Extended Data Fig. 5: Characterization of stereoprobe-protein interactions.
From: Multi-tiered chemical proteomic maps of tryptoline acrylamide–protein interactions in cancer cells
a, b, Cysteine-directed ABPP data showing stereoselective liganding of PLEK_C250 in Ramos cells by WX-01-06 (a) and WX-02-26 (b) in Ramos cells. c, Protein-directed ABPP data showing stereoselective enrichment of PLEK by WX-01-06 and blockade of this enrichment by WX-02-26. d, Gel-ABPP data demonstrating stereoselective engagement of recombinant WT-PLEK, but not a C250A-PLEK mutant by WX-01-06 (5 µM, 1 h). e, AlphaFold-predicted structure of PLEK showing location of C250 (red) relative to the IP5 binding pocket (blue). f, Cysteine-directed ABPP data showing stereoselective liganding of C210/213 of NFU1 by WX-01-12 and WX-02-46. g, Protein-directed ABPP data showing stereoselective enrichment of NFU1 by WX-01-12 and blockade of this enrichment by WX-02-46. h, Competitive gel-ABPP data showing stereoselective blockade of WX-01-12 reactivity with recombinant WT-NFU1 by WX-02-46 (20 µM, 1 h pre-treatment). i, Gel-ABPP data demonstrating engagement of WT-NFU1 and the C213A-NFU1 mutant, but not the C210A-NFU1 mutant, by WX-01-12. j, CellTiter-Glo data showing pH-dependent impairment in SW480 cell growth by WX-01-12 (5 µM, 72 h). Data are mean values ± s.d. from three independent experiments. One-way ANOVA with Dunnett’s multiple comparison, **P = 0.0023, ****P < 0.0001. k, l, Cysteine-directed ABPP data showing stereoselective liganding of TYMS_C195 by WX-01-07 (k) and WX-02-36 (l) in Ramos cells. m, Protein-directed ABPP data showing stereoselective enrichment of recombinant TYMS by WX-01-07 (5 µM, 1 h) and blockade of this enrichment by WX-02-36 (20 µM, 1 h pre-treatment). n, o, Gel-ABPP data showing stereoselective engagement of WT-TYMS, but not a C195-TYMS mutant by WX-01-07 (n) and stereoselective blockade of this engagement by WX-02-36 (o). p, Bar graph showing enantioselective enrichment of TMX1/4, but not TMX2/3 by WX-01-09 from protein-directed ABPP experiments in Ramos cells. For each TMX protein, the signal intensity in WX-01-11-treated cells was set to a value of 1. q, Cysteine-directed ABPP data showing stereoselective liganding of TMX4_C64/67 by WX-01-09 in Ramos cells. r, s, Gel-ABPP data demonstrating engagement of recombinant WT-TMX1 and C59A- and C205A-TMX1 mutants, but not C56A- or C56A/C59A TMX1 mutants (r), and recombinant WT-TMX4 and C67A- and C213A-TMX4 mutants, but not C64A- or C64A/C67A-TMX4 mutants (s) by WX-01-09 (5 µM, 1 h). t, Gel-ABPP confirming stereoselective engagement of recombinant TMX1 and TMX4, but not TMX2 and TMX3, by WX-01-09. u, v, Competitive gel-ABPP data showing concentration-dependent and enantioselective blockade of WX-01-09 (5 µM, 1 h) engagement of TMX1 (u) and TMX4 (v) by WX-02-16 (1 h pre-treatment). Top, representative gel-ABPP data; bottom, quantification of gel-ABPP. Data represent 2 biological replicates. The red asterisk in r represents alkyne liganded and rhodamine tagged species of TMX1 (this corresponds to the signal seen in gel-ABPP above the IB). Proteomic data presented in a-c, g, k- m, and p-q are mean values ± s.d. of n = 4 biological replicates. For d, h, i, n-o, r- t experiments were performed in transfected HEK293T cells as described in Fig. 4b, and data are from a single experiment representative of two experiments; IB = anti-Flag immunoblot, UT= untransfected cells.
