Drug interaction mapping with proximity dependent enzyme recruiting chimeras

Abstract

Proximity dependent labeling using engineered enzymes has been used extensively to identify protein-protein interactions, and map protein complexes in-vitro and in-vivo. Here, we extend the use of engineered promiscuous biotin ligases to the identification of small molecule protein targets. Chimeric bi-functional chemical probes are used to effectively recruit tagged biotin ligases for proximity dependent labeling of target and target interactors. The broad applicability of this approach is demonstrated with probes developed from a multi-kinase inhibitor, a bromodomain targeting moiety, and an FKBP targeting molecule. While complementary to traditional chemo-proteomic strategies such as photo-affinity labeling (PAL), and activity-based protein profiling (ABPP), this approach is a useful addition to the target ID toolbox with opportunities for tunability based on the inherent labeling efficiencies of different engineered enzymes and control over the enzyme cellular localization.

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

“…These features make the BioTAC system readily accessible for broad application. During the preparation of this manuscript, a preprint describing a related approach for identifying targets of small molecules via SNAP-and Halo-tagging of TurboID was also disclosed 29 . Whilst these systems differ from the BioTAC system in their reported specificity and have not yet been benchmarked for interactome-detection, their successful implementation across a range of ligands highlights the robustness of using proximity labeling to interrogate small molecule targets 29 .…”

“…During the preparation of this manuscript, a preprint describing a related approach for identifying targets of small molecules via SNAP-and Halo-tagging of TurboID was also disclosed 29 . Whilst these systems differ from the BioTAC system in their reported specificity and have not yet been benchmarked for interactome-detection, their successful implementation across a range of ligands highlights the robustness of using proximity labeling to interrogate small molecule targets 29 . Next, we show the BioTAC system can identify molecular glue pairs that previously evaded detection, in a single experiment using Trametinib as a model system.…”

Measuring Ligand‐bound Protein Complexes with Proximity Labeling: A Practical Guide

“…Several other publications developing similar methods have also recently been disclosed. [8][9][10] These systems employ diverse ligandability tags, including SNAP-tag and Halo-tag, different proximity labeling enzymes (e. g. TurboID), evaluate a wide range of molecules with targets in the nucleus and cytoplasm identified using both targeted and untargeted constructs. [8][9][10] While Kwak et existing bifunctional tag-based recruiter molecules they would like to evaluate.…”

“…[8][9][10] These systems employ diverse ligandability tags, including SNAP-tag and Halo-tag, different proximity labeling enzymes (e. g. TurboID), evaluate a wide range of molecules with targets in the nucleus and cytoplasm identified using both targeted and untargeted constructs. [8][9][10] While Kwak et existing bifunctional tag-based recruiter molecules they would like to evaluate. [8][9][10] Although the chemical properties of each tag-binder are different, all variants have been proven to produce bifunctionals with sufficient target engagement by these studies.…”

“…[8][9][10] While Kwak et existing bifunctional tag-based recruiter molecules they would like to evaluate. [8][9][10] Although the chemical properties of each tag-binder are different, all variants have been proven to produce bifunctionals with sufficient target engagement by these studies. (Nevertheless, we always recommend a cellular target engagement assay is used for newly generated molecules to ensure success of the project.)…”