NRX-0492 selectively degrades BTK via a cereblon (CRBN)- and proteasome-dependent mechanism. (A) Chemical structure of NRX-0492. (B) Binding of NRX-0492 to BTK WT, C481S, and T474I mutant BTK measured in a FRET competition assay. Data represent average values and standard deviations from 3 experiments. (C) Crystal structure of kinase domain of WT BTK in gray cartoon bound to the “hook” of NRX-0492 in yellow sticks. Functional kinase motives are colored for orientation: c-Helix in pink, P-loop in orange, activation loop in green, and gate/hinge in cyan. Mainchain atoms that engage in hydrogen bonding interaction are shown in sticks and the corresponding hinge region residues are labeled in black. The hydrogen bonding interactions are indicated with dashed black lines. Sidechains of mutational hotspots T474 and C481 are shown in sticks and labeled in red. (D) TMD8 cells were pretreated with MLN4924, MG132, harness, or hook at the indicated concentrations for 1 hour. Cells were then treated with NRX-0492 for an additional 4 hours, and BTK levels were quantified by total-BTK homologous time–resolved fluorescence. Each experiment was performed in triplicate. Graphs represent mean ± SD. (E) TMD8 cells were treated for 6 hours with 50 nM NRX-0492 or DMSO in triplicate. Samples were analyzed with tandem mass tag mass spectrometry. Results are graphed in a volcano plot with a statistical significance threshold of P < .01 indicated by the horizontal dotted line.