. This work validates RNF5 as a drug target for CF, providing evidence to support its druggability., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Myotonic dystrophy type 1 (DM1) is a progressive neuromuscular disease caused by expanded CUG repeats, which misregulate RNA metabolism through several RNA-binding proteins, including CUG-binding protein/CUGBP1 elav-like factor 1 (CUGBP1/CELF1) and muscleblind 1 protein. Mutant CUG repeats elevate CUGBP1 and alter CUGBP1 activity via a glycogen synthase kinase 3β (GSK3β)-cyclin D3-cyclin D-dependent kinase 4 (CDK4) signaling pathway. Inhibition of GSK3β corrects abnormal activity of CUGBP1 in DM1 mice [human skeletal actin mRNA, containing long repeats ( HSA LR ) model]. Here, we show that the inhibition of GSK3β in young HSA LR mice prevents development of DM1 muscle pathology. Skeletal muscle in 1-yr-old HSA LR mice, treated at 1.5 mo for 6 wk with the inhibitors of GSK3, exhibits high fiber density, corrected atrophy, normal fiber size, with reduced central nuclei and normalized grip strength. Because CUG-GSK3β-cyclin D3-CDK4 converts the active form of CUGBP1 into a form of translational repressor, we examined the contribution of CUGBP1 in myogenesis using Celf1 knockout mice. We found that a loss of CUGBP1 disrupts myogenesis, affecting genes that regulate differentiation and the extracellular matrix. Proteins of those pathways are also misregulated in young HSA LR mice and in muscle biopsies of patients with congenital DM1. These findings suggest that the correction of GSK3β-CUGBP1 pathway in young HSA LR mice might have a positive effect on the myogenesis over time.-Wei, C., Stock, L., Valanejad, L., Zalewski, Z. A., Karns, R., Puymirat, J., Nelson, D., Witte, D., Woodgett, J., Timchenko, N. A., Timchenko, L. Correction of GSK3β at young age prevents muscle pathology in mice with myotonic dystrophy type 1.

Altered glycolytic flux in cancer cells (the "Warburg effect") causes their proliferation to rely upon elevated glutamine metabolism ("glutamine addiction"). This requirement is met by the overexpression of glutaminase C (GAC), which catalyzes the first step in glutamine metabolism and therefore represents a potential therapeutic target. The small molecule CB-839 was reported to be more potent than other allosteric GAC inhibitors, including the parent compound bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl (BPTES), and is in clinical trials. Recently, we described the synthesis of BPTES analogs having distinct saturated heterocyclic cores as a replacement for the flexible chain moiety, with improved microsomal stability relative to CB-839 and BPTES. Here, we show that one of these new compounds, UPGL00004, like CB-839, more potently inhibits the enzymatic activity of GAC, compared with BPTES. We also compare the abilities of UPGL00004, CB-839, and BPTES to directly bind to recombinant GAC and demonstrate that UPGL00004 has a similar binding affinity as CB-839 for GAC. We also show that UPGL00004 potently inhibits the growth of triple-negative breast cancer cells, as well as tumor growth when combined with the anti-vascular endothelial growth factor antibody bevacizumab. Finally, we compare the X-ray crystal structures for UPGL00004 and CB-839 bound to GAC, verifying that UPGL00004 occupies the same binding site as CB-839 or BPTES and that all three inhibitors regulate the enzymatic activity of GAC via a similar allosteric mechanism. These results provide insights regarding the potency of these inhibitors that will be useful in designing novel small-molecules that target a key enzyme in cancer cell metabolism., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Alkaline Phosphatases (APs) play a key role in maintaining a ratio of phosphate to inorganic pyrophosphate (P i /PP i ) and thus regulate extracellular matrix calcification during bone formation and growth. Among different isozymes of AP, aberrant increase in the level of tissue non-specific alkaline phosphatase (TNAP) is strongly associated with vascular calcification and end-stage renal diseases. In this context, we synthesized a novel series of fluorinated pyrimidone derivatives, i.e., 2-bromo-7-trifluoromethyl-5-oxo-5H-1,3,4-thiadiazolepyrimidones. The bromine functionality was further used for derivatisation by nucleophilic aromatic substitution using amines as nucleophiles as well as by Palladium catalysed Suzuki-Miyaura reactions. The synthesized derivatives were found potent but non-selective inhibitors of both isozymes of AP. Arylated thiadiazolopyrimidones exhibited stronger inhibitory activities than 2-amino-thiadiazolopyrimidones. The binding modes and possible interactions of the most active inhibitor within the active site of the enzyme were observed by molecular docking studies., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Glutaminase (GLS), which is responsible for the conversion of glutamine to glutamate, plays a vital role in up-regulating cell metabolism for tumor cell growth and is considered to be a valuable therapeutic target for cancer treatment. Based on this important function of glutaminase in cancer, several GLS inhibitors have been developed in both academia and industry. Most importantly, Calithera Biosciences Inc. is actively developing the glutaminase inhibitor CB-839 for the treatment of various cancers, and it is currently being evaluated in phase 1 and 2 clinical trials. In this review, recent efforts to develop small molecule glutaminase inhibitors that target glutamine metabolism in both preclinical and clinical studies are discussed. In particular, more emphasis is placed on CB-839 because it is the only small molecule GLS inhibitor being studied in a clinical setting. The inhibition mechanism is also discussed based on X-ray structure studies of thiadiazole derivatives present in glutaminase inhibitor BPTES. Finally, recent medicinal chemistry efforts to develop a new class of GLS inhibitors are described in the hopes of providing useful information for the next generation of GLS inhibitors., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

α-Amylase is a target for type-2 diabetes mellitus treatment. However, small molecule inhibitors of α-amylase are currently scarce. In the course of developing small molecule α-amylase inhibitors, we designed and synthesized thiadiazole quinoline analogs (1-30), characterized by different spectroscopic techniques such as 1 HNMR and EI-MS and screened for α-amylase inhibitory potential. Thirteen analogs 1, 2, 3, 4, 5, 6, 22, 23, 25, 26, 27, 28 and 30 showed outstanding α-amylase inhibitory potential with IC 50 values ranges between 0.002±0.60 and 42.31±0.17μM which is many folds better than standard acarbose having IC 50 value 53.02±0.12μM. Eleven analogs 7, 9, 10, 11, 12, 14, 15, 17, 18, 19 and 24 showed good to moderate inhibitory potential while seven analogs 8, 13, 16, 20, 21 and 29 were found inactive. Our study identifies novel series of potent α-amylase inhibitors for further investigation. Structure activity relationship has been established., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The dCTP pyrophosphatase 1 (dCTPase) is involved in the regulation of the cellular dNTP pool and has been linked to cancer progression. Here we report on the discovery of a series of 3,6-disubstituted triazolothiadiazoles as potent dCTPase inhibitors. Compounds 16 and 18 display good correlation between enzymatic inhibition and target engagement, together with efficacy in a cellular synergy model, deeming them as a promising starting point for hit-to-lead development.

Experiments were performed on the snails trained in conditioned food aversion for 3 days. Injection of TDZD-8 (glycogen synthase kinase-3 inhibitor, 2 mg/kg) in combination with reminder (presentation of a conditioned food stimulus) led to memory impairment developing 3 days after inhibitor/reminder exposure and followed by spontaneous recovery in 10 days. Injections of TDZD-8 in a dose of 4 or 20 mg/kg before reminder were shown to cause amnesia that persisted for more than 10 days. Memory recovery during repeated training was observed at the earlier period than after initial training. The impairment of memory reconsolidation by TDZD-8 after training of snails for 1 day was less pronounced than under standard training conditions (3 days). The effect of a glycogen synthase kinase-3 inhibitor during memory reconsolidation is probably followed by impairment of memory retrieval and/or partial loss, which can be compensated spontaneously or after repeated training.

1,3,4-Thiadiazole derivatives bearing Schiff base moieties were designed, synthesized, and their tyrosinase inhibitory activities were evaluated. Some compounds displayed potent tyrosinase inhibitory activities, especially, 4-(((5-mercapto-1,3,4-thiadiazol-2-yl)-imino)methyl)-2-methoxy-phenol (14) exhibited superior inhibitory effect to the other compounds with an IC 50 value of 0.036μM. The structure-activity relationships (SARs) were preliminarily discussed and docking studies showed compound 14 had strong binding affinity to mushroom tyrosinase. Hydroxy might be the active groups. The inhibition kinetics study revealed that compounds (13 and 14) inhibited tyrosinase by acting as uncompetitive inhibitors. The LD 50 value of the compound 14 was 5000mg/kg., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Metabolic reprogramming has been described as a hallmark of transformed cancer cells. In this study, we examined the role of the glutamine (Gln) utilization pathway in acute myeloid leukemia (AML) cell lines and primary AML samples. Our results indicate that a subset of AML cell lines is sensitive to Gln deprivation. Glutaminase (GLS) is a mitochondrial enzyme that catalyzes the conversion of Gln to glutamate. One of the two GLS isoenzymes, GLS1 is highly expressed in cancer and encodes two different isoforms: kidney (KGA) and glutaminase C (GAC). We analyzed mRNA expression of GLS1 splicing variants, GAC and KGA, in several large AML datasets and identified increased levels of expression in AML patients with complex cytogenetics and within specific molecular subsets. Inhibition of glutaminase by allosteric GLS inhibitor bis-2-(5-phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethyl sulfide or by novel, potent, orally bioavailable GLS inhibitor CB-839 reduced intracellular glutamate levels and inhibited growth of AML cells. In cell lines and patient samples harboring IDH1/IDH2 (Isocitrate dehydrogenase 1 and 2) mutations, CB-839 reduced production of oncometabolite 2-hydroxyglutarate, inducing differentiation. These findings indicate potential utility of glutaminase inhibitors in AML therapy, which can inhibit cell growth, induce apoptosis and/or differentiation in specific leukemia subtypes.

The glycogen-synthase-kinase 3 (GSK-3) is an important target in drug discovery. This enzyme is involved in the signaling pathways of type 2 diabetes, neurological disorders, cancer, and other diseases. Therefore, inhibitors of GSK-3 are promising drug candidates for the treatment of a broad range of diseases. Here we report pannorin ( 1 ), alternariol ( 2 ), and alternariol-9-methylether ( 3 ) to be promising inhibitors of the isoform GSK-3β showing sub-μM IC 50 values. The in vitro inhibition is in the range of the known highly active GSK-3β inhibitor TDZD-8. Compounds 1 - 3 have a highly oxygenated benzocoumarin core structure in common, which suggests that this may be a new structural feature for efficient GSK-3β inhibition., Competing Interests: The authors declare no conflict of interest.

A novel set of GAC (kidney glutaminase isoform C) inhibitors able to inhibit the enzymatic activity of GAC and the growth of the triple negative MDA-MB-231 breast cancer cells with low nanomolar potency is described. Compounds in this series have a reduced number of rotatable bonds, improved ClogPs, microsomal stability and ligand efficiency when compared to the leading GAC inhibitors BPTES and CB-839. Property improvements were achieved by the replacement of the flexible n-diethylthio or the n-butyl moiety present in the leading inhibitors by heteroatom substituted heterocycloalkanes., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Hyperuricemia is a risk factor for not only gout, but also to a variety of disorders that affect the vital organ systems of the human body. The xanthine oxidase (XO) is the key enzyme in the production of uric acid and its inhibition can inhibit hyperuricemia. Although, XO inhibitor allopurinol is widely prescribed antigout agent but its use is not without any side effects. Previously, we described the synthesis of four novel thiadiazolopyrimidin-5-one analogues as effective XO inhibitors and molecular docking studies also confirmed this. When these analogues were tested in potassium oxonate treated rats, their serum uric acid and creatinine levels were dropped significantly from 4.85±0.03 mg/dl to 1.21±0.01 mg/dl and 0.92±0.02 mg/dl to 0.40±0.02 mg/dl respectively. Among the pyrimidine analogues tested, 6a was most potent. Histological examinations of both liver and kidney tissues exhibited severe necrosis in oxonate treated rats and pyrimidine analogues could significantly attenuate this with a correlative inhibitory profile of hepatic XO from the same rats. Our results demonstrate antihyperuricemic effect of novel thiadiazolopyrimidin-5-one analogues in oxonate treated rats, which can be further explored not only as antigout therapeutics but also in other systems where hyperuricemia is the driving cause of the disease., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Plasmodium falciparum (Pf) like most other organisms, has a sophisticated antioxidant system, part of which includes glutathione reductase (GR). GR works by recycling toxic glutathione disulfide to glutathione, thereby reducing reactive oxygen species and making a form of glutathione (GSH) the parasite can use. Inhibition of this enzyme in Pf impedes parasite growth. In addition, it has been confirmed that PfGR is not identical to human GR. Thus, PfGR is an excellent target for antimalarial drug development. A functional assay utilizing liquid chromatography-mass spectrometry was developed to specifically identify and evaluate inhibitors of PfGR. Using recombinant PfGR enzyme and 1,4-naphthoquinone (1) as a reference compound and 4-nitrobenzothiadiazole (2) and methylene blue (3) as additional compounds, we quantified the concentration of GSH produced compared with a control to determine the inhibitory effect of these compounds. Our results coincide with that presented in literature: compounds 1-3 inhibit PfGR with IC50 values of 2.71, 8.38, and 19.23 µm, respectively. Good precision for this assay was exhibited by low values of intraday and interday coefficient of variation (3.1 and 2.4%, respectively). Thus, this assay can be used to screen for other potential inhibitors of PfGR quickly and accurately., (Copyright © 2015 John Wiley & Sons, Ltd.)

Background: New therapeutics are needed for neglected tropical diseases including Human African trypanosomiasis (HAT), a progressive and fatal disease caused by the protozoan parasites Trypanosoma brucei gambiense and T. b. rhodesiense. There is a need for simple, efficient, cost effective methods to identify new molecules with unique molecular mechanisms of action (MMOAs). The mechanistic features of a binding mode, such as competition with endogenous substrates and time-dependence can affect the observed inhibitory IC50, and differentiate molecules and their therapeutic usefulness. Simple screening methods to determine time-dependence and competition can be used to differentiate compounds with different MMOAs in order to identify new therapeutic opportunities., Methodology/principal Findings: In this work we report a four point screening methodology to evaluate the time-dependence and competition for inhibition of GSK3β protein kinase isolated from T. brucei. Using this method, we identified tideglusib as a time-dependent inhibitor whose mechanism of action is time-dependent, ATP competitive upon initial binding, which transitions to ATP non-competitive with time. The enzyme activity was not recovered following 100-fold dilution of the buffer consistent with an irreversible mechanism of action. This is in contrast to the T. brucei GSK3β inhibitor GW8510, whose inhibition was competitive with ATP, not time-dependent at all measured time points and reversible in dilution experiments. The activity of tideglusib against T. brucei parasites was confirmed by inhibition of parasite proliferation (GI50 of 2.3 μM)., Conclusions/significance: Altogether this work demonstrates a straightforward method for determining molecular mechanisms of action and its application for mechanistic differentiation of two potent TbGSK3β inhibitors. The four point MMOA method identified tideglusib as a mechanistically differentiated TbGSK3β inhibitor. Tideglusib was shown to inhibit parasite growth in this work, and has been reported to be well tolerated in one year of dosing in human clinical studies. Consequently, further supportive studies on the potential therapeutic usefulness of tideglusib for HAT are justified.

Chemical modulation of a formerly disclosed DGAT-1 inhibitor resulted in the identification of a compound with a suitable profile for preclinical development. Optimisation of solubility is discussed and a PK/PD study is presented., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Antimitotic agents are widely used in cancer chemotherapy but the numerous side effects and the onset of resistance limit their clinical efficacy. Therefore, with the purpose of discovering more selective and efficient anticancer agents to be administered alone or in combination with traditional drugs, we synthesized a large library of 1,3,4-thiadiazoline analogues, maintaining the pharmacophoric structure of an antiproliferative compound known as K858: this is a new inhibitor of kinesin Eg5, able to induce the mitotic arrest in colorectal cancer cells and in xenograft ovarian cancer cells. We screened 103 compounds to assess their antiproliferative activity on PC3 prostate cancer cell line. Two derivatives, compounds 32 (corresponding to K858) and 33, have shown to be the most effective against prostate tumor cells and also towards two melanoma cell lines (SK-MEL-5 and SK-MEL-28) at low micromolar concentrations, confirming the pharmacological activity of this scaffold and revealing the potential role of 1,3,4-thiadiazolines in the management of cancer., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

Excessive angiogenesis contributes to numerous diseases, including cancer and blinding retinopathy. Antibodies against vascular endothelial growth factor (VEGF) have been approved and are widely used in clinical treatment. Our previous studies using SRPIN340, a small molecule inhibitor of SRPK1 (serine-arginine protein kinase 1), demonstrated that SRPK1 is a potential target for the development of antiangiogenic drugs. In this study, we solved the structure of SRPK1 bound to SRPIN340 by X-ray crystallography. Using pharmacophore docking models followed by in vitro kinase assays, we screened a large-scale chemical library, and thus identified a new inhibitor of SRPK1. This inhibitor, SRPIN803, prevented VEGF production more effectively than SRPIN340 owing to the dual inhibition of SRPK1 and CK2 (casein kinase 2). In a mouse model of age-related macular degeneration, topical administration of eye ointment containing SRPIN803 significantly inhibited choroidal neovascularization, suggesting a clinical potential of SRPIN803 as a topical ointment for ocular neovascularization. Thus SRPIN803 merits further investigation as a novel inhibitor of VEGF., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

A series of novel thiadiazole amide derivatives have been synthesized and evaluated for inhibitory activities against Cdc25B and PTP1B. Most of them showed inhibitory activities against Cdc25B (IC50=1.18-8.01 μg/mL) and PTP1B (IC50=0.85-8.75 μg/mL), respectively. Moreover, compounds 5b and 4l were most potent with IC50 values of 1.18 and 0.85 μg/mL for Cdc25B and PTP1B, respectively, compared with reference drugs Na3VO4 (IC50=0.93 μg/mL) and oleanolic acid (IC50=0.85 μg/mL). The results of selectivity experiments showed that the target compounds were selective inhibitors against PTP1B and Cdc25B. Enzyme kinetic experiments demonstrated that compound 5k was a specific inhibitor with the typical characteristics of a mixed inhibitor., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Phosphoinositides are low abundance membrane phospholipids that have key roles in signaling, membrane trafficking, and cytoskeletal dynamics in all cells. Until recently, strategies for robust and quantitative development of pharmacological tools for manipulating phosphoinositide levels have focused selectively on PI(3,4,5)P3 due to the importance of this lipid in growth factor signaling and cell proliferation. However, drugs that affect levels of other phosphoinositides have potential therapeutic applications and will be powerful research tools. Here, we describe methodology for the high-throughput screening of small molecule modulators of the inositol 5-phosphatases, which dephosphorylate PI(4,5)P2 (the precursor for PI(3,4,5)P3) and PI(3,4,5)P3). We developed three complementary in vitro activity assays, tested hit compounds on a panel of 5-phosphatases, and monitored efficacy toward various substrates. Two prominent chemical scaffolds were identified with high nanomolar/low micromolar activity, with one class showing inhibitory activity toward all 5-phosphatases tested and the other selective activity toward OCRL and INPP5B, which are closely related to each other. One highly soluble OCRL/INPP5B-specific inhibitor shows a direct interaction with the catalytic domain of INPP5B. The efficacy of this compound in living cells was validated through its property to enhance actin nucleation at the cell cortex, a PI(4,5)P2 dependent process, and to inhibit PI(4,5)P2 dephosphorylation by OCRL (both overexpressed and endogenous enzyme). The assays and screening strategies described here are applicable to other phosphoinositide-metabolizing enzymes, at least several of which have major clinical relevance. Most importantly, this study identifies the first OCRL/INPP5B specific inhibitor and provides a platform for the design of more potent inhibitors of this family of enzymes.

Glutamine serves as an important source of energy and building blocks for many tumor cells. The first step in glutamine utilization is its conversion to glutamate by the mitochondrial enzyme glutaminase. CB-839 is a potent, selective, and orally bioavailable inhibitor of both splice variants of glutaminase (KGA and GAC). CB-839 had antiproliferative activity in a triple-negative breast cancer (TNBC) cell line, HCC-1806, that was associated with a marked decrease in glutamine consumption, glutamate production, oxygen consumption, and the steady-state levels of glutathione and several tricarboxylic acid cycle intermediates. In contrast, no antiproliferative activity was observed in an estrogen receptor-positive cell line, T47D, and only modest effects on glutamine consumption and downstream metabolites were observed. Across a panel of breast cancer cell lines, GAC protein expression and glutaminase activity were elevated in the majority of TNBC cell lines relative to receptor positive cells. Furthermore, the TNBC subtype displayed the greatest sensitivity to CB-839 treatment and this sensitivity was correlated with (i) dependence on extracellular glutamine for growth, (ii) intracellular glutamate and glutamine levels, and (iii) GAC (but not KGA) expression, a potential biomarker for sensitivity. CB-839 displayed significant antitumor activity in two xenograft models: as a single agent in a patient-derived TNBC model and in a basal like HER2(+) cell line model, JIMT-1, both as a single agent and in combination with paclitaxel. Together, these data provide a strong rationale for the clinical investigation of CB-839 as a targeted therapeutic in patients with TNBC and other glutamine-dependent tumors.

It is believed that glycogen synthase kinase-3 hyperphosphorylates tau protein in progressive supranuclear palsy (PSP). The Tau Restoration on PSP (TAUROS) trial assessed the glycogen synthase kinase-3 inhibitor tideglusib as potential treatment. For the magnetic resonance imaging (MRI) substudy reported here, we assessed the progression of brain atrophy. TAUROS was a multinational, phase 2, double-blind, placebo-controlled trial in patients with mild-to-moderate PSP who were treated with oral tideglusib (600 mg or 800 mg daily) or with placebo for 1 year. A subset of patients underwent baseline and 52-week MRI. Automated, observer-independent, atlas-based, and mask-based volumetry was done on high-resolution, T1-weighted, three-dimensional data. For primary outcomes, progression of atrophy was compared both globally (brain, cerebrum) and regionally (third ventricle, midbrain, pons) between the active and placebo groups (Bonferroni correction). For secondary outcomes, 15 additional brain structures were explored (Benjamini & Yekutieli correction). In total, MRIs from 37 patient were studied (placebo group, N = 9; tideglusib 600 mg group, N = 19; tideglusib 800 mg group, N = 9). The groups compared well in their demographic characteristics. Clinical results showed no effect of tideglusib over placebo. Progression of atrophy was significantly lower in the active group than in the placebo group for the brain (mean ± standard error of the mean: -1.3% ± 1.4% vs. -3.1% ± 2.3%, respectively), cerebrum (-1.3% ± 1.5% vs. -3.2% ± 2.1%, respectively), parietal lobe (-1.6% ± 1.9% vs. -4.1% ± 3.0%, respectively), and occipital lobe (-0.3% ± 1.8% vs. -2.7% ± 3.2%, respectively). A trend toward reduced atrophy also was observed in the frontal lobe, hippocampus, caudate nucleus, midbrain, and brainstem. In patients with PSP, tideglusib reduced the progression of atrophy in the whole brain, particularly in the parietal and occipital lobes., (© 2014 International Parkinson and Movement Disorder Society.)

It is believed that glycogen synthase kinase-3 (GSK-3) hyperphosphorylates tau protein in progressive supranuclear palsy (PSP). The Tau Restoration on PSP (TAUROS) study was a double-blind, placebo-controlled, randomized trial to assess the efficacy, safety, and tolerability of tideglusib, a GSK-3 inhibitor, as potential treatment for PSP. The study enrolled 146 PSP patients with mild-to-moderate disease who were randomized to receive once-daily 600 mg tideglusib, 800 mg tideglusib, or placebo (ratio, 2:2:1) administered orally over 52 weeks. The primary endpoint was the change from baseline to week 52 on the PSP rating scale. Secondary endpoints were safety and tolerability of tideglusib, changes in motor function (the Timed Up and Go Test), cognition (Dementia Rating Scale-2, Frontal Assessment Battery, verbal fluency), apathy (Starkstein scale), activities of daily living (Schwab and England scale; Unified Parkinson's Disease Rating Scale, part II), quality of life (EuroQol), and Global Clinical Assessment. Brain atrophy on magnetic resonance imaging and several biomarkers in plasma and cerebrospinal fluid also were examined. No significant differences were detected in the primary or secondary endpoints at week 52 between placebo and either dose of tideglusib. Tideglusib was safe, with the exception of some asymptomatic, transient, and reversible transaminase elevations (mainly alanine aminotransferase) in 9% of patients, and diarrhea in 13% of patients. Tideglusib was generally well tolerated but it did not show clinical efficacy in patients with mild-to-moderate PSP., (© 2014 International Parkinson and Movement Disorder Society.)

O-Perbenzoylated 4-phenyl-[C-(β-d-glucopyranosyl)formaldehyde]semicarbazone was prepared in the reaction of O-perbenzoylated β-d-glucopyranosyl cyanide and 4-phenylsemicarbazide in the presence of Raney-Ni. Acylation of O-perbenzoylated C-(β-d-glucopyranosyl)formaldehyde semicarbazone furnished the corresponding 4-acyl-[C-(β-d-glucopyranosyl)formaldehyde]semicarbazones. The reaction of O-perbenzoylated C-(β-d-glucopyranosyl)formaldehyde semicarbazone with the corresponding thiosemicarbazide resulted in O-perbenzoylated C-(β-d-glucopyranosyl)formaldehyde thiosemicarbazone and its 4-phenyl derivative. Acylation of O-perbenzoylated C-(β-d-glucopyranosyl)formaldehyde thiosemicarbazone provided the corresponding 4-acyl-2-acylamino-5-(β-d-glucopyranosyl)-Δ(2)-1,3,4-thiadiazolidines. Oxidative transformations of these precursors gave O-protected 2-(β-d-glucopyranosyl)-5-substituted-amino-1,3,4-oxa- and -thiadiazoles. The O-benzoyl protecting groups were removed under base-catalysed transesterification conditions. The C-glucopyranosyl heterocyclic compounds proved inactive against rabbit muscle glycogen phosphorylase b, however, the semicarbazones showed moderate inhibition (best inhibitor was 4-phenyl-[C-(β-d-glucopyranosyl)formaldehyde]semicarbazone (Ki=29μM)., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Human cells utilize a variety of complex DNA repair mechanisms in order to combat constant mutagenic and cytotoxic threats from both exogenous and endogenous sources. The RecQ family of DNA helicases, which includes Bloom helicase (BLM), plays an important function in DNA repair by unwinding complementary strands of duplex DNA as well as atypical DNA structures such as Holliday junctions. Mutations of the BLM gene can result in Bloom syndrome, an autosomal recessive disorder associated with cancer predisposition. BLM-deficient cells exhibit increased sensitivity to DNA damaging agents indicating that a selective BLM inhibitor could be useful in potentiating the anticancer activity of these agents. In this work, we describe the medicinal chemistry optimization of the hit molecule following a quantitative high-throughput screen of >355,000 compounds. These efforts lead to the identification of ML216 and related analogs, which possess potent BLM inhibition and exhibit selectivity over related helicases. Moreover, these compounds demonstrated cellular activity by inducing sister chromatid exchanges, a hallmark of Bloom syndrome., (Published by Elsevier Ltd.)

Context: Triosephosphate isomerase (TIM) is a ubiquitous enzyme that has been targeted for the discovery of small molecular weight compounds with potential use against Trypanosoma cruzi, the causative agent of Chagas disease. We have identified a new selective inhibitor chemotype of TIM from T. cruzi (TcTIM), 1,2,4-thiadiazol-5(4H)-one., Objective: Study the mechanism of TcTIM inhibition by a 1,2,4-thiadiazol derivative., Methods: We performed the biochemical characterization of the interaction of the 1,2,4-thiadiazol derivative with the wild-type and mutant TcTIMs, using DOSY-NMR and MS experiments. Studies of T. cruzi growth inhibition were additionally carried out., Results and Conclusion: At low micromolar concentrations, the compound induces highly selective irreversible inactivation of TcTIM through non-covalent binding. Our studies indicate that it interferes with the association of the two monomers of the dimeric enzyme. We also show that it inhibits T. cruzi growth in culture.

GGT (γ-glutamyl transpeptidase) is an essential enzyme for maintaining cysteine homoeostasis, leukotriene synthesis, metabolism of glutathione conjugates and catabolism of extracellular glutathione. Overexpression of GGT has been implicated in many pathologies, and clinical inhibitors of GGT are under development for use in the treatment of asthma, cancer and other diseases. Inhibitors are generally characterized using synthetic GGT substrates. The present study of uncompetitive inhibitors of GGT, has revealed that the potency with which compounds inhibit GGT activity in the standard biochemical assay does not correlate with the potency with which they inhibit the physiological reaction catalysed by GGT. Kinetic studies provided insight into the mechanism of inhibition. Modifications to the sulfobenzene or distal benzene ring of the uncompetitive inhibitor OU749 affected activity. One of the most potent inhibitors was identified among a novel group of analogues with an amine group para on the benzosulfonamide ring. New more potent uncompetitive inhibitors of the physiological GGT reaction were found to be less toxic than the glutamine analogues that have been tested clinically. Development of non-toxic inhibitors is essential for exploiting GGT as a therapeutic target.

Protein arginine methyltransferases (PRMTs) play an important role in diverse biological processes. Among the nine known human PRMTs, PRMT3 has been implicated in ribosomal biosynthesis via asymmetric dimethylation of the 40S ribosomal protein S2 and in cancer via interaction with the DAL-1 tumor suppressor protein. However, few selective inhibitors of PRMTs have been discovered. We recently disclosed the first selective PRMT3 inhibitor, which occupies a novel allosteric binding site and is noncompetitive with both the peptide substrate and cofactor. Here we report comprehensive structure-activity relationship studies of this series, which resulted in the discovery of multiple PRMT3 inhibitors with submicromolar potencies. An X-ray crystal structure of compound 14u in complex with PRMT3 confirmed that this inhibitor occupied the same allosteric binding site as our initial lead compound. These studies provide the first experimental evidence that potent and selective inhibitors can be created by exploiting the allosteric binding site of PRMT3.