Protein kinases control most biological processes by regulating nearly every signal transduction pathway. Aberrant kinase activity causes many diseases including cancer and autoimmunity. Therefore, kinases are attractive targets for the regulation of these diseases by small molecule inhibitors. Since kinase activity plays such a fundamental role in biology, it is vital to our understanding of cellular signaling to both measure and regulate cellular kinase activity. Here, I describe new chemical tools to measure kinase activity in cells. In addition, I discovered the mechanism allowing a new class of multi-targeted inhibitors to potently inhibit distantly related kinases.

Using structure-guided design, I developed a chemical genetic system capable of measuring cellular kinase activity. I synthesized a series of 6-acrylamido-4-anilinoquinazoline irreversible kinase inhibitors that potently and selectively target rationally designed kinases bearing two selectivity elements that are not found together in any wild-type kinase: an electrophile-targeted cysteine residue and a glycine gatekeeper residue. Cocrystal structures of two irreversible quinazoline inhibitors bound to either EGFR or engineered c-Src show covalent inhibitor binding to the targeted cysteine. Based on these structures, we developed a fluorescent derivative—an affinity probe—to report the fraction of kinase necessary for cellular signaling. Using this probe, I quantitated the relationship between EGFR stimulation by EGF and its downstream outputs—Akt and Erkl/2.

Kinase inhibitors hold great promise as anti-cancer therapeutics, but the first generation of selective kinase drugs have only modest clinical efficacy. To test whether inhibiting many kinases simultaneously overcome these modest effects, our lab developed a series of pyrazolopyrimidine inhibitors that inhibit both tyrosine kinases and phosphoinositide kinases in cancer cell lines. It was unclear how these inhibitors achieve dual-potency, since tyrosine kinases and phosphoinositide kinases share little sequence and structural similarity. By solving the cocrystal structures of these inhibitors bound to c-Src and comparing these structures to complexes of inhibitor-bound phophatidylinositol-3-OH kinase pilOγ, I uncovered striking structural elements that allow these unique inhibitors to bind to two distantly related kinase families.