Eukaryotic protein kinases are one of the most important classes of human proteins, and a great deal of research has focused on the development of small molecule inhibitors as biological probes for the determination of their cellular function or as therapeutics for the treatment of disease, such as cancer. The need for new selective inhibitors and a better understanding of the selectivities of existing small molecules is readily apparent. Towards the goal of better understanding protein kinases and the molecules that inhibit them, I have developed a split-protein-based approach for the investigation of these kinase-small molecule interactions. Employing split-firefly luciferase as a reporter domain, we engineered a three-hybrid system capable of determining kinase inhibition through competitive interactions between an active site-directed ligand and a small molecule of interest. This method measures luciferase activity as a function of ligand binding, as opposed to the more traditional assays which quantify kinase activity directly, and alleviates the laborious process of protein purification. The model kinase PKA and the promiscuous ligand staurosporine were used in an initial test case to successfully validate the general design principles of our assay. The modular nature inherent to the assay's design enabled us to adapt it to roughly 300 additional protein kinases and two different ligands. We were able to establish a protocol for rapidly ascertaining the inhibition of a kinase by a library of 80 commercially available kinase inhibitors in a 96-well, high-throughput format. This protocol was then systematically applied to the AGC group of kinases to observe patterns of inhibition across similarly related kinases. We have further shown how these results might be correlated with the sequence identity between kinases to better anticipate inhibitor promiscuity. Finally, we were able to illustrate how a kinase-centric approach could be applied to correlate alterations to the kinase domain with changes in luminescence. This has use for the interrogation of different modes of inhibition as well as in identifying the specific determinants of inhibitor binding. In total, these efforts represent the optimization of a new, general platform for determining kinase inhibitor selectivity across the kinome, and it could potentially be applied universally to the interrogation of protein-ligand interactions.

The interactions between protein-protein, protein-nucleic acid, and protein-small molecules are central to biological processes and are key for the design of new therapeutics. Rapid and easy to implement methodologies are needed that enable the interrogation of these interactions in a complex cellular context. Towards this goal, I have utilized the concept of split-protein reassembly, also called protein complementation, for the creation of a variety of sensor architectures that enable the interrogation of protein-nucleic acid, protein-protein, and protein-small molecule interactions. Utilizing the enzymatic split-reporter β-lactamase and existing zinc finger design strategies we applied our recently developed technology termed SEquence-Enabled Reassembly (SEER) towards the creation of a sensor capable of the specific detection of the CryIA transgene. Additionally, the split β-lactamase reporter was utilized for the sitespecific determination of DNA methylation at cytosine residues that is involved in epigenetic regulation. This method, dubbed mCpG-SEER, enabled the direct detection of femtomole levels of dsDNA methylation in sequence specific manner. In a separate endeavor, we have developed and optimized the first cell-free split-reporter systems for GFP, split β-lactamase, and firefly luciferase for the successful dsDNA-dependent reassembly of the various reporters. Our cell free in vitro translation systems eliminates previous bottlenecks encountered in split-reporter technologies such as laborious transfection/cell culture or protein purification. Capitalizing on the ease of use and speed afforded by this new technology we describe the sensitive detection of protein-protein, protein-nucleic acid, and protein-small molecule interactions and inhibitors thereof. In a related area, we have applied this rapid cell-free split-firefly luciferase assay to the specific interrogation of a large class of helix-receptor protein-protein interactions. We have built a panel consisting of the clinically relevant Bcl-2 family of proteins, hDM2, hDM4, and p53 and interrogated the specificity of helix-receptor interactions as well as the specificity of peptide and small-molecule inhibitors of these interactions. Finally, we describe the further applications of our cell-free technology to the development of a large number of general split-firefly luciferase sensors for the detection of ssRNA sequences, the detection of native proteins, the evaluation of protease activity, and interrogation of enzyme-inhibitor interactions. The new methodologies provided in this study provides a general and enabling methodology for the rapid interrogation of a wide variety of biomolecular interactions and their antagonists without the limitations imposed by current in vitro and in vivo approaches.