Affinity selection-mass spectrometry (AS-MS) screening of kinesin spindle protein (KSP) followed by enzyme inhibition studies and temperature-dependent circular dichroism (TdCD) characterization was utilized to identify a series of benzimidazole compounds. This series also binds in the presence of Ispinesib, a known anticancer KSP inhibitor in phase I/II clinical trials for breast cancer. TdCD and AS-MS analyses support simultaneous binding implying existence of a novel non-Ispinesib binding pocket within KSP.
View Article and Find Full Text PDFMEK1 is a member of the MAPK signal transduction pathway that responds to growth factors and cytokines. We have determined that the kinase domain spans residues 35-382 by proteolytic cleavage. The complete kinase domain has been crystallized and its X-ray crystal structure as a complex with magnesium and ATP-gammaS determined at 2.
View Article and Find Full Text PDFMEK1 is a member of the MAPK signal transduction pathway that responds to growth factors and cytokines. A wealth of information about the enzymatic activity of MEK1, its domain functions, and inhibitor action is available; however, the thermodynamic properties of the interaction between MEK1 and ligands, such as nucleotides and non-ATP-competitive inhibitors, have not been reported. This study describes the thermodynamic parameters for the binding interactions of MEK1, nucleotides, and non-ATP-competitive inhibitor complexes using temperature-dependent circular dichroism (TdCD) and isothermal titration calorimetry (ITC).
View Article and Find Full Text PDFKinases exist in either a high or low activity form depending on the phosphorylation state of the activating lip. These two different forms of the same kinase may adopt different conformations that affect not only activity but also inhibitor binding and the ability to crystallize the protein. Therefore, isolation of homogenous preparations of the phosphorylated and non-phosphorylated versions of a kinase is critical for accurate biophysical measurements of activity, stability and ligand binding as well as for protein crystallization.
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