REVIEW article

Front. Pharmacol.

Sec. Experimental Pharmacology and Drug Discovery

Volume 16 - 2025 | doi: 10.3389/fphar.2025.1593844

This article is part of the Research TopicExploring Small Molecule Probes: Advancements and Applications in Pharmacological ResearchView all 5 articles

Utilizing small molecules to probe and harness the proteome by pooled protein tagging with ligandable domains

Provisionally accepted
  • Rutgers, The State University of New Jersey - Busch Campus, Piscataway, United States

The final, formatted version of the article will be published soon.

Multifunctional ligand-binding domains have revolutionized cell biology by enabling the precise visualization and manipulation of fused proteins of interest through small molecule probes. Employing these domains is labor-intensive and low-throughput, limiting studies to only small subsets of proteins at a time. However, advancements in high-throughput technologies like pooled protein tagging have enabled the tagging of proteins across the entire proteome with ligand-binding domains. This allows for the generation of complex cell libraries where each cell expresses a different protein fused to a generic, ligandable handle. These libraries unlock opportunities to explore the proteome with a versatile toolbox of small molecule probes designed to interact with the fused tags, allowing researchers to visualize protein localization, induce protein misfolding, manipulate protein-protein interactions, modulate protein stability, and more in a scalable, systematic manner. This review explores recent studies employing multifunctional domains at proteome scale, delving into how the associated chemical probes have been employed to enable insights into endogenous protein function, cellular processes, and disease mechanisms. Additional multifunctional ligand-binding domains are discussed that can be used in pooled protein tagging, as well as their potential strengths and weaknesses. We also discuss potential applications in drug discovery such as high-throughput screening for therapeutic targets with insights for bifunctional ligand optimization. By integrating pooled protein tagging with ligand-binding domains and chemical probes, we highlight how the fusion of chemical biology and functional genomics is paving the way for innovative research avenues and transformative advances in cell biology and pharmacology.

Keywords: Protein tagging, Pooled tagging, SPOTLITES, HaloTag, Induced proximity, Hydrophobic tagging, Protein degradation

Received: 14 Mar 2025; Accepted: 03 Jun 2025.

Copyright: © 2025 Larrimore and Serebrenik. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Yevgeniy Vladimirovich Serebrenik, Rutgers, The State University of New Jersey - Busch Campus, Piscataway, United States

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