Photoactive molecular probes for protein kinases: Development of selective photoligands for lympocyte specific kinase (Lck)
This thesis describes the development of a chemical approach for selective labeling of protein kinases, regardless of their chemical reactivities, using photoactive molecular probes. Selectivity toward target proteins was attained based on the well-known fact that two modest-affinity ligands, when tethered through an appropriate linker, can make a bidentate ligand with high affinity and selectivity. Our probes were designed as bidentate ligands containing adenine, which targets the catalytic site of protein kinases, and benzophenone, which targets hydrophobic pockets in the vicinity of the catalytic site. In addition to its role as a recognition unit, benzophenone serves as a photocrosslinker to covalently label target proteins. Binding selectivity studies revealed that our probes, despite their simple structure, selectively tag certain proteins out of crude protein mixtures. In addition, it was shown that probe's selectivity can be modulated via modifications of the probe's chirality as well as the distance between adenine and benzophenone.
Our methodology was successfully employed for the development of a library of photoactive probes for selective tagging of Src-family of kinases. During this study, we identified a selective photoactive probe for Lck, a Src-family kinase involved in lymphocyte proliferation and differentiation. This probe exhibited selectivity toward Lck both in a mixture of commercial kinases and in lysate from Jurkat cells, and was shown to interact with Lck through both its adenine and benzophenone units. Structural motifs crucial for the recognition by Lck were identified as the chirality of the probe's peptidic backbone as well as the distance between adenine and benzophenone and their locations within the probe. The probe-Lck complex was characterized by a series of photolabeling and mass spectrometric analyses, which showed that the benzophenone covalently binds to Leu384 residue located near the catalytic site of Lck. A structural model of probe-Lck complex was subsequently obtained and utilized for structure-affinity relationship studies. These studies resulted in improved labeling of Lck and revealed the importance of linker flexibility for efficient labeling by our probe. Further studies are required to fully characterize the affinity and selectivity of our probes and understand the recognition process in probe-Lck complex.
0490: Organic chemistry