Development of Lysine-Targeted Probes for Protein Kinases

Abstract

Lysine is one of the most common amino acids in the proteome, but relatively few probes have been designed to label specific lysine. Properties of lysine such as its low intrinsic nucleophilicity and its ubiquity has made it a challenge to develop probes that label active site lysine in protein kinases. In this thesis, I describe the design, synthesis, and testing of three different lysine-targeted type I kinases probes based on different scaffolds. Two of these probes are fluorosulfates (Probe 1 and Probe 2) displayed from either a quinazoline or pyridine-pyrimidine scaffold. The third (Probe 3) is a sulfonyl fluoride probe with a trans- cyclooctyne (TCO) click handle, which is based on the previously developed probe XO44 probe. Based on lysate labeling experiments and sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, neither alkyne-containing nor TCO-containing versions of Probe 1 and Probe 2 are able to specifically label protein kinases. Lysate labelingexperiments followed by liquid chromatography tandem mass spectrometry (LC/MS/MS) demonstrate that Probe 3 can specifically label at least 7 protein kinases. Together, these results represent a comprehensive analysis of lysine-targeted type I kinases probes. The supplementary spreadsheets include the whole proteomic data for Probe 3 LC/MS/MS pull-down experiment (‘Proteomic data for Probe 3’); the protein kinases with non-significant label free intensities difference between the ‘Probe 3’ group and the ‘Probe 3 + Competitor 2’ group (‘Non-significant difference PKs’); the protein kinases only contain one label free intensity in the ‘Probe 3’ group (‘Non-representative PKs’); the protein kinases labeled by Probe 3 (‘Labeled PKs’).