Kulesskiy, Evgeny

University of Helsinki (Finland)



Development of small molecule inhibitors of the endocytic cytoskeleton


Clathrin-mediated endocytosis (CME) is the primary cellular route of cell surface receptors uptake, which in turn regulate the strength and specificity of downstream signalling. Changes in CME have been linked to increasing of cancer cell survival, proliferation and migration. FCHSD2 protein is a key activator of actin polymerization during CME. Importantly, it has been linked to chemotheraphy resistance and other diseases such as diabetes. FCHSD2 is recruited to clathrin-coated pits (CCPs) by intersectin via an SH3-SH3 interaction. Such interaction might be an interesting therapeutic target for inhibition of cancer cell metastasis and chemoresistance. Here, we are presenting two biochemical assays for a compound screening for a molecule that can break the interaction between FCHSD2 and Intersectin. The first assay is based on a split Nanoluc-luciferase where the second SH3 domain of FCHSD2 (F2S2) and the fourth SH3 domain of Intersectin (ITSd) were fused to Large and Small Nanoluc fragments respectively. After initial screens of Specs Consortium collection (30 000 compounds) and MicroSource Spectrum library (2 000 compounds), we found 148 hits which showed significant inhibition of luminescent signal. To exclude compounds that directly affect on Nanoluc luminescence a second screen was performed using the split-FAST tag fluorescent labelling system. This confirmatory screen yield 9 compounds that will be followed up for their specificity, mechanism of inhibition and cellular effects.

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