Chemical Approaches for Interrogating Fundamental Biomedical Processes

Project Summary The development and application of novel chemical tools can provide fundamental insights intobiomedical processes. The research program outlined in this proposal focuses on three core areas: 1) thedevelopment and application of chemosensors for phospho-regulatory enzymes, 2) new methodologies forconstructing designer signaling networks, and 3) fundamental studies of protein misfolding. Protein phosphorylation plays a central role in cellular signaling. Consequently, the enzymes thatmaintain the phosphoproteome, protein kinases and protein phosphatases, are considered key drug targets inhuman disease. Currently, indirect proxies are used to estimate activity perturbations of kinases andphosphatases associated with disease development. Although useful, these proxies do not provide a directmeasure of enzymatic activity, leading to inaccurate estimates of kinase and phosphatase activity. As a result,a clear understanding of the role of kinase and phosphatase activity perturbations during the development andprogression of human disease is lacking. Therefore, there is a critical need for the development and applicationof chemical tools to directly quantify kinase and phosphatase activity in human disease states. In this proposal,we will leverage a phosphorylation-sensitive amino acid to construct a panel of kinase and phosphataseactivity probes and apply this panel to develop longitudinal activity profiles of signaling changes during humandisease progression. The finely tuned activity of signaling proteins is essential for normal physiology. Indeed, perturbationsin the activity of signaling proteins are central to the development of human disease. Unfortunately, the fieldstill lacks a unified approach to modulating the activity of multiple signaling proteins simultaneously in livingsystems in order to model human disease. To address this critical need, we will leverage split-proteinreassembly in order to define a lexicon of standardized parts for fine-tuning the activity of signaling nodes inliving cells. In the long-term, this set of mix-and-match parts will be utilized to model human disease states andidentify potential drug targets. Lastly, our laboratory will investigate the fundamental aspects of protein misfolding, which is nowrecognized as a central pathological mechanism in numerous human disease states. The current lack ofapproaches to assess protein misfolding and aggregation in living systems has created a critical need for thedevelopment of novel methodologies to address this gap. By leveraging a novel, luminescence-based assayfor protein misfolding and aggregation our laboratory will assess the molecular determinants of proteinaggregation in living cells. In addition, we will utilize this approach to identify molecules capable interfering withprotein aggregation.