Extended Data Fig. 6: Characterization of tryptoline acrylamide stereoprobe-LIMK1 interactions.
From: Multi-tiered chemical proteomic maps of tryptoline acrylamide–protein interactions in cancer cells
a, Cysteine-directed ABPP data showing enantioselective liganding of C349 of LIMK1 by alkyne stereoprobes WX-01-10 and WX-01-11 in Ramos cells. b, Cysteine-directed ABPP data showing lack of engagement of LIMK1_C349 by WX-02-26 and WX-02-36. c, Protein-directed ABPP data showing enantioselective enrichment of LIMK1 by WX-01-10 and WX-01-11 that is not blocked by WX-02-26 and WX-02-36, respectively. d, Gel-ABPP data demonstrating engagement of recombinant WT-LIMK1, but not the C349A-LIMK1 mutant by WX-01-11 (5 µM, 1 h). Experiments were performed in transfected HEK293T cells as described in Fig. 4b. e, Sequence alignment of LIMK1 and LIMK2 showing conserved residues (yellow) that are proximal (<15 Å) to the LIMK1-restricted stereoprobe-liganded cysteine C349 (red). f, Crystal structure of LIMK1 (PDB: 8AAU) showing C349 (red) in a pocket adjacent to the ATP (blue) binding site. Highlighted in yellow are residues conserved between LIMK1 and LIMK2 located within 15 Å of LIMK1 C349. g, Enantioselective and concentration-dependent enhancement of BRET signal in an LIMK1 NanoBRET kinase assay by WX-01-10 and WX-01-11 compared to their respective enantiomers WX-01-12 and WX-01-09. Data were generated in HEK293T cells transiently expressing LIMK1-nanoLuciferase fusion protein, where cells were treated with 0.5 µM of the NanoBRET K-10 tracer and different concentrations of stereoprobes for 3 h (data represent mean values for one experiment setup in triplicates). h, NanoBRET kinase assay showing that WX-01-11 increases signals for LIMK1 with the general kinase NanoBRET probe K-10, with the largest effect observed at lower concentrations of the NanoBRET probe. Data represent mean values for one experiment setup in triplicates. i, Enantioselective increase in BRET signal by WX-01-11 in WT- but not C349A-LIMK1 mutant cells. The ATP-binding pocket kinase inhibitor HG-9-91-01 decreased NanoBRET signals for both WT- and C349A-LIMK1 mutant (data represent mean values ± s.d., n = 3 independent experiments). j, Immunoblot of LIMK1-Nanoluc protein expressed in HEK293T cells showing that alkyne stereoprobes do not affect LIMK1 expression under conditions where they increase NanoBRET signals. For a-c, data represent mean values ± s.d. for n = 4 biological replicates. For d, g-h, j data are from a single experiment representative of two independent experiments with similar results. For d, j, IB = anti-Flag immunoblot, and UT= untransfected cells.
