Interfering with protein interaction of phosphoinositide-3-kinase gamma and of store-operated calcium channels
Zusammenfassung der Projektergebnisse
PI3-kinase γ-mediated generation of phosphatidylinositol 3,4,5-trisphosphate (PIP3) triggers a recruitment of signalling proteins to the plasma membrane. Taking advantage of a fluorescent fusion protein of a PIP3-binding PH domain (GRP1(PH)-GFP), we set up a high-content analysis to screen the Spectrum Collection (2,000 drugs and biologically active compounds) and the ChemBionet Collection (16,670 chemically diverse drug-like compounds) for inhibitors of PI3-kinase γ-mediated signalling. After validation of primary hits, we focused on 5 compounds that displayed a reasonable potency of PI3 kinase γ inhibition (IC50 = 6-50 µM). However, none of them acted in an isotype-specific manner, indicating that inhibition either relies on an ATP-competitive or otherwise poorly specific mechanism that is unrelated to an interference with protein-protein interactions. We then focused the project on cation channels that are regulated by mechanisms that result in the depletion of intracellular Ca2+ stores. Whereas interference with the Stim1:ORAI1-mediated Ca2+ entry pathway was not achieved, identification of novel modulators of canonical TRP channels was successful. In brief: screening the Spectrum Collection, the ChemBionet collection, or plant secondary metabolites revealed a number of potent and isotype-prevalent or -selective TRP channel modulators. Further hit optimisation was achieved in the case of TRPC6- and TRPM3-inhibiting compounds, and corresponding biological activities of the inhibitors were verified at the organ level (TRPC6) or in vivo (TRPM3). These modulators are valuable tools to acutely modulate and thereby probe for TRP channel-related responses or to develop experimental therapies.
Projektbezogene Publikationen (Auswahl)
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(2012) HaCaT keratinocytes exhibit a cholesterol and plasma membrane viscosity gradient during directed migration. Exp Cell Res., 318, 809-818
Klein A.S., Schaefer M., Korte T., Herrmann A., Tannert A.
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(2012). Novel pharmacological TRPC inhibitors block hypoxia-induced vasoconstriction. Cell Calcium, 51, 194-206
Urban N., Hill K., Wang L., Kuebler W.M., Schaefer M.
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(2013). Citrus fruit and fabacea secondary metabolites potently and selectively block TRPM3. Br J Pharmacol., 168, 1835-1850
Straub I., Mohr F., Stab J., Konrad M., Philipp S.E., Oberwinkler J., Schaefer M.
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(2013). Flavanones that selectively inhibit TRPM3 attenuate thermal nociception in vivo. Mol Pharmacol., 84, 736-750
Straub I., Krügel U., Mohr F., Teichert J., Rizun O., Konrad M., Oberwinkler J., Schaefer M.
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(2014) Cholesterol sensitises the transient receptor potential channel TRPV3 to lower temperatures and activator concentrations. Cell Calcium, 55, 59-68
Klein A.S., Tannert A., Schaefer M.
