Monday, April 29, 2024
4/29/2024
ABSTRACT Development of covalent drugs for cancer therapy relies heavily on identifying the full spectrum of on- and off- targets within the proteome, along with the specific site of inhibitor binding to each target. Currently we cannot predict the set of targetable cysteines for a given inhibitor nor the potential for covalent labeling of off-target proteins, which can lead to unintended toxicities. These limitations impede 'target discovery' during development of new covalent inhibitors. Similarly, once a specific protein target has been identified as being selectively bound by an inhibitor-candidate of interest, it is difficult to ascertain the fraction of target molecules in a cell or tissue that are covalently bound by the compound ('target occupancy'). Target discovery and occupancy data can be combined with toxicology and pharmacokinetic results to build more informative risk profiles to ensure that only the most promising drug candidates are advanced and ultimately nominated for clinical trials. The objective of our interdisciplinary team (Justin Kim, chemistry and chemical biology; Jarrod Marto, protein biochemistry and chemical proteomics) is to synthesize and test first-generation reagents designed to advance covalent inhibitor target discovery and target occupancy studies. Achieving our objectives will validate our approach and pave the way for an intensive and iterative campaign of probe synthesis paired with chemoproteomic analysis to systematically optimize our reagents. In the fullness of time we envision a complete suite of chemoproteomic assay-specific reagents to facilitate target discovery and target occupancy studies. Aim 1. To optimize new bioorthogonal reagents for proteome-wide inhibitor target identification. In this aim we will further develop our bioorthogonal chemistry and rationally expand our library of alkylating scaffolds suitable for cysteine profiling experiments. We will systematically assess the performance of our probes for target discovery using recently characterized covalent inhibitors of kinases and deubiquitinating enzymes. Aim 2. To develop multiplexed reporter probes to rapidly quantify covalent inhibitor target occupancy. We will leverage selective fragmentation of our reporter probe scaffold to develop high-sensitivity, high-through- put targeted mass spectrometry assays to quantify the occupancy of covalent inhibitors on their primary protein target. We will carefully assess the performance of our cysteine alkylating probes as universal reagents for pull- down-based (PD) target occupancy analysis, including linearity, accuracy, and limits of detection/quantification. Successful completion of our objectives will provide an initial set of reagents with an extensible framework to support future probe optimization campaigns by us and others in the field. Collectively the output of our program will accelerate development of new covalent drugs for cancer therapy.
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