Controlling Protein Complexation and Cellular Localization using Novel Molecular Therapeutics

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PROPOSAL SUMMARY I propose to conduct two major research projects. PROJECT 1: Develop Synthetic Peptide Prenylation Boxes (PPB’s) for application in Membrane-Localizing Ligands to Manipulate the Spatial and Temporal Location of Proteins in Cells. I will describe an entirely new and innovative molecular approach to facilitate protein-membrane anchorage, negating the typical shortcomings of this strategy such as poor water solubility and aggregation. The global objective is to design and develop a ‘warhead’ that is recognized by native prenylating enzymes, undergoes prenylation, and creates a membrane anchor in situ. We have coined this ‘warhead’ a Peptide Prenylation Box (PPB). We propose to incorporate this motif into a variety of small molecule, protein ligands to further our understanding of the fundamental science involved in protein-membrane anchorage and explore its functional utility and limitations. In the same field, we will investigate the use of coordination complex ligands in anchoring phosphorylated (activated) proteins to the lipid membrane. This proposed work represents a significant shift in the direction of this branch of molecular recognition and offers a more creative approach to a difficult challenge.PROJECT 2: Developing turn-on, ratiometric fluorescent sensors selective for di-phosphorylated protein motifs. The second project entails the development of a turn-on, ratiometric fluorescent sensor, selective for proximal diphosphorylated residues on a protein target. Our preliminary work has developed the first fluorometric sensor, based on pyrene-mediated excimer formation, that selectively detects pYpY over pY containing peptide/proteins. Our objective is to better understand this modality and optimize sensors for gel-based and solution-based quantification of important diphosphorylated proteins. While non-specific detection of mono-phosphorylated sites (pY, pS and pT) using commercial dyes (Pro-Q Diamond) has been employed to study the phospho-proteome, they do not selectively detect a subset of proteins, whose activation requires pXpX motif phosphorylation (where X = Y, T, and S). Initial studies in our lab have identified an innovative fluorescent sensor, based on excimer emission signal (480 nm), that selectively detects pYpY over pY-containing peptide/proteins. Our objective is to expand the application of these novel sensors to pXpX containing phosphoproteins, optimize structures to confer greater selectivity, potency, and enhanced sensitivity limits for application in 96-well and on solid supports such as SDS-PAGE gel and nitrocellulose membrane.Overall Summary. We will build on our growing knowledge of synthetic ligands to develop elegant PPB-protein-membrane anchors for selectively membrane localizing target proteins. This proposed work represents a significant shift in the direction of this branch of molecular recognition and offers a more creative approach to a difficult challenge. Our pXpX excimer-based sensors have yielded very promising sensitivity and selectivity profiles for phosphorylated protein states, which we expect to improve by an order of magnitude with higher potency binding scaffolds for ultimate application in SDS-PAGE gel assay and 96-well plate assay format.