Journal: bioRxiv

Article Title: Leveraging the BAF chromatin remodeling complex for targeted transcriptional rewiring in cancer

doi: 10.64898/2026.03.30.715217

Figure Lengend Snippet: a , In vitro TR-FRET ternary complex formation assay. The interaction between biotinylated SMARCA4 bromodomain (100 nM) and FITC-labeled BCL6 BTB domain (500 nM) was measured with increasing concentrations of TRIP1. The biotinylated SMARCA4 bromodomain was captured with streptavidin-terbium cryptate donor (SA-Tb, 2 nM), and TR-FRET signal was monitored upon complex formation. The resulting TR-FRET ratio was normalized to the DMSO vehicle control; data represent mean ± SD, n = 2 independent replicates. b , TR-FRET competition assay. The interaction between biotinylated SMARCA2/4 bromodomains (100 nM) and FITC-labeled BCL6 BTB domain (500 nM) was measured in the presence of 100 nM TRIP1 and increasing concentrations of BCL6 or SMARCA ligand. The resulting TR-FRET ratio was background subtracted and normalized to the DMSO vehicle control; data represent mean ± SD, n = 2 independent replicates. c , SplitHalo assay for BCL6-SMARCA2 interaction. Schematic of the in-cell splitHalo assay to probe induced protein-protein interactions (top). A HaloTag enzyme is split into two complementing parts, cpHalo and Hpep3 peptide, which only assemble into a functional self-labeling HaloTag enzyme when actively brought into proximity and supplied with TAMRA dye. HEK293T cells co-expressing BCL6-cpHalo and SMARCA2(BD)-Hpep3 were treated with a dilution series of TRIP1 or negative controls and incubated with compound and the covalent HaloTag dye TAMRA for 3 hours (bottom). Data is normalized to DMSO vehicle control, which corresponds to the baseline signal upon TAMRA addition; data represent mean ± SD, n = 3 independent replicates. d , SplitHalo competition assay for TRIP1-induced BCL6-SMARCA2 interaction. Schematic of the in-cell splitHalo competition assay to probe the inhibition of induced protein-protein interactions (top). Cells are pre-incubated with excess amounts of protein ligands to saturate binding pockets and prevent or reduce ternary complex formation. HEK293T cells co-expressing BCL6-cpHalo and SMARCA2(BD)-Hpep3 were pre-treated with a dilution series of SMARCA or BCL6 ligand for 30 minutes before adding 1 µM TRIP1 and TAMRA dye for 3 hours (bottom). Data is normalized to TRIP1 with DMSO vehicle control without ligand addition, corresponding to maximum ternary complex formation; data represent mean ± SD, n = 3 independent replicates. e , f , BCL6 transcriptional reporter competition. KARPAS-422 cells expressing a BCL6 transcriptional reporter were pre-treated with DMSO, the BCL6 ligand ( e ), or SMARCA ligand ( f ) for 8 hours, followed by co-treatment with 0.5 µM of TRIP1 for 24 hours. Reporter activity is normalized to DMSO vehicle control without TRIP1 co-treatment; data represent mean ± SD, n = 6 independent replicates. g , CaspaseGlo 3/7 apoptosis pre-degradation. KARPAS-422 cells were pre-treated with DMSO or SMARCA degrader (ACBI1) for 8 hours, followed by co-treatment with 1 µM of TRIP1 for 16 hours. Caspase 3/7 activity is normalized to DMSO vehicle control without TRIP1 co-treatment; data represent mean ± SD, n = 6 independent replicates.

Article Snippet: For treatment with test compounds, drug dilutions were prepared in DMEM with a final concentration of 100 nM TAMRA dye (MedChemExpress, HY-D2270).

Techniques: In Vitro, Tube Formation Assay, Labeling, Control, Competitive Binding Assay, Protein-Protein interactions, Functional Assay, Expressing, Incubation, Inhibition, Binding Assay, Activity Assay

Journal: Journal of Medicinal Chemistry

Article Title: Tunable Aromatic Sulfoxides and Sulfones as Cysteine-Targeting Warheads: Exploring the Structure–Reactivity Relationship

doi: 10.1021/acs.jmedchem.5c03536

Figure Lengend Snippet: Biological evaluation of alkyne probes in living cells. (a) Dose-dependent labeling of cellular BTK. Ramos cells were treated with 10–500 nM probe for 1 h, and the resulting cell lysates were modified by click reaction with TAMRA-N 3 . Proteins were separated by SDS-PAGE, and the gel was imaged with ChemiDoc imaging system at two channels (green LED, 605/50 filter for TAMRA, red LED 695/50 filter for MW markers). Images were merged to generate the composite image. The blue arrow indicates the BTK band. The gels were subsequently stained with SimplyBlue for total protein visualization. Full gel images are presented in Figures S13–S15 . (b) Cellular protein labeling profiles of the probes, together with ibrutinib competition, confirms BTK binding. Ramos cells were pretreated with either DMSO or 1 μM ibrutinib for 30 min, followed by treatment with 100 nM probe for 1 h. The resulting cell lysates were modified and analyzed as in (a). The blue arrow indicates the BTK band. (c) Volcano plot obtained from TMT-based quantitative proteomics analysis of the pull-down performed with Ibr-2 . Ramos cells were treated with 250 nM Ibr-2 , lysed, and conjugated to biotin-N 3 . Proteins with a log 2 fold-change >1 compared to the DMSO control and an adjusted p -value <0.05 were considered significantly enriched (highlighted in blue and annotated). An enlarged version of the volcano plot is presented in Figure S19 . (d) Ibr-2 does not affect BTK activity in Ramos cells, as measured by BTK autophosphorylation. Cells were pretreated with either DMSO or 1 μM ibrutinib for 30 min, followed by treatment with 100 nM Ibr-2 for 1 h. Thereafter, the cells were washed before BCR-stimulation with antihuman IgM (10 μg/mL) for 10 min. Proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with antibodies against phospho-BTK (Tyr223), total BTK, and β-actin.

Article Snippet: Ibrutinib and TAMRA-N 3 were purchased from MedChemExpress.

Techniques: Labeling, Modification, SDS Page, Imaging, Staining, Binding Assay, Quantitative Proteomics, Control, Activity Assay