Covalent Targeting of Splicing in T Cells

Despite significant interest in therapeutic targeting of splicing, few chemical probes are available for the proteins involved in splicing. Here, we show that elaborated stereoisomeric acrylamide chemical probe EV96 and its analogues lead to a selective T cell state-dependent loss of interleukin 2-inducible T cell kinase (ITK) by targeting one of the core splicing factors SF3B1. Mechanistic investigations suggest that the state-dependency stems from a combination of differential protein turnover rates and availability of functional mRNA pools that can be depleted due to extensive alternative splicing. We further introduce a comprehensive list of proteins involved in splicing and leverage both cysteine- and protein-directed activity-based protein profiling (ABPP) data with electrophilic scout fragments to demonstrate covalent ligandability for many classes of splicing factors and splicing regulators in primary human T cells. Taken together, our findings show how chemical perturbation of splicing can lead to immune state-dependent changes in protein expression and provide evidence for the broad potential to target splicing factors with covalent chemistry. Expanded primary human T cells were pelleted (600 g, 5 min, 4 °C) and re-suspended in fresh T cell media at 2 x 106 cells/mL for compound treatment. T cells were treated with WX-02-23 (5 µM), WX-02-43 (5 µM), PladB (10 nM)) or DMSO control in 6-well plates (2 mL/well, 4 x 106 cells/treatment) with or without CD3/CD28 stimulation for 8 h. For treatment conditions that required T cell activation, plates were pre-coated with 1.5 mL PBS containing CD3 (5 µg/mL, BioXCell) and CD28 (2 µg/mL, BioXCell) overnight at 4 °C or for 2 h at 37 °C and washed with PBS (3 mL, 3 times) prior to treatment.