A significant portion of the human proteome (greater than 50%) remains uncharacterized and an even greater fraction of these proteins lack covalent, selective probes for functional analysis. High-Throughput Screening (HTS) and Fragment-Based Ligand Discovery (FBLD) have both had success in the development of new probes and inhibitors but are challenged by prerequisites for biological insight and structural information about a target. To address the need for global identification of new sites for protein modulation, we have begun to develop a chemoproteomic platform for Fragment-Based Ligand Discovery in Proteomes (FBLDiP). We propose to use this platform to discover novel ligand-binding sites and fragments that modulate protein activity at these sites in a massively parallel fashion that will access traditionally 'undruggable'targets. FBLDiP simultaneously reports the site of labeling and the structure activity relationship (SAR) of fragment binding at thousands of cysteines within the proteome. We have already begun to implement the FBLDiP platform and have uncovered novel ligand-binding sites in many cancer relevant proteins, including the epigenetic enzyme PRMT1, the transcription factor NF-?B, the oncogenic adaptor protein CRKL, cancer metabolism enzyme IDH1 and the poorly characterized kinase ZAK. The synthesis of a cysteine-reactive fragment library is ongoing and we anticipate the production of a 100+ compound library. Biological evaluation of the effects of ligand-binding on selected identified proteins has begun and preliminary data supports that the FBLDiP methodology is effective for discovery of novel ligand-binding sites and probes for these sites. Guided by docking and structural biology, we will generate potent, selective inhibitors at novel ligand-binding sites in cancer relevant targets. Inhibitor synthesis will be guided by SAR profiles for targets of interest from FBLDiP screening and secondary gel-based protein profiling. We anticipate that the FBLDiP methodology will uncover a wide range of new sites for protein modulation and lead inhibitors to target these sites. As a whole, this research offers an exciting new means to identify ligand-binding sites and to guide the synthesis of inhibitors for important and untargeted, biologically-relevant proteins.

Public Health Relevance

Small molecule probes offer a potent means to investigate protein function;most proteins in the human proteome lack such selective chemical probes. The goal of this project is to address the gap between target identification and inhibitor discovery through the development of a new chemoproteomic platform for Fragment-Based Ligand Discovery in Proteomes (FBLDiP) and the application of this platform for the discovery of novel ligand-binding sites. This work has the potential to elucidate novel sites for protein modulation i previously untargeted proteins and to guide the synthesis of selective probes and chemotherapeutic agents.

National Institute of Health (NIH)
Postdoctoral Individual National Research Service Award (F32)
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Special Emphasis Panel (ZRG1)
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Barski, Oleg
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Scripps Research Institute
La Jolla
United States
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