Extended Data Fig. 6: Covalent binding of AB1 to TbCLK1.
From: Targeting the trypanosome kinetochore with CLK1 protein kinase inhibitors
a, Summary of thermal shift assay of TbCLK1 and TbCLK1 C215A proteins incubated with DMSO, AB0 or AB1 compounds. Data represents mean ± standard deviation (n = 3 technical replicates) for △Tm. Note the significant increase in Tm for AB1 compound bound to CLK1 but not CLK1 C215A. The Tm was calculated by fitting the fluorescence vs. temperature data to the Boltzmann equation and represents the point where half the protein is unfolded. b, Mass spectrum chromatograms of TbCLK1 (left) and TbCLK1 C215A proteins (right) incubated with DMSO (control), AB1 or AB2 compounds. Y-axis and X-axis represent the counts and deconvoluted Mass (amu) respectively. Calculated differences in base mass and the difference in mass after treatment is shown in the table. Note the increase in mass of 475 Da only for AB1 treatment with TbCLK1 protein, which corresponds to the mass of AB1. No increase in the mass was seen for DMSO or AB2 in both CLK1 and CLK1 C215A protein. Treatment of CLK1 C215A with AB1 also did not result in an increase in mass, clearly showing covalent interaction of the Michael acceptor with C215 of TbCLK1 (n = 1).
