Fluorescently Or Spin-Labeled Kinases For Rapid Screening And Identification Of Novel Kinase Inhibitor Scaffolds

A method of generating a mutated kinase suitable for the screening of kinase inhibitors comprising

• (a) replacing solvent exposed amino acids having a free thiol or amino group, if any, present in a kinase of interest outside the activation loop or amino acids having a free thiol or amino group at an unsuitable position within the activation loop with an amino acid not having a free thiol or amino group;
• (b) mutating an amino acid in the activation loop of said kinase of interest to an amino acid having a free thiol or amino group if no amino acid having a free thiol or amino group is present in the activation loop;
• (c) labeling the kinase of interest with a thiol- or amino-reactive fluorophore sensitive to polarity changes in its environment, a thiol-reactive spin label, an isotope or an isotope-enriched thiol- or amino-reactive label such that said fluorophore, spin label, isotope or isotope-enriched label does not inhibit the catalytic activity of the kinase and/or does not interfere with the stability of the kinase;
• (d) contacting the kinase obtained in step (c) with a known inhibitor of said kinase;
• and
• (e) recording the fluorescence emission signal at one or more wavelengths or a spectrum of said fluorescently labeled kinase of step (c) and (d) upon excitation or
• (e)′ recording the EPR or NMR spectra of said spin-labeled kinase of step (c) and (d);
• (f) comparing the fluorescence emission spectra recorded in step (e) or the EPR or NMR spectra recorded in step (e)′;
• wherein a difference in the fluorescence intensity at least one wavelength, preferably at the emission maximum, and/or a shift in the fluorescence emission wavelength in the spectra of said fluorescently labeled kinase obtained in step (e), or an alteration in the EPR or NMR spectra of said spin-labeled or isotope-labeled kinase obtained in step (e)′ indicates that the kinase is suitable for the screening for kinase inhibitors.