Journal: Journal of Medicinal Chemistry

Article Title: Discovery of 2-(3-Benzamidopropanamido)thiazole-5-carboxylate Inhibitors of the Kinesin HSET (KIFC1) and the Development of Cellular Target Engagement Probes

doi: 10.1021/acs.jmedchem.2c01591

Figure Lengend Snippet: (A) Images showing the distribution of Cy5-induced fluorescence from tetrazine-Cy5 (Txz-Cy5) with and without the addition of TCO probe 40 compared to the HSET intensities identified by indirect immunofluorescence using an Alexa-488 fluorophore in DLD1 4N and DLD1 HSET KO cell lines. The overlay shows the mitotic pole areas identified by staining for pericentrin (red), the HSET intensity as measured by indirect immunofluorescence using an Alexa-488 fluorophore (green) and the nucleus stained with DAPI (blue). (B) Images showing the effects on the fluorescence of increasing the concentration of 36 , which outcompeted the TCO probe 40 . (C) Plot showing the decreasing Cy5 signal intensity measured at the mitotic pole areas (defined by pericentrin staining), normalized to % control, with the increasing concentration of 36 . (D) Plot showing the increasing HSET 488 signal measured at the mitotic pole areas (defined by pericentrin staining), normalized to % control, with the increasing concentration of 36 . (E) Plot showing the ratio of the corrected click probe Cy5 signal intensity (subtracting KO cell signal) the over total HSET protein 488 signal, normalized to % control, with the increasing concentration of 36 .

Article Snippet: Therefore, to correctly depict the competitive effect of increasing concentrations of 36 , we plotted the ratio of the corrected click probe Cy5 signal intensity (subtracting KO cell signal) over the total HSET protein 488 signal, which was normalized to % control ( Figure E) The tetrazine-Cy5 dye alone did not produce any signal in either HSET-expressing or HSET-nonexpressing cells, while minimal localization of 40 to the mitotic spindle was observed in HSET-nonexpressing cells ( Figure A).

Techniques: Fluorescence, Immunofluorescence, Staining, Concentration Assay