This document summarizes the development of a series of 1,2,3,4-tetrahydroisoquinolin-1-ones as novel antagonists of G protein-coupled receptor 40 (GPR40). High-throughput screening identified initial hits that showed high GPR40 inhibition and low human liver microsomal clearance but high rat liver microsomal clearance. Optimization focused on reducing rat clearance while maintaining GPR40 potency. This led to the identification of compound 15i, which showed low rat in vitro and in vivo clearance, moderate GPR40 inhibition, and satisfactory pharmacokinetic parameters in rats.
Journal of natural products volume 64 issue, take -- triterpene saponins from...MỐc MOn
This document summarizes research on triterpene saponins isolated from Vietnamese ginseng (Panax vietnamensis) and their ability to protect liver cells. Two new dammarane-type saponins were isolated and identified, along with nine known saponins and one known sapogenin. One of the known saponins, majonoside R2, showed strong protective effects against chemically-induced liver cell death in mice. This demonstrates that the hepatoprotective properties of Vietnamese ginseng are due to dammarane saponins containing an ocotillol-type side chain, characteristic of this plant species.
This study compared the efficiency of 8 elutable affinity tags for purifying proteins from E. coli, yeast, Drosophila, and HeLa extracts. The tags included 2 protein tags (GST and MBP) and 6 peptide tags. Results showed the tags differed substantially in purity, yield, and cost. The HIS tag provided good yields but only moderate purity from E. coli extracts and poorer purification from other extracts. The Strep II tag appeared to be an excellent candidate overall due to producing high purity material in good yields at a moderate cost. The choice of tag depends on experimental requirements around yield, purity and cost.
The document describes using peptidomics to monitor protease inhibition in vivo by analyzing peptides as surrogates for protease activity. Peptidomics allows the comprehensive analysis of endogenous peptides from biological samples. The study demonstrates inhibiting different proteases in rats, including DPPIV and FXA, and analyzing changes in peptide levels. Several novel protease substrates were identified, including an ITM2B peptide for DPPIV. Collagen peptide levels were also strongly affected by DPPIV inhibition, indicating its importance in collagen metabolism. Peptidomics can thus non-invasively monitor protease activity and inhibition in vivo by analyzing surrogate peptide markers.
New Cayman Chemical Products - Sept 17th, 2013Cayman Chemical
This document summarizes new products introduced by Cayman Chemical from September 9-13, 2013. It describes several assay kits for studying bromodomain interactions, renal function, and catalase activity. Antibodies and recombinant proteins involved in DNA damage response and epigenetic regulation are also highlighted. A variety of natural products and inhibitors are noted including curcumin analogs, sPLA2 inhibitors, and receptor antagonists. New dyes, indicators, and forensic standards are briefly outlined.
This document summarizes the design, synthesis, and testing of novel non-covalent thrombin inhibitors featuring P3-heterocycles and P1-bicyclic arginine surrogates. A series of 44 inhibitors (NC1-NC44) were developed using a strategy that included: (1) optimizing P4-aromatic groups for potency and selectivity, (2) linking P4 to P3 heterocycles via short linkers, and (3) surveying diverse weakly basic bicyclic P1 groups to restore potency. Several inhibitors showed potent and selective thrombin inhibition, with some displaying good oral bioavailability in dogs.
This document summarizes the development of efficient protocols for synthesizing 1,2,3,4-tetrahydroisoquinolin-1-ones. Several methods were developed, including the use of Mitsunobu reactions, copper-catalyzed arylations, and SNAr reactions to install various substituents on the core scaffold. These methods proved to be versatile, efficient, and amenable to parallel synthesis, allowing for SAR exploration across different regions of the molecule.
This document describes the identification of a new series of potent histone deacetylase (HDAC) inhibitors. The researchers designed substituted 2-piperazinyl-5-pyrimidylhydroxamic acids using a multicomponent Petasis reaction to introduce chemical diversity. Compounds in the new series exhibited HDAC inhibitory activity in the low nanomolar range and inhibited tumor cell growth similarly. The new compounds showed improved solubility over previous generations, making them promising starting points for developing new HDAC inhibitor drugs. Stereochemistry and substitution of the phenyl ring had little effect on inhibitory potency. The increased solubility of the compounds represents an important improvement for drug development.
Journal of natural products volume 64 issue, take -- triterpene saponins from...MỐc MOn
This document summarizes research on triterpene saponins isolated from Vietnamese ginseng (Panax vietnamensis) and their ability to protect liver cells. Two new dammarane-type saponins were isolated and identified, along with nine known saponins and one known sapogenin. One of the known saponins, majonoside R2, showed strong protective effects against chemically-induced liver cell death in mice. This demonstrates that the hepatoprotective properties of Vietnamese ginseng are due to dammarane saponins containing an ocotillol-type side chain, characteristic of this plant species.
This study compared the efficiency of 8 elutable affinity tags for purifying proteins from E. coli, yeast, Drosophila, and HeLa extracts. The tags included 2 protein tags (GST and MBP) and 6 peptide tags. Results showed the tags differed substantially in purity, yield, and cost. The HIS tag provided good yields but only moderate purity from E. coli extracts and poorer purification from other extracts. The Strep II tag appeared to be an excellent candidate overall due to producing high purity material in good yields at a moderate cost. The choice of tag depends on experimental requirements around yield, purity and cost.
The document describes using peptidomics to monitor protease inhibition in vivo by analyzing peptides as surrogates for protease activity. Peptidomics allows the comprehensive analysis of endogenous peptides from biological samples. The study demonstrates inhibiting different proteases in rats, including DPPIV and FXA, and analyzing changes in peptide levels. Several novel protease substrates were identified, including an ITM2B peptide for DPPIV. Collagen peptide levels were also strongly affected by DPPIV inhibition, indicating its importance in collagen metabolism. Peptidomics can thus non-invasively monitor protease activity and inhibition in vivo by analyzing surrogate peptide markers.
New Cayman Chemical Products - Sept 17th, 2013Cayman Chemical
This document summarizes new products introduced by Cayman Chemical from September 9-13, 2013. It describes several assay kits for studying bromodomain interactions, renal function, and catalase activity. Antibodies and recombinant proteins involved in DNA damage response and epigenetic regulation are also highlighted. A variety of natural products and inhibitors are noted including curcumin analogs, sPLA2 inhibitors, and receptor antagonists. New dyes, indicators, and forensic standards are briefly outlined.
This document summarizes the design, synthesis, and testing of novel non-covalent thrombin inhibitors featuring P3-heterocycles and P1-bicyclic arginine surrogates. A series of 44 inhibitors (NC1-NC44) were developed using a strategy that included: (1) optimizing P4-aromatic groups for potency and selectivity, (2) linking P4 to P3 heterocycles via short linkers, and (3) surveying diverse weakly basic bicyclic P1 groups to restore potency. Several inhibitors showed potent and selective thrombin inhibition, with some displaying good oral bioavailability in dogs.
This document summarizes the development of efficient protocols for synthesizing 1,2,3,4-tetrahydroisoquinolin-1-ones. Several methods were developed, including the use of Mitsunobu reactions, copper-catalyzed arylations, and SNAr reactions to install various substituents on the core scaffold. These methods proved to be versatile, efficient, and amenable to parallel synthesis, allowing for SAR exploration across different regions of the molecule.
This document describes the identification of a new series of potent histone deacetylase (HDAC) inhibitors. The researchers designed substituted 2-piperazinyl-5-pyrimidylhydroxamic acids using a multicomponent Petasis reaction to introduce chemical diversity. Compounds in the new series exhibited HDAC inhibitory activity in the low nanomolar range and inhibited tumor cell growth similarly. The new compounds showed improved solubility over previous generations, making them promising starting points for developing new HDAC inhibitor drugs. Stereochemistry and substitution of the phenyl ring had little effect on inhibitory potency. The increased solubility of the compounds represents an important improvement for drug development.
Analyzing ligand and small molecule binding activity of solubilized myszkaJohannesdedooper
This document describes a study that used biosensor technology to analyze the binding activity of solubilized G protein-coupled receptors (GPCRs). Specifically, it analyzed the binding of natural ligands and small molecules to the chemokine receptors CXCR4 and CCR5. Both receptors were solubilized from cell pellets and captured on an antibody surface for analysis. The solubilized receptors maintained high-affinity binding of chemokines and allowed characterization of binding kinetics for novel small molecule inhibitors of CCR5. This demonstrated that the solubilized receptors retained native binding properties, making them useful for biophysical studies and structural analysis.
Recombinant proteins are manipulated forms of proteins produced in large quantities through genetic engineering techniques. The document discusses how recombinant DNA technology is used to modify gene sequences and produce proteins in specialized vectors. It provides several examples of recombinant human proteins that have replaced animal-derived versions in medicine, such as recombinant human insulin and growth hormone. The production of recombinant Bowman-Birk inhibitor in E. coli is described as a case study, outlining the cloning of the gene and expression of the recombinant protein. Common protein purification methods are also summarized, such as affinity chromatography, ion exchange chromatography, gel filtration, and centrifugation.
Human: Thank you for the summary. You captured the key details about recombinant proteins and provided relevant examples
1) The authors designed and synthesized fluorescent oligonucleotide and nucleotide substrates containing 30-(4-methylumbelliferone)-phosphate that are efficiently cleaved by the DNA repair enzyme tyrosyl-DNA phosphodiesterase 1 (Tdp1).
2) Cleavage of these substrates by Tdp1 generates the fluorescent 4-methylumbelliferone molecule, allowing kinetic analysis of Tdp1 activity through fluorescence detection.
3) Kinetic characterization of Tdp1 using the new fluorescent substrates yielded values similar to those obtained using standard gel-based assays and previous oligonucleotide substrates, validating this new high-throughput fluorescent assay for studying Tdp1 function.
This document describes a thesis project on the heterologous expression, purification, and characterization of cinnamoyl coenzyme A reductase (CCR) from Leucaena leucocephala. CCR plays a key role in lignin biosynthesis in plants by catalyzing the reduction of hydroxycinnamoyl-CoA thioesters to the corresponding aldehydes. The goal of the project is to better understand the structure and function of CCR to determine how altering its expression affects lignin content and quality in plants, which has applications for the paper industry by enabling more efficient delignification. The CCR gene from L. leucocephala was cloned into expression vectors and expressed in E. coli.
This study describes the structure-based design and evaluation of novel peptide inhibitors of thrombin-induced platelet aggregation. Researchers designed tetrapeptide analogs of a lead thrombin inhibitor by replacing the P3 residue with various unnatural amino acids like D-3,3-di-phenylalanine. All inhibitors competitively inhibited thrombin and improved inhibition of platelet aggregation. Compounds containing D-3,3-di-phenylalanine at P3 completely inhibited thrombin-induced platelet aggregation at concentrations of 300-60 nM. These novel thrombin inhibitors could be used as scaffolds to develop treatments for acute coronary syndrome by inhibiting thrombin-mediated platelet aggregation.
This document describes the purification and characterization of a trypanothione-glutathione thioltransferase enzyme from Trypanosoma cruzi. Key findings include:
1) A 52 kDa protein was purified from T. cruzi using affinity chromatography columns containing either S-hexylglutathione or a trypanothione disulfide analogue as ligands.
2) Partial amino acid sequencing showed this protein is identical to one encoded by the previously described TcAc2 cDNA.
3) While it does not have significant glutathione or trypanothione transferase activity, it was found to catalyze thiol-disulfide exchange between dihydrotrypanothione and glutathione dis
This document summarizes a study that evaluated the effects of an extract from Aphanizomenon flos-aquae (AFA) enriched for a novel ligand for human L-selectin on stem cell physiology. The study found that AFA contains a ligand composed of two proteins that binds to L-selectin. It inhibits the fucoidan-induced expression of CXCR4 on bone marrow stem cells, indicating it blocks L-selectin. Consumption of an AFA extract enriched in the ligand resulted in a transient 25% increase in circulating stem cells in humans. The extract has potential as a stem cell mobilizer by modulating the CXCR4/SDF-1 axis through L-selectin
This study investigated the role of the amino and carboxyl terminal regions of cytosolic serine hydroxymethyltransferase (SHMT) in subunit assembly and catalysis. Six N-terminal and two C-terminal deletion mutants were constructed from a full-length SHMT cDNA clone and expressed in E. coli. The two shortest N-terminal deletion mutants (lacking the first 6 and 14 residues) were purified and found to be catalytically active, but the 14-residue mutant had decreased thermal stability compared to the full enzyme. The 14-residue mutant also predominantly formed dimers rather than tetramers and had reduced ability to reconstitute activity after removal of the cofactor. These results demonstrate that the N-terminal region plays
This document describes a new method for quantifying poly(3-hydroxybutyrate) (PHB) in microbial cells using headspace solid-phase microextraction (SPME) coupled with gas chromatography. The method involves either methanolyzing or hydrolyzing PHB in samples to form methyl 3-hydroxybutyrate (Me-3-HB) or crotonic acid, respectively. These products are then extracted using SPME and analyzed by gas chromatography. The new SPME-based methods provide accurate results, are easier to perform than existing methods, and avoid use of hazardous chlorinated solvents. The document compares the new methods to the commonly used methanolysis/chloroform method and finds excellent agreement between all
Daily changes in the phosphoproteome of the dinoflagellate LingulodiniumCIMMYT
This document summarizes a study that analyzed changes in protein phosphorylation in the dinoflagellate Lingulodinium polyedra over a daily light-dark cycle using two approaches. Two proteins, Rad24 and a light harvesting complex protein, were found to have higher phosphorylation at night and may be involved in DNA repair and balancing energy transfer between photosystems, respectively. A total of 45 phosphorylated proteins were identified, some relating to RNA binding and polyketide synthesis. The study provides insight into biochemical changes underlying rhythmic behavior in Lingulodinium.
This study evaluated the effects of an extract from Aphanizomenon flos-aquae (AFA) containing a novel ligand for human L-selectin on stem cell physiology. Methods showed AFA contains a ligand composed of two proteins that binds to L-selectin. In vitro, AFA reduced L-selectin antibody binding and inhibited fucoidan-induced CXCR4 expression on stem cells, indicating it is an L-selectin blocker. In vivo, consumption of an AFA extract enriched in the ligand resulted in a transient 25% increase in circulating stem cells within 60 minutes, returning to baseline by 3-4 hours later.
Proteomic Strategies for purification of lactate dehydrogenase ...Gaurav Dwivedi
The document summarizes proteomic strategies for purifying lactate dehydrogenase (LDH) from chicken muscle. Key steps included homogenizing muscle tissue, removing debris via centrifugation, precipitating proteins with ammonium sulfate, dialyzing to remove salts, and purifying LDH using blue sepharose affinity chromatography. SDS-PAGE and activity assays confirmed the isolation of a single 35 kDa protein band corresponding to LDH. The multi-step process successfully extracted and purified LDH from chicken muscle for further study.
This document describes the design, synthesis, and evaluation of 4-[(4-cyano-2-arylbenzyloxy)-(3-methyl-3H-imidazol-4-yl)methyl]benzonitriles as potent and selective farnesyltransferase (FTase) inhibitors. A previous compound, A315493, was a potent FTase inhibitor but also inhibited geranylgeranyltransferase-I (GGTase-I). The authors designed new compounds by moving the naphthyl group of A315493 to improve selectivity. Compound 16 was found to have improved selectivity while maintaining potency. Further structure-activity relationship studies led to the discovery of compound 64
Poster demonstrating the results from the development/verification project for the quantitation of pyridoxal 5-phosphate and 4-pyridoxic acid in human plasma.
Society of Toxicology Presentation Annual Meeting 2011lothargoretzki
The document describes a multiplex assay called the MIILIPLEX MAG Human OXPHOS 6-Plex Panel that enables the simultaneous detection of 5 oxidative phosphorylation complexes and nicotinamide nucleotide transhydrogenase as a mechanistic indicator for drug-induced mitochondrial toxicity. The assay was able to detect mitochondrial toxicity in HepG2 cells induced by various drug classes including antibiotics, antiviral drugs, and anti-diabetes drugs by showing reductions in specific complexes. The data from the assay correlated with established complex activity and oxygen consumption assays, demonstrating it is a suitable novel safety screening tool to identify off-target effects on mitochondrial respiration during drug development.
The document summarizes characterization of the H134C mutant of the Thermus thermophilus Rieske protein. Key findings include:
1) The H134C mutant substitutes one ligating histidine for a cysteine, changing the iron-sulfur cluster ligation from 2Cys-2His to 3Cys-1His.
2) Studies using UV-visible spectroscopy, circular dichroism, and reaction with diethyl pyrocarbonate (DEPC) show the H134C mutant is stable across a wide pH range, unlike the similar mitochondrial protein mitoNEET.
3) Reaction of H134C with DEPC results in modification of the cluster but no reduction,
My Doctoral Thesis, titled "Advanced Oxidation Processes applied to mineralize Paracetamol, Chloroxylenol, Ibuprofen and Diclofenac in aqueous medium".
KM409 and KM456 are allosteric modulators of the serotonin transporter hSERT that were investigated. KM456 is a partial, non-competitive inhibitor of hSERT, suggesting it binds to an allosteric site. KM409 has a biphasic effect on hSERT, activating it at low concentrations but inhibiting at higher concentrations. A mutation at residue E493D disrupted KM456's inhibitory effect, indicating its importance for allosteric binding. A biotinylation assay was developed to study the interaction of KM409 and KM456 with hSERT residues, which could provide insight into their therapeutic potential for mental health disorders.
1) Researchers developed anthranilic acid sulfonamides as inhibitors of methionine aminopeptidase-2 (MetAP2), a potential cancer therapy target. Initial compounds had micromolar affinity for MetAP2 but also extensively bound to human serum albumin (HSA), limiting cellular activity.
2) Using protein crystal structures of compounds bound to MetAP2 and HSA, researchers designed modifications to reduce HSA binding by adding a positively charged tertiary amine to the compounds. This reduced the HSA shift in potency while maintaining MetAP2 inhibition.
3) Various linkers connecting the amine to the core structure were evaluated. Compounds with an alkenyl linker
The children in the classroom set up a pretend restaurant called the "Spicy Food Restaurant" where they explored different roles like cooks, waiters, and customers. They served pretend spicy foods like sausages and pancakes. The children then made signs, menus, and props to enhance their role playing. They also visited a real cafeteria to observe different roles and setups before making and serving real pancakes in their restaurant.
Nikita Zaytsev was a defender for the Siberia hockey team who was born in Moscow in 1991 and studied hockey in a youth club. From 2009 to 2013, Nikita protected the honor of the Siberian team and became captain at the end of the 2013 season. In 2011, Nikita became world champion as a member of the Russian national youth hockey team. In 2013, Nikita began playing for the CSKA hockey club, which was a loss for his previous team Siberia.
Analyzing ligand and small molecule binding activity of solubilized myszkaJohannesdedooper
This document describes a study that used biosensor technology to analyze the binding activity of solubilized G protein-coupled receptors (GPCRs). Specifically, it analyzed the binding of natural ligands and small molecules to the chemokine receptors CXCR4 and CCR5. Both receptors were solubilized from cell pellets and captured on an antibody surface for analysis. The solubilized receptors maintained high-affinity binding of chemokines and allowed characterization of binding kinetics for novel small molecule inhibitors of CCR5. This demonstrated that the solubilized receptors retained native binding properties, making them useful for biophysical studies and structural analysis.
Recombinant proteins are manipulated forms of proteins produced in large quantities through genetic engineering techniques. The document discusses how recombinant DNA technology is used to modify gene sequences and produce proteins in specialized vectors. It provides several examples of recombinant human proteins that have replaced animal-derived versions in medicine, such as recombinant human insulin and growth hormone. The production of recombinant Bowman-Birk inhibitor in E. coli is described as a case study, outlining the cloning of the gene and expression of the recombinant protein. Common protein purification methods are also summarized, such as affinity chromatography, ion exchange chromatography, gel filtration, and centrifugation.
Human: Thank you for the summary. You captured the key details about recombinant proteins and provided relevant examples
1) The authors designed and synthesized fluorescent oligonucleotide and nucleotide substrates containing 30-(4-methylumbelliferone)-phosphate that are efficiently cleaved by the DNA repair enzyme tyrosyl-DNA phosphodiesterase 1 (Tdp1).
2) Cleavage of these substrates by Tdp1 generates the fluorescent 4-methylumbelliferone molecule, allowing kinetic analysis of Tdp1 activity through fluorescence detection.
3) Kinetic characterization of Tdp1 using the new fluorescent substrates yielded values similar to those obtained using standard gel-based assays and previous oligonucleotide substrates, validating this new high-throughput fluorescent assay for studying Tdp1 function.
This document describes a thesis project on the heterologous expression, purification, and characterization of cinnamoyl coenzyme A reductase (CCR) from Leucaena leucocephala. CCR plays a key role in lignin biosynthesis in plants by catalyzing the reduction of hydroxycinnamoyl-CoA thioesters to the corresponding aldehydes. The goal of the project is to better understand the structure and function of CCR to determine how altering its expression affects lignin content and quality in plants, which has applications for the paper industry by enabling more efficient delignification. The CCR gene from L. leucocephala was cloned into expression vectors and expressed in E. coli.
This study describes the structure-based design and evaluation of novel peptide inhibitors of thrombin-induced platelet aggregation. Researchers designed tetrapeptide analogs of a lead thrombin inhibitor by replacing the P3 residue with various unnatural amino acids like D-3,3-di-phenylalanine. All inhibitors competitively inhibited thrombin and improved inhibition of platelet aggregation. Compounds containing D-3,3-di-phenylalanine at P3 completely inhibited thrombin-induced platelet aggregation at concentrations of 300-60 nM. These novel thrombin inhibitors could be used as scaffolds to develop treatments for acute coronary syndrome by inhibiting thrombin-mediated platelet aggregation.
This document describes the purification and characterization of a trypanothione-glutathione thioltransferase enzyme from Trypanosoma cruzi. Key findings include:
1) A 52 kDa protein was purified from T. cruzi using affinity chromatography columns containing either S-hexylglutathione or a trypanothione disulfide analogue as ligands.
2) Partial amino acid sequencing showed this protein is identical to one encoded by the previously described TcAc2 cDNA.
3) While it does not have significant glutathione or trypanothione transferase activity, it was found to catalyze thiol-disulfide exchange between dihydrotrypanothione and glutathione dis
This document summarizes a study that evaluated the effects of an extract from Aphanizomenon flos-aquae (AFA) enriched for a novel ligand for human L-selectin on stem cell physiology. The study found that AFA contains a ligand composed of two proteins that binds to L-selectin. It inhibits the fucoidan-induced expression of CXCR4 on bone marrow stem cells, indicating it blocks L-selectin. Consumption of an AFA extract enriched in the ligand resulted in a transient 25% increase in circulating stem cells in humans. The extract has potential as a stem cell mobilizer by modulating the CXCR4/SDF-1 axis through L-selectin
This study investigated the role of the amino and carboxyl terminal regions of cytosolic serine hydroxymethyltransferase (SHMT) in subunit assembly and catalysis. Six N-terminal and two C-terminal deletion mutants were constructed from a full-length SHMT cDNA clone and expressed in E. coli. The two shortest N-terminal deletion mutants (lacking the first 6 and 14 residues) were purified and found to be catalytically active, but the 14-residue mutant had decreased thermal stability compared to the full enzyme. The 14-residue mutant also predominantly formed dimers rather than tetramers and had reduced ability to reconstitute activity after removal of the cofactor. These results demonstrate that the N-terminal region plays
This document describes a new method for quantifying poly(3-hydroxybutyrate) (PHB) in microbial cells using headspace solid-phase microextraction (SPME) coupled with gas chromatography. The method involves either methanolyzing or hydrolyzing PHB in samples to form methyl 3-hydroxybutyrate (Me-3-HB) or crotonic acid, respectively. These products are then extracted using SPME and analyzed by gas chromatography. The new SPME-based methods provide accurate results, are easier to perform than existing methods, and avoid use of hazardous chlorinated solvents. The document compares the new methods to the commonly used methanolysis/chloroform method and finds excellent agreement between all
Daily changes in the phosphoproteome of the dinoflagellate LingulodiniumCIMMYT
This document summarizes a study that analyzed changes in protein phosphorylation in the dinoflagellate Lingulodinium polyedra over a daily light-dark cycle using two approaches. Two proteins, Rad24 and a light harvesting complex protein, were found to have higher phosphorylation at night and may be involved in DNA repair and balancing energy transfer between photosystems, respectively. A total of 45 phosphorylated proteins were identified, some relating to RNA binding and polyketide synthesis. The study provides insight into biochemical changes underlying rhythmic behavior in Lingulodinium.
This study evaluated the effects of an extract from Aphanizomenon flos-aquae (AFA) containing a novel ligand for human L-selectin on stem cell physiology. Methods showed AFA contains a ligand composed of two proteins that binds to L-selectin. In vitro, AFA reduced L-selectin antibody binding and inhibited fucoidan-induced CXCR4 expression on stem cells, indicating it is an L-selectin blocker. In vivo, consumption of an AFA extract enriched in the ligand resulted in a transient 25% increase in circulating stem cells within 60 minutes, returning to baseline by 3-4 hours later.
Proteomic Strategies for purification of lactate dehydrogenase ...Gaurav Dwivedi
The document summarizes proteomic strategies for purifying lactate dehydrogenase (LDH) from chicken muscle. Key steps included homogenizing muscle tissue, removing debris via centrifugation, precipitating proteins with ammonium sulfate, dialyzing to remove salts, and purifying LDH using blue sepharose affinity chromatography. SDS-PAGE and activity assays confirmed the isolation of a single 35 kDa protein band corresponding to LDH. The multi-step process successfully extracted and purified LDH from chicken muscle for further study.
This document describes the design, synthesis, and evaluation of 4-[(4-cyano-2-arylbenzyloxy)-(3-methyl-3H-imidazol-4-yl)methyl]benzonitriles as potent and selective farnesyltransferase (FTase) inhibitors. A previous compound, A315493, was a potent FTase inhibitor but also inhibited geranylgeranyltransferase-I (GGTase-I). The authors designed new compounds by moving the naphthyl group of A315493 to improve selectivity. Compound 16 was found to have improved selectivity while maintaining potency. Further structure-activity relationship studies led to the discovery of compound 64
Poster demonstrating the results from the development/verification project for the quantitation of pyridoxal 5-phosphate and 4-pyridoxic acid in human plasma.
Society of Toxicology Presentation Annual Meeting 2011lothargoretzki
The document describes a multiplex assay called the MIILIPLEX MAG Human OXPHOS 6-Plex Panel that enables the simultaneous detection of 5 oxidative phosphorylation complexes and nicotinamide nucleotide transhydrogenase as a mechanistic indicator for drug-induced mitochondrial toxicity. The assay was able to detect mitochondrial toxicity in HepG2 cells induced by various drug classes including antibiotics, antiviral drugs, and anti-diabetes drugs by showing reductions in specific complexes. The data from the assay correlated with established complex activity and oxygen consumption assays, demonstrating it is a suitable novel safety screening tool to identify off-target effects on mitochondrial respiration during drug development.
The document summarizes characterization of the H134C mutant of the Thermus thermophilus Rieske protein. Key findings include:
1) The H134C mutant substitutes one ligating histidine for a cysteine, changing the iron-sulfur cluster ligation from 2Cys-2His to 3Cys-1His.
2) Studies using UV-visible spectroscopy, circular dichroism, and reaction with diethyl pyrocarbonate (DEPC) show the H134C mutant is stable across a wide pH range, unlike the similar mitochondrial protein mitoNEET.
3) Reaction of H134C with DEPC results in modification of the cluster but no reduction,
My Doctoral Thesis, titled "Advanced Oxidation Processes applied to mineralize Paracetamol, Chloroxylenol, Ibuprofen and Diclofenac in aqueous medium".
KM409 and KM456 are allosteric modulators of the serotonin transporter hSERT that were investigated. KM456 is a partial, non-competitive inhibitor of hSERT, suggesting it binds to an allosteric site. KM409 has a biphasic effect on hSERT, activating it at low concentrations but inhibiting at higher concentrations. A mutation at residue E493D disrupted KM456's inhibitory effect, indicating its importance for allosteric binding. A biotinylation assay was developed to study the interaction of KM409 and KM456 with hSERT residues, which could provide insight into their therapeutic potential for mental health disorders.
1) Researchers developed anthranilic acid sulfonamides as inhibitors of methionine aminopeptidase-2 (MetAP2), a potential cancer therapy target. Initial compounds had micromolar affinity for MetAP2 but also extensively bound to human serum albumin (HSA), limiting cellular activity.
2) Using protein crystal structures of compounds bound to MetAP2 and HSA, researchers designed modifications to reduce HSA binding by adding a positively charged tertiary amine to the compounds. This reduced the HSA shift in potency while maintaining MetAP2 inhibition.
3) Various linkers connecting the amine to the core structure were evaluated. Compounds with an alkenyl linker
The children in the classroom set up a pretend restaurant called the "Spicy Food Restaurant" where they explored different roles like cooks, waiters, and customers. They served pretend spicy foods like sausages and pancakes. The children then made signs, menus, and props to enhance their role playing. They also visited a real cafeteria to observe different roles and setups before making and serving real pancakes in their restaurant.
Nikita Zaytsev was a defender for the Siberia hockey team who was born in Moscow in 1991 and studied hockey in a youth club. From 2009 to 2013, Nikita protected the honor of the Siberian team and became captain at the end of the 2013 season. In 2011, Nikita became world champion as a member of the Russian national youth hockey team. In 2013, Nikita began playing for the CSKA hockey club, which was a loss for his previous team Siberia.
The document summarizes an extra low profile remote-controlled dozer called the MVD XLP. It was designed for uses like underground mining, construction, and military missions. It is a small, low profile machine that is controlled remotely by an operator and can assist with tasks like cleaning rock slopes after blasting. It can work continuously in difficult conditions and handle 50-120 tons of ore per hour, increasing productivity and safety. Testing has shown it performs well in real mine conditions.
The document discusses the benefits of exercise for mental health. Regular physical activity can help reduce anxiety and depression and improve mood and cognitive function. Exercise causes chemical changes in the brain that may help protect against mental illness and improve symptoms.
This document summarizes the key aspects of School 21, a free school in London for students aged 4-18. It discusses the school's core values of integrity and humanity. It also outlines 6 attributes to prepare students for the 21st century: small class sizes, a focus on language and oracy, collaboration between primary and secondary, technology integration, authentic assessment, and real-world learning. The school aims to close achievement gaps and create a student-centered, innovative approach through its curriculum, teaching, and collaboration between staff. It discusses moving beyond "best practices" to develop each student and create a supportive community.
This document summarizes various marketing campaigns conducted on the Tvidi.ru website between 2010-2011. It describes contests, games, polls, and branded pages created in partnership with Cartoon Network, Nivea, IKEA, Justice clothing, Ambre Solaire sunscreen, MTS mobile provider, Disney movies, and Orbit gum. The campaigns aimed to promote brands and media to children through interactive content on the website.
This document summarizes the development of efficient protocols for the synthesis of 1,2,3,4-tetrahydroisoquinolin-1-ones. Specifically, it describes:
1) A concise 5-step synthesis of a key phenol intermediate from 4-benzyloxybenzaldehyde.
2) Three parallel arrays utilizing the phenol intermediate to explore structure-activity relationships, including a Mitsunobu reaction, copper-catalyzed arylation, and nucleophilic aromatic substitution.
3) The need to further optimize the key 5-step synthesis to enable more diverse SAR exploration of the chemical series.
A series of (2R)-2-ethylchromane-2-carboxylic acid derivatives were synthesized and evaluated for their PPARγ and PPARα agonist activities with the goal of discovering a novel dual agonist. A structure-activity relationship was developed that led to the identification of compound 48 as a potent, selective, and structurally novel PPARγ/α dual agonist. Compound 48 showed substantial antihyperglycemic and hypolipidemic effects in animal models of type 2 diabetes and dyslipidemia.
This document summarizes a study that investigated the effects of o-phenylenediamine (oPD) exposure on biochemical parameters in the liver and brain of zebrafish. Zebrafish were exposed to 1ppm and 5ppm of oPD for 15 days. Biological oxygen demand tests found moderate pollution in water treated with both concentrations of oPD. Exposure resulted in increased liver oxidative stress and alterations in liver detoxification enzymes and brain monoamine oxidase activity, suggesting oPD toxicity affects multiple organ systems. The study aims to understand the chronic effects and toxicity mechanism of oPD in aquatic animals like zebrafish.
The document discusses using the Burmese python as a model to study lipid homeostasis. After eating, pythons show a doubling in size of major organs and a 52-fold increase in triglycerides without signs of lipotoxicity. This suggests a more efficient mechanism of lipid homeostasis than humans. The nuclear receptor PPARα is identified as a master regulator of lipid homeostasis and is the gene of interest. Methods are described to isolate RNA from python liver, synthesize cDNA, validate and test primers for PPARα and other genes, and perform quantitative real-time PCR to analyze gene expression levels at different time points after feeding.
This study investigated the role of glycerol biosynthesis in lifespan extension from hyperosmotic conditions in C. elegans. The authors found that both 10x peptone and 5% sorbitol conditions increased wild type C. elegans lifespan by around 35%, but this lifespan extension was absent in gpdh-1;gpdh-2 mutant worms, which are unable to accumulate glycerol. Future experiments will determine if overexpressing glycerol biosynthesis is sufficient to extend lifespan without osmotic stress. This supports the role of glycerol accumulation, mediated by glycerol 3-phosphate dehydrogenase, in the longevity response to hyperosmotic stress in C. elegans.
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2. Author's personal copy
Bioorganic & Medicinal Chemistry Letters 19 (2009) 2400–2403
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and SAR of 1,2,3,4-tetrahydroisoquinolin-1-ones as novel
G-protein-coupled receptor 40 (GPR40) antagonists
Paul S. Humphries *, John W. Benbow, Paul D. Bonin, David Boyer, Shawn D. Doran, Richard K. Frisbie,
David W. Piotrowski, Gayatri Balan, Bruce M. Bechle, Edward L. Conn, Kenneth J. Dirico,
Robert M. Oliver, Walter C. Soeller, James A. Southers, Xiaojing Yang
Pfizer Global R&D, Eastern Point Road, Groton, CT 06340, USA
a r t i c l e i n f o a b s t r a c t
Article history: The development of a series of novel 1,2,3,4-tetrahydroisoquinolin-1-ones as antagonists of G protein-
Received 4 February 2009 coupled receptor 40 (GPR40) is described. The synthesis, in vitro inhibitory values for GPR40, in vitro
Revised 16 March 2009 microsomal clearance and rat in vivo clearance data are discussed. Initial hits displayed high rat
Accepted 20 March 2009
in vivo clearances that were higher than liver blood flow. Optimization of rat in vivo clearance was
Available online 25 March 2009
achieved and led to the identification of 15i, whose rat oral pharmacokinetic data is reported.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Diabetes
GPR40
Tetrahydroisoquinolinones
Ligand efficiency
Type 2 diabetes (T2D), the most commonly occurring form of
diabetes, is a condition in which the body resists the insulin that
is produced by the pancreas and may fail to make enough insulin
to maintain normal glucose levels.1 The incidence of T2D has in-
creased worldwide in recent years, largely because of growing
rates of obesity, and is likely to grow to greater than 366 million
by the year 2030.2
One of the recently characterized G-protein-coupled receptor
(GPCR) families is the GPR40–43 family,3 comprising GPR40, 41
and 43.4 These three family members share $ 30–40% sequence Figure 1. Initial hits from high-throughput screening.
identity. Three independent groups have identified GPR40 as a
fied compounds 1 and 2 (Fig. 1) as antagonists of GPR40. These hits
receptor for medium- (C6–C12) and long-chain (C14–C24) fatty
were extremely attractive based on their high ligand efficiency,8
acids (FAs).5 GPR40 is preferentially expressed in the pancreas with
low human liver microsomal (HLM) clearance and promising selec-
elevated levels reported in the islets and also in the pancreatic
tivity in an initial selectivity panel. The above properties motivated
b-cell lines.6 GPR40-deficient b-cells secrete less insulin in
an initiation of hit-to-lead chemistry to explore the activity of this
response to FAs, and loss of GPR40 protects mice from obesity-in-
class of compounds as GPR40 antagonists.
duced hyperinsulinemia, increased hepatic glucose output,
Initial efforts involved exploration of the SAR of the terminal
yperglycemia and glucose intolerance.7 Conversely, overexpression
cyclohexyl and phenyl moieties of 1 and 2. In order to do this in
of GPR40 in b-cells of mice leads to impaired cell function, hypoin-
an efficient manner, a concise synthesis of phenol intermediate 7
sulinemia and diabetes. These results suggest that GPR40 plays a
was required. Intermediate 7 could then be utilized for late stage
critical role in linking obesity and T2D.
diversification and efficient SAR exploration of this region of the
Efforts toward the identification of 1,2,3,4-tetrahydroisoquino-
molecule.
lin-1-ones as novel GPR40 antagonists are reported here. A high-
Phenol 7 was accessed in a straightforward fashion following
throughput screen (HTS) of the Pfizer compound collection identi-
the five step protocol below (Scheme 1). 4-Benzyloxybenzaldehyde
3 was condensed with propylamine to afford imine 4 in excellent
* Corresponding author. Tel.: +1 860 705 0559. yield. Treatment of imine 4 with homophthalic anhydride resulted
E-mail address: phumphri@gmail.com (P.S. Humphries). in the formation of cis-lactam 5 in moderate yield along with minor
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.082
3. Author's personal copy
P. S. Humphries et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2400–2403 2401
Table 1
GPR40 activity, human and rat liver microsome data for antagonists 1, 2 and 8–10
Compound R/Ar/HetAr GPR40 IC50 HLM Cl (lL/ RLM Cl (lL/
(nM)a min/kg) min/kg)
1 — 10 <12 274
2 — 12 <9 97
8a Me 2100 <8 <14
8b CH2Ph 96 <8 <16
9a 3,5-Di-Me–Ph 2 21 46
9b 3,4-Di-Me–Ph 3 <8 <14
10a 29 <8 <14
a
Values are means of two or more independent experiments.
0
Scheme 1. Reagents and conditions: (a) C3H7NH2, 4 Å MS, CH2Cl2, rt, 16 h, 100%; (b)
A dent in vitro clearance of this series was required, while maintain-
homophthalic anhydride, CH3CN, 60 °C, 16 h, 50%; (c) AcOH, 120 °C, 16 h, 95%; (d) ing our GPR40 potency and excellent human in vitro clearance.
MeI, K2CO3, Me2CO, rt, 16 h, 90%; (e) BBr3, CH2Cl2, rt, 16 h, 90%. Significant reduction in MW (e.g., 8a) resulted in a concomitant
drop-off in potency, but interestingly this compound possessed
low rat microsomal clearance. Benzyl substitution (e.g., 8b) re-
sulted in a compound with moderate GPR40 activity and low hu-
man and rat in vitro clearance. Biaryl ethers (e.g., 9b and 10a)
also provided excellent potency and in vitro clearance.
Compound 8b was then selected for rat pharmacokinetic (PK)
studies.15 Administration to male Sprague–Dawley (SD) rats sur-
prisingly resulted in iv clearance that was greater than liver blood
flow (82.8 mL/min/kg). In order to see whether this high rat iv
clearance held true across the series, a number of other compounds
were tested and all possessed high rat in vivo clearance.
In an effort to better understand the reason for the higher than
predicted in vivo rat clearance, four compounds were cassette
Table 2
Rat in vivo clearance and biliary excretion results
Structure Rat iv Cl (mL/min/kg) Percent dose in bile (%)
111 13.9
Scheme 2. Reagents and conditions: (a) ROH, PPh3, DIAD, THF, DMF, 80 °C, 16 h. (b) 0
LiOH, THF, MeOH, H2O, rt, 16 h; (c) ArB(OH)2, Et3N, Cu(OAc)2, Py, 1,2-DCE, DMF, 4 ÅA
MS, air, 50 °C, 16 h; (d) 2-Br-HetAr, CuI (cat.), ligand 1 from ref.14 (cat.), K3PO4,
MeCN, 100 °C, 16 h.
84.5 22.9
amounts of trans-lactam 6.9 The thermodynamically less stable iso-
mer 5 could be epimerized under acidic conditions, resulting in the
exclusive formation of trans-lactam 6 in excellent yield.10 Esterifi-
136 69.9
cation of acid 6 with iodomethane afforded the intermediate
methyl ester, which was subsequently debenzylated utilizing bor-
on tribromide to yield the required phenol intermediate 7.11
Phenol 7 underwent a Mitsunobu reaction, followed by saponi-
fication, to afford required products 8 in a parallel fashion (Scheme
2).12 Diaryl ethers 9 were also accessed from phenol 7 utilizing ele- 32.7 64.6
gant methodology developed by Evans.13 SNAr reaction of phenol 7
with 2-bromoheteroaryls yielded products 10 via a copper-cata-
lyzed Ullman-type coupling.14
All the compounds were tested in a GPR40 FLIPR calcium mobi-
lization assay.5 Simultaneously, compounds were screened in hu-
man (HLM) and rat liver microsome (RLM) assays. The effect of
different terminal substitution (e.g., replacements for cyclohexyl
and phenyl moieties in 1 and 2) was studied first, and these results
are summarized in Table 1. Both compounds 1 and 2 have good
GPR40 activity and low human microsomal clearance, but high
rat microsomal clearance. Knowing that the desired pre-clinical Scheme 3. Reagents and conditions: (a) MeOH, rt, 16 h then DMF, homophthalic
diabetic model study would be in a rat, improvements in the ro- anhydride, rt, 16 h then 1 N aq NaOH, rt, 16 h.
4. Author's personal copy
2402 P. S. Humphries et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2400–2403
Table 3 Table 4
GPR40 activity and ligand efficiency for antagonists 2 and 15 GPR40 activity and ligand efficiency for antagonists 15
Compound R1 R2 GPR40 IC50 (nM)a RLM Cl Compound R1 R2 GPR40 IC50 (nM)a RLM Cl
(lL/min/kg) (lL/min/kg)
2 4-PhO$-Ph Et 12 97
15a 4-PhO-Ph Me 156 27 15f 3,4-di-Cl–Ph 13 <14
15b 4-PhO-Ph CH2(c-C3H5) 4 120
15c 3,4-Di-Cl–Ph C3H7 89 <14 15g 3,4-di-Cl–Ph CH2CH(Et)2 10 27
15d 3-Cl–Ph C3H7 228 <14
15h CH2CH(Et)2 10 <18
15e C3H7 119 18
15i 20 <14
a
Values are means of two or more independent experiments.
a
Values are means of two or more independent experiments.
dosed in a bile duct cannulated (BDC) rat study (Table 2).15 A wide
variation in the percentage of dose found in the bile (even with
compounds that possessed low rat in vitro clearance and good
structurally similar compounds) was observed, which may indicate
GPR40 activity (Table 4).
that (i) biliary excretion is not a series-wide issue, and (ii) this set
Based on good GPR40 activity, good in vitro permeability (CaCo-
of compounds suffers from high in vivo clearance for multiple
2 AB = 12.0 and BA = 18.7 Â 10À6 cm/sec), human in vitro clearance
reasons.
(HLM Cl < 8.0 lL/min/mg) and rat in vitro clearance (RLM
In parallel to the above rat BDC study, one representative com-
Cl < 14.1 lL/min/mg) 15i was selected for rat PK studies.15 Admin-
pound, 12, was chosen for a dog iv PK study in order to assess
istration to male SD rats resulted in satisfactory PK parameters – iv
whether this was a rodent specific phenomenon.15 Acid 12 dis-
clearance = 4.3 mL/min/kg, Vd = 1.1 L/kg and iv half-life of 5.4 h.
played low dog microsomal clearance (DLM Cl = 2.99 lL/min/mg)
In summary, a novel series of 1,2,3,4-tetrahydroisoquinolin-1-
and low dog in vivo clearance (7.4 mL/min/kg). The fact still re-
ones have been identified as antagonists of GPR40. Initial hits dis-
mained that we required access to a tool GPR40 antagonist that
played high rat in vivo clearances that were higher than liver blood
displayed low rat in vivo clearance and thus needed to break into
flow. Optimization of rat in vivo clearance was achieved and led to
different chemical space.
the identification of 15i, which showed satisfactory PK parameters.
Readily accessible regions of the molecule were varied in paral-
lel to modulate metabolism and reduce clearance, while retaining
potency. In order to achieve this, optimization of the chemistry Acknowledgments
in Scheme 1 was required in order to allow for it to be utilized in
a number of parallel arrays (Scheme 3). Two of the most immediate The authors gratefully acknowledge all our colleagues in the
challenges were the solubility of the reagents/products in the GPR40 program for their technical support in the evaluation of
existing solvents and the requirement for Dean-Stark conditions the compounds presented in this manuscript. We also thank Yue
on formation of the imine (e.g., 4) Solutions to these challenges in- Chen for the rat BDC study and Bruce Lefker and Joseph Warmus
volved utilizing methanol as the solvent of choice for imine forma- for stimulating discussions and feedback on this manuscript.
tion. The poor solubility of homophthalic anhydride necessitated
the use of DMF as the solvent for the formation of the cis- and References and notes
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