A biotin targeting chimera (BioTAC) system to map small molecule interactomes in situ

Abstract

Understanding how small molecules bind to specific protein complexes in living cells is critical to understanding their mechanism-of-action. Unbiased chemical biology strategies for direct readout of protein interactome remodelling by small molecules would provide advantages over target-focused approaches, including the ability to detect previously unknown ligand targets and complexes. However, there are few current methods for unbiased profiling of small molecule interactomes. To address this, we envisioned a technology that would combine the sensitivity and live-cell compatibility of proximity labelling coupled to mass spectrometry, with the specificity and unbiased nature of chemoproteomics. In this manuscript, we describe the BioTAC system, a small-molecule guided proximity labelling platform that can rapidly identify both direct and complexed small molecule binding proteins. We benchmark the system against µMap, photoaffinity labelling, affinity purification coupled to mass spectrometry and proximity labelling coupled to mass spectrometry datasets. We also apply the BioTAC system to provide interactome maps of Trametinib and analogues. The BioTAC system overcomes a limitation of current approaches and supports identification of both inhibitor bound and molecular glue bound complexes.

Fig. 1. The BioTAC system enables rapid, accurate small molecule target-ID.

A Schematic depicting the components of the BioTAC system. B Example molecule, Cpd 1, annotated with key functional groups. C Immunoblot analysis of BRD4 enrichment following treatment of HEK293 cells transiently transfected with miniTurboFKBP12^F36V with the indicated compounds and 100 µM biotin at the 30 min timepoint (input = sample processing control, n = 3 biological replicates, Supplementary Fig. 1I, J). D Scatterplot displaying relative FC of streptavidin-enriched protein abundance following treatment of HEK293 cells transiently transfected with miniTurboFKBP12^F36V with either DMSO or 1 µM Cpd 1 plus the indicated concentration of (+)-JQ1 competitor and 100 µM biotin at the 30 min timepoint. Only proteins with P-value < 0.05 (two-sample moderated T test) in both conditions depicted. FC fold-change, complete datasets in Supplementary Data 1, plotted individually in Supplementary Fig. 4. Source data are provided as a Source Data file.

Fig. 2. The BioTAC system enables rapid, accurate small molecule interactome-ID.

A Time-course proteomics using data-dependent acquisition methods of streptavidin-enriched biotinylated proteins isolated from HEK293 cells transiently transfected with miniTurboFKBP12^F36V and treated with the indicated compounds and 100 µM biotin, demonstrating enrichment and competition of known direct targets (30 min) and complexed proteins (60 min, 4 h). Only proteins with P-value < 0.05 (two-sample moderated T-test) in both conditions depicted. FC = fold-change, complete datasets in Supplementary Data 1, plotted individually in Supplementary Fig. 4. High-confidence hits are defined as those that are enriched >2-fold in both Cpd 1/ DMSO and Cpd 1/Cpd 1 + 10x (+)-JQ1, where P < 0.05, plotted upper-right quadrant. B Proteomics using data-independent acquisition methods of streptavidin-enriched biotinylated proteins isolated from HEK293 cells transiently transfected with miniTurboFKBP12^F36V and treated with the indicated compounds and 100 µM biotin for 30 min, demonstrating enrichment and competition of known direct targets and complexed proteins. Only proteins with P-value < 0.05 (two-sample moderated T test) in both conditions depicted. FC = fold-change, complete datasets in Supplementary Data 1. C Percent enrichment of reference interactors (Lambert et al.) in the hits identified from datasets depicted in panels A and B vs. the percent enrichment of reference interactors in 5000 random protein sets of equivalent size from the human transcriptome, showing significant enrichment by Cpd 1 (>41 standard deviations away from mean of random chance). D Gene Ontology analysis, showing significant enrichment of biological processes associated with known BET-protein function in the datasets depicted in panels A and B. Source data are provided as a Source Data file.

Fig. 3. Linker Exit Vector Diversification Identifies Core and Extended Interactors.

A Chemical structure of (+)-JQ1 bifunctional with alternative linker attachment site. B Immunoblot analysis of BRD4 enrichment following treatment of HEK293 cells transiently transfected with miniTurboFKBP12^F36V with the indicated compounds and 100 µM biotin at the 30 min timepoint (input = sample processing control, n = 2 biological replicates, Supplementary Fig. 5B). C Venn diagram showing statistically significant hits from DIA BioTAC experiments at the 30 min time point using Cpd 1 (blue) or at the 4 h time point from Cpd 2 (purple). Overlapping hits are listed below. D, E Proteomics using data-independent acquisition methods of streptavidin-enriched biotinylated proteins isolated from HEK293 cells transiently transfected with miniTurboFKBP12^F36V and treated with DMSO, 1 µM Cpd 2, plus the indicated compounds and 100 µM biotin for 4 h, demonstrating enrichment and competition of known direct targets and complexed proteins. Only proteins with P-value < 0.05 (two-sample moderated T test) in both conditions depicted. FC fold-change, complete datasets in Supplementary Data 1. Source data are provided as a Source Data file.

Fig. 4. The BioTAC system is an extensible strategy for live-cell target-ID.

A Chemical Structure of Cpd 3. B Immunoblot analysis of Aurora A enrichment following treatment of HEK293 cells transiently transfected with miniTurboFKBP12^F36V with the indicated compounds and 100 µM biotin at the 4 h timepoint (input = sample processing control, n = 2 biological replicates. C, D Proteomics using data-independent acquisition methods of streptavidin-enriched biotinylated proteins isolated from HEK293 cells transiently transfected with miniTurboFKBP12^F36V and treated with DMSO, 1 µM Cpd 3, plus the indicated compounds and 100 µM biotin for 4 h, demonstrating enrichment and competition of known direct targets and complexed proteins. Only proteins with P-value < 0.05 (two-sample moderated T test) in both conditions depicted. FC fold-change, complete datasets in Supplementary Data 1. Source data are provided as a Source Data file.

Fig. 5. The BioTAC system enables detection of non-degrader molecular glue interactions.

A Schematic depicting how the BioTAC system can detect molecular glue interactions. B Chemical structure of trametinib bifunctional molecule Cpd 4. C Immunoblot analysis of MEK1 and KSR1 enrichment following treatment of HEK293 cells transiently transfected with miniTurboFKBP12^F36V with the indicated compounds and 100 µM biotin at the 4 h timepoint, and streptavidin-based enrichment, showing successful enrichment and competition with trametinib (input = sample processing control, n = 2 biological replicates). D Immunoblot analysis of mKSR1 enrichment following treatment of HEK293 cells transiently transfected with miniTurbo-FKBP12^F36V and mKSR1, with the indicated compounds and 100 µM biotin at the 4 h timepoint, and streptavidin-based enrichment, showing successful enrichment and competition with trametinib (input = sample processing control, n = 3 biological replicates, see Supplementary Fig. 7D, E) In C and D two KSR1 isoforms are observed, produced by alternative splicing. KSR1-L (102 KDa) corresponds to the expected MW of Uniprot Q8IVT5-1 (canonical sequence), KSR1-S (87 KDa) corresponds to the expected MW of variant with residues 1–137 missing, Uniprot Q8IVT5-3, and −4. Our data indicate both isoforms can complex with trametinib-bound MEK1. E Proteomics using data-independent acquisition methods of streptavidin-enriched biotinylated proteins isolated from HEK293 cells transiently transfected with miniTurboFKBP12^F36V and treated with either DMSO or 1 µM Cpd 4 plus the indicated compounds and 100 µM biotin for 4 h, demonstrating enrichment and competition of known direct targets and complexed proteins. Only proteins with P-value < 0.05 (two-sample moderated T test) in both conditions depicted. FC = fold-change, complete datasets in Supplementary Data 1. F Chemical structure of trametiglue bifunctional molecule Cpd 5. G Proteomics using data-independent acquisition methods of streptavidin-enriched biotinylated proteins isolated from HEK293 cells transiently transfected with miniTurboFKBP12^F36V and treated with either DMSO or 1 µM Cpd 5, plus the indicated compounds and 100 µM biotin for 4 h, demonstrating enrichment and competition of known direct targets and complexed proteins. Only proteins with P-value < 0.05 (two-sample moderated T test) in both conditions depicted. FC fold-change, complete datasets in Supplementary Data 1. Source data are provided as a Source Data file.