scholarly journals Assessment of direct binding interaction between CD36 and its potential lipid ligands using a peptide mimic of the receptor labeled with a fluorophore

2021 ◽  
Vol 42 (5) ◽  
pp. 181-191
Author(s):  
Satoshi TSUZUKI ◽  
Yusaku KIMOTO ◽  
Masayuki YAMASAKI ◽  
Tatsuya SUGAWARA ◽  
Yuki MANABE ◽  
...  
Keyword(s):  
Binding Interaction ◽  
Peptide Mimic ◽  
Direct Binding

scholarly journals Xanthine-Based Photoaffinity Probes Allow Assessment of Ligand Engagement by TRPC5 Channels

2020 ◽  
Author(s):  
Claudia Bauer ◽  
Aisling Minard ◽  
Isabelle Pickles ◽  
Matthew Burnham ◽  
Nikil Kapur ◽  
...  
Keyword(s):  
Small Molecule ◽  
Drug Targets ◽  
Direct Evidence ◽  
Binding Mode ◽  
Chemical Probes ◽  
Binding Interaction ◽  
Biological Studies ◽  
Photoaffinity Probes ◽  
Direct Binding ◽  
Trpc5 Channels

TRPC1/4/5 cation channels are emerging drug targets for the treatment of, amongst others, central nervous system (CNS) disorders, kidney disease, and cardiovascular and metabolic disease. Various small-molecule TRPC1/4/5 modulators have been reported, including highly potent xanthine derivatives that can distinguish between specific TRPC1/4/5 tetramers. However, there is a paucity of information about their binding mode, which limits the ability to develop them further as chemical probes of specific TRPC1/4/5 channels for use in fundamental biological studies and drug discovery programmes. Here, we report the development of a set of potent xanthine-based photoaffinity probes that functionally mimic the xanthines Pico145 and AM237, respectively. Using these probes, we have developed a quantitative photoaffinity labelling protocol for TRPC5 channels. Our results provide the first direct evidence that xanthines modulate TRPC5 channels through a direct binding interaction with TRPC5 protein, and the first quantitative method for the assessment of binding interactions of TRPC5 and small molecules. Our method may allow the study of the mode-of-action of other TRPC1/4/5 modulators, and the identification of small molecule binding sites of TRPC1/4/5 channels.

scholarly journals Xanthine-Based Photoaffinity Probes Allow Assessment of Ligand Engagement by TRPC5 Channels

2020 ◽  
Author(s):  
Claudia Bauer ◽  
Aisling Minard ◽  
Isabelle Pickles ◽  
Matthew Burnham ◽  
Nikil Kapur ◽  
...  
Keyword(s):  
Small Molecule ◽  
Drug Targets ◽  
Direct Evidence ◽  
Binding Mode ◽  
Chemical Probes ◽  
Binding Interaction ◽  
Biological Studies ◽  
Photoaffinity Probes ◽  
Direct Binding ◽  
Trpc5 Channels

TRPC1/4/5 cation channels are emerging drug targets for the treatment of, amongst others, central nervous system (CNS) disorders, kidney disease, and cardiovascular and metabolic disease. Various small-molecule TRPC1/4/5 modulators have been reported, including highly potent xanthine derivatives that can distinguish between specific TRPC1/4/5 tetramers. However, there is a paucity of information about their binding mode, which limits the ability to develop them further as chemical probes of specific TRPC1/4/5 channels for use in fundamental biological studies and drug discovery programmes. Here, we report the development of a set of potent xanthine-based photoaffinity probes that functionally mimic the xanthines Pico145 and AM237, respectively. Using these probes, we have developed a quantitative photoaffinity labelling protocol for TRPC5 channels. Our results provide the first direct evidence that xanthines modulate TRPC5 channels through a direct binding interaction with TRPC5 protein, and the first quantitative method for the assessment of binding interactions of TRPC5 and small molecules. Our method may allow the study of the mode-of-action of other TRPC1/4/5 modulators, and the identification of small molecule binding sites of TRPC1/4/5 channels.

scholarly journals PIP4Ks Suppress Insulin Signaling Through a Catalytic-Independent Mechanism

2018 ◽  
Author(s):  
Diana G. Wang ◽  
Marcia N. Paddock ◽  
Mark R. Lundquist ◽  
Janet Y. Sun ◽  
Oksana Mashadova ◽  
...  
Keyword(s):  
Insulin Signaling ◽  
Pi3k Pathway ◽  
Binding Interaction ◽  
Subsequent Increase ◽  
Catalytic Function ◽  
Insulin Dependent ◽  
Direct Binding ◽  
Novel Strategy ◽  
Phosphatidylinositol 4

SUMMARYInsulin stimulates conversion of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) to phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3), which mediates downstream cellular responses. PI(4,5)P2 is produced by phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks) and byphosphatidylinositol-5-phosphate 4-kinases (PIP4Ks). Here we show that deletion of the three genes that encode PIP4Ks (PIP4K2A, PIP4K2B and PIP4K2C) in vitro results in a paradoxical increase in PI(4,5)P2 and a subsequent increase in insulin-stimulated production of PI(3,4,5)P3. Surprisingly, reintroduction of either wild-type or kinase-dead forms of the PIP4Ks restored cellular PI(4,5)P2 levels and insulin stimulation of the PI3K pathway. These effects are explained by an increase in PIP5K activity upon deletion of PIP4Ks, which we demonstrate can suppress PIP5K activity in vitro through a direct binding interaction. Collectively, our work reveals an important non-catalytic function of PIP4Ks in suppressing PIP5K-mediated PI(4,5)P2 synthesis and insulin-dependent conversion to PI(3,4,5)P3 by PI3K enzymes and suggests that pharmacological depletion of PIP4K enzymes using emerging degrader technologies could represent a novel strategy for stimulating insulin signaling.

Prothrombin Residues 473 to 487 Contribute To Factor Va Binding in the Prothrombinase Complex.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1720-1720
Author(s):  
Subramanian Yegneswaran ◽  
Rolf M. Mesters ◽  
Jose A. Fernandez ◽  
John H. Griffin
Keyword(s):  
Binding Sites ◽  
Synthetic Peptides ◽  
Spectroscopic Data ◽  
Active Sites ◽  
Inhibitory Peptide ◽  
Binding Interaction ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Direct Binding ◽  
Prothrombinase Activity

Abstract To identify sequences in prothrombin (fII) involved in assembly of the prothrombinase complex (fXa:factor Va:fII:phospholipids), synthetic peptides based on fII sequences were prepared and screened for their ability to inhibit fXa-induced clotting of normal plasma. The fII peptide comprising residues 473–487 (designated PT473–487 that are homologous to chymotrypsin residues 149D-163) potently inhibited plasma clotting assays and prothrombinase activity with 50% inhibition observed at 12 microM and 10 microM peptide, respectively. Prothrombinase inhibition by PT473–487 was fVa-dependent and sequence-specific since the peptide did not inhibit fII activation in the absence of fVa and a scrambled sequence peptide, PT473–487SCR, was not inhibitory. Peptide PT473–487 also inhibited cleavage at Arg271 in meizo-thrombin, showing that it similarly inhibited fXa cleavage at both Arg320 and Arg271. Peptide PT473–487 did not inhibit the amidolytic activities of fXa or thrombin, suggesting that the peptide did not alter the integrity of their active sites. To determine if PT473–487 interacted directly with fVa, fluorescein labeled-fVa (Fl-fVa) was prepared. When PT473–487 was titrated into samples containing phospholipid-bound Fl-fVa, the peptide increased fluorescein anisotropy (EC50 at 3 microM peptide) whereas the control peptide PT473–487SCR peptide did not alter the anisotropy, suggesting a direct binding interaction between PT473–487 and Fl-fVa. These functional and spectroscopic data suggest that fII residues 473–487 provide fVa binding sites and mediate interactions between fVa and fII in the prothrombinase complex that contribute to cleavages at both Arg271 and Arg320 by fXa.

scholarly journals Identification of PDE6D as a potential target of sorafenib via PROTAC technology

2020 ◽  
Author(s):  
Yang Li ◽  
Qingshi Meng ◽  
Ping Wang ◽  
Xiaolei Liu ◽  
Qiuyu Fu ◽  
...  
Keyword(s):  
Mechanism Of Action ◽  
Kinase Inhibitor ◽  
Target Identification ◽  
Binding Interaction ◽  
Cellular Targets ◽  
Potential Target ◽  
Cellular Target ◽  
Anti Cancer ◽  
Different Types ◽  
Direct Binding

AbstractThe identification of unknown target of a multi-kinase inhibitor sorafenib is important to better understand the mechanism of action of this drug in anti-cancer and anti-fibrotic treatments. Here, we report the combination of PROTAC technique with quantitative proteomic analysis to identify the unknown cellular targets of sorafenib. Sorafenib-based PROTAC can strongly degrade a non-kinase target PDEδ in different types of cells. We also confirmed the direct binding interaction of PDEδ with sorafenib by CETSA and SPR assays. Together, our research suggests that PDEδ is a new potential target of sorafenib, and PROTAC technology may be a promising approach for cellular target identification of bioactive compounds of interest.

scholarly journals Xanthine-Based Photoaffinity Probes Allow Assessment of Ligand Engagement by TRPC5 Channels

2020 ◽  
Author(s):  
Claudia Bauer ◽  
Aisling Minard ◽  
Isabelle Pickles ◽  
Matthew Burnham ◽  
Nikil Kapur ◽  
...  
Keyword(s):  
Small Molecule ◽  
Drug Targets ◽  
Direct Evidence ◽  
Binding Mode ◽  
Chemical Probes ◽  
Binding Interaction ◽  
Biological Studies ◽  
Photoaffinity Probes ◽  
Direct Binding ◽  
Trpc5 Channels

TRPC1/4/5 cation channels are emerging drug targets for the treatment of, amongst others, central nervous system (CNS) disorders, kidney disease, and cardiovascular and metabolic disease. Various small-molecule TRPC1/4/5 modulators have been reported, including highly potent xanthine derivatives that can distinguish between specific TRPC1/4/5 tetramers. However, there is a paucity of information about their binding mode, which limits the ability to develop them further as chemical probes of specific TRPC1/4/5 channels for use in fundamental biological studies and drug discovery programmes. Here, we report the development of a set of potent xanthine-based photoaffinity probes that functionally mimic the xanthines Pico145 and AM237, respectively. Using these probes, we have developed a quantitative photoaffinity labelling protocol for TRPC5 channels. Our results provide the first direct evidence that xanthines modulate TRPC5 channels through a direct binding interaction with TRPC5 protein, and the first quantitative method for the assessment of binding interactions of TRPC5 and small molecules. Our method may allow the study of the mode-of-action of other TRPC1/4/5 modulators, and the identification of small molecule binding sites of TRPC1/4/5 channels.

scholarly journals A systematic analysis of gene-gene interaction in multiple sclerosis

2020 ◽  
Author(s):  
Lotfi Slim ◽  
Clément Chatelain ◽  
Hélène de Foucauld ◽  
Chloé-Agathe Azencott
Keyword(s):  
Multiple Sclerosis ◽  
Association Studies ◽  
Gene Interaction ◽  
Genome Wide Association Studies ◽  
Binding Interaction ◽  
Systematic Analysis ◽  
Gene Pairs ◽  
Epistasis Analysis ◽  
Multiple Sclerosis Disease ◽  
Direct Binding

AbstractMultiple sclerosis is a complex autoimmune disease which genetic basis has been extensively investigated through genome wide association studies. So far, the conducted studies have detected a number of loci independently associated with the disease but few have investigated the interaction between distant loci, or epistasis. In the present work, we perform a gene level epistasis analysis of multiple sclerosis GWAS from the Wellcome Trust Case Control Consortium 2. We systematically study the epistatic interactions between all pairs of genes within 19 multiple sclerosis disease maps from the MetaCore pathway database. We report 4 gene pairs with epistasis involving missense variants, and 117 gene pairs with epistasis mediated by eQTLs. Our epistasis analysis is able to retrieve known interactions linked to multiple sclerosis: direct binding interaction between GLI-I and SUFU, involved in oligodendrocyte precursor cells differentiation, and regulation of IP10 transcription by NF-κB, thus validating the potential of epistasis analysis to reveal biological interaction with relevance in a disease specific context.

Calcium and the Fc Receptor on Human Platelets

1987 ◽  
Vol 57 (03) ◽  
pp. 298-301
Author(s):  
William F Clark ◽  
Gerald J M Tevaarwerk ◽  
Bruce D Reid ◽  
Suzanne Hall ◽  
Anita Caveney ◽  
...  
Keyword(s):  
Specific Binding ◽  
Normal Subjects ◽  
Human Platelets ◽  
Fc Receptor ◽  
Normal Male ◽  
Scatchard Analysis ◽  
Normal Female ◽  
Apparent Difference ◽  
Binding Data ◽  
Direct Binding

SummaryWe have described the calcium dependence of the IgG Fc receptor (Fc-R) on human platelets by analyzing the direct binding of radiolabelled Fc fragments, monomers and dimers of IgG. Specific binding to platelets was undetectable at 37° C in a calcium-free preparation but readily detected when calcium was restored. Scatchard analysis of the binding data for the calcium-restored platelets permitted calculation of the available Fc-R and the Ka of binding for the different IgG ligands. The mean Ka of binding for 12 normal subjects varied from 107 to 108 L/M, with an equal receptor number measured by Fc fragments and dimers of IgG, but a lesser amount for monomeric IgG. There was no apparent difference in Fc-R number for platelets from 6 normal male versus 6 normal female subjects.At 4° C binding was detectable for dimers and polymers of IgG in a calcium-free preparation and this was markedly increased with recalcification. Thus, our data are consistent with an Fc receptor population on human platelets whose avidity for binding is significantly enhanced in a calcium-restored medium.

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