Evaluation of a Pooling Chemoproteomics Strategy with an FDA-Approved Drug Library

Chemoproteomics is a key platform for characterizing
the mode of
action for compounds, especially for targeted protein degraders such
as proteolysis targeting chimeras (PROTACs) and molecular glues. With
deep proteome coverage, multiplexed tandem mass tag-mass spectrometry
(TMT-MS) can tackle up to 18 samples in a single experiment. Here,
we present a pooling strategy for further enhancing the throughput
and apply the strategy to an FDA-approved drug library (95 best-in-class
compounds). The TMT-MS-based pooling strategy was evaluated in the
following steps. First, we demonstrated the capability of TMT-MS by
analyzing more than 15 000 unique proteins (> 12 000
gene products) in HEK293 cells treated with five PROTACs (two BRD/BET
degraders and three degraders for FAK, ALK, and BTK kinases). We then
introduced a rationalized pooling strategy to separate structurally
similar compounds in different pools and identified the proteomic
response to 14 pools from the drug library. Finally, we validated
the proteomic response from one pool by reprofiling the cells via
treatment with individual drugs with sufficient replicates. Interestingly,
numerous proteins were found to change upon drug treatment, including
AMD1, ODC1, PRKX, PRKY, EXO1, AEN, and LRRC58 with 7-hydroxystaurosporine;
C6orf64, HMGCR, and RRM2 with Sorafenib; SYS1 and ALAS1 with Venetoclax;
and ATF3, CLK1, and CLK4 with Palbocilib. Thus, pooling chemoproteomics
screening provides an efficient method for dissecting the molecular
targets of compound libraries.