Understanding the Enzyme-inhibitor Interaction within the Substrate Pocket of Protein Tyrosine Kinases.

Protein phosphorylation occurs through enzymatic transfer of the gamma-phosphate of adenosine triphosphate (ATP) to the hydroxyl moieties of the amino acid residues serine, threonine, and tyrosine. This post-translational modification is catalyzed by a group of enzymes classified as protein kinases. The phosphotransfer event initiates signal transduction cascades that are instrumental in the regulation of diverse cell functions. Aberrant protein kinase activity has been associated with various disease states. Because of this, academic as well as pharmaceutical efforts have been aimed at the understanding of kinase activity and understanding the related disease states. The work described herein focuses specifically on the development and evaluation of protein tyrosine kinase inhibitors in hopes to glean further knowledge regarding the substrate-binding region of the protein tyrosine kinase, c-Src. Chapter 2 will discuss the use of a privileged scaffold, the biphenyl moiety, to screen for potential substrate-competitive inhibitors of the protein tyrosine kinase, c-Src. A 300 member library of biphenyl-containing compounds was synthesized and evaluated. From this study one compound, BiPH 65, emerged as a low micromolar affinity, substrate-competitive inhibitor of c-Src (IC50: 60.4 micromolar). Thorough evaluation of the compound revealed inhibitory activity not only against the kinase domain of the protein, but also against the physiologically relevant three domain c-Src (IC50: 17.2 micromolar), as well as the clinically relevant T338M c-Src (IC50: 26.4 micromolar). Chapter 3 examines the use of an inhibitor peptide to arrive at truncated,tetrafluorinated peptidomimetic small molecule inhibitors of c-Src. Although initially promising, these compounds were eventually found to bind competitively with ATP. Lastly, Chapter 4 details the study performed using a purchased fragment library. This fragment library was screened with hopes to elucidate a new scaffold on which to further optimize and design substrate-competitive inhibitors. However, we were happily surprised to find a compound that data suggest binds allosterically to the protein tyrosine kinase, c-Src. Although the lead compound, [1], does not inhibit cell growth of a human colon carcinoma cell line, compound [1c], the methyl ester analog of [1], inhibits cell growth of a human breast cancer cell line (GI50: 23.5 micromolar).PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/99913/1/smsantos_1.pd