Post translational modifications (PTMs) such as phosphorylation, glycosylation and methylation play a central and ubiquitous role in cellular signal transduction, and the enzymes that catalyze these reactions such as kinases and methyltransferases are the targets of intense drug discovery efforts for virtually every therapeutic area, with the most intense focus on cancer. Specific detection of posttranslational modifications (PTMs) in cell extracts is a fundamental technical challenge facing drug discovery and proteomics researchers working in this area. Though many site-specific antibodies are available, they often are not selective enough to differentiate between closely related PTM sites, leading to unreliable results. Moreover, the immunocytochemical or Western blot methods used to detect PTMs are difficult to perform in an automated, high throughput fashion, which discourages their use for identifying potential drug molecules using high throughput screening (HTS). To overcome these technical barriers, we propose to develop a generic platform for rapid, in vitro development of assays for highly specific, homogenous detection of PTMs that leverages the extensive pool of available PTM antibodies. Phase I feasibility will include: a) increasing the specificity of PTM immunodetection methods using peptides from phage display libraries that specifically recognize antibody-phosphoprotein complexes and b) demonstrating homogenous detection of the trivalent immune complex using a new single label method called Quenched Resonance Energy Transfer. In Phase II, BellBrook will develop panels of """"""""High Throughput-PTM"""""""" assays for many of the most therapeutically relevant kinase and methyltransferase pathways and commercialize them as HTS cellular assay kits with a simple lyse-and-detect format. These products could have a significant impact on drug discovery for cancer and other diseases by enabling large scale screening for kinase and methyltransferase inhibitors in the physiological context of intact cells using the most immediate endpoint of target enzyme activity: a specific PTM. Additionally, we will investigate how the specificity and detection enhancements of the High Throughput-PTM platform can be applied to improve proteomic methods, such as histochemical detection of PTMs, and thus accelerate efforts to map PTMs in healthy and diseased tissues.
The function of most proteins in the cell is regulated by covalent modifications, and aberrant modifications underlie many disease pathologies, especially cancer. We propose to develop detection methods to accelerate the identification of drug molecules that prevent specific aberrant protein modifications without affecting normal ones.
