Chemical probe

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A chemical probe is a well-characterized small molecule that selectively modulates the function or abundance of a specific protein of interest [1] [2] . Chemical probes enable reversible or irreversible perturbation of biological pathways and are widely used to investigate protein function, dissect fundamental biological processes in cells and organisms, and validate molecular targets [1] [2] . While early definitions for chemical probes, including criteria for potency, selectivity, and cellular activity, focused on reversible intracellular inhibitors, guidelines for other types  chemical probes have been defined, encompassing agonists and antagonists, covalent inhibitors, and targeted protein degraders, such as PROTACs and molecular glues, the latter of which promote protein degradation rather than inhibiting activity [1] .

Contents

Chemical probes differ from drugs. Drugs are optimised for clinical properties such as pharmacokinetics and safety, whereas chemical probes are optimised for high target selectivity, potency, and demonstrated cellular target engagement to help ensure that observed phenotypes arise directly from target modulation (https://www.science.org/content/blog-post/chemical-probes-versus-drugs). Incorrect use of poorly selective or unsuitable small molecules can lead to misleading biological conclusions and irreproducible research [3] .

Unlike genetic perturbation technologies such as CRISPR–Cas editing and RNA interference, chemical probes provide rapid, reversible, and tunable controls of protein activity, enabling the study of essential genes, multifunctional proteins, and protein scaffolding roles [4] .

History

Small molecules have long been used as mechanistic tools in biochemistry and pharmacology [5] . Following the publication of the human genome, the main challenge shifted from gene identification to understanding protein function, and selective chemical probes helped stimulate research on understudied targets [6] . Systematic efforts to define chemical probes emerged in the 2010s, when concerns about irreproducibility and misuse of poorly selective inhibitors were highlighted [2] . In response, expert communities developed guidelines and resources, including the Chemical Probes Portal, https://en.wikipedia.org/wiki/Chemical_Probes_Portal), to provide recommendations and best-practice guidance.  

Advances in genetics [7] [6] , structural biology, covalent chemistry, and targeted protein degradation have since expanded the types and applications of chemical probes, including targeted protein degraders such as PROTACs and molecular glues [5] [8] .

Types of Chemical Probes

Chemical probes can act through several mechanisms [8] :

TypeMechanism
Classical modulatorsSuppress target activity (commonly for enzymes such as kinases)
Covalent inhibitorsForm irreversible bonds to the target protein [8]
Targeted degraders (PROTACs, molecular glues, dTAGs)Recruit ubiquitin ligases to degrade target proteins rather than inhibiting them [8] [9]
Activators / Agonists Enhance or mimic target activity, including receptor agonists and allosteric activators

Notes:

Applications

Chemical probes are used to characterize proteins and pathways across diverse experimental systems, including mammalian cells, microbes, animal and plant models [12] . Including:  

Roles in drug discovery

Chemical probes can serve as starting points, providing initial structural scaffolds for drug design for medicinal chemistry optimisation and development of clinically suitable molecules. They play a key role in early-stage drug discovery, particularly during target validation. As tool compounds, they help determine whether modulating a specific protein produces a disease-relevant phenotype, thereby assessing whether a target is viable as a therapeutic target [14] [5] [1] . By exposing cells or organisms to selective probes, researchers can uncover resistance mechanisms and compensatory network behaviour that may compromise potential drug efficacy. Chemical probes also enable the identification of biomarkers that report target engagement in biological systems.

Quality criteria

Standards are recommended to help ensure that chemical probes deliver reliable biological insights [5] [15] [16] (https://www.chemicalprobes.org /).

Guidelines include:

  1. Biochemical potency – often sub-100nM (IC₅₀/Ki/Kd) in assays using purified protein.
  2. Selectivity – typically ≥30-fold over related homologues, supported by broader profiling across protein families.
  3. Target engagement and cellular activity – demonstrated through biochemical, biophysical, or proteomics-based assays below 1µM, without nonspecific toxicity or off-target effects.
  4. Properties – solubility and absence of assay interference or general cytotoxicity.
  5. Availability of controls – ideally a matched inactive analogue and, where feasible, an orthogonal active probe with a different chemical scaffold. Negative controls should be used alongside probes to confirm target-specific effects.

Community resources

Several open-access initiatives provide guidance and tools for selecting chemical probes [17] :

These resources provide validated chemical probes and usage guidelines that support reproducible and mechanistic research.

See also

Reference

  1. 1 2 3 4 5 Müller, Susanne; Sanfelice, Domenico; Workman, Paul (2025-03-10). "Probing cancer with small-molecule tools-Progress and challenges". Cancer Cell. 43 (3): 323–327. doi:10.1016/j.ccell.2025.02.003. ISSN   1878-3686. PMID   40020670.
  2. 1 2 3 Arrowsmith, Cheryl H.; Audia, James E.; Austin, Christopher; Baell, Jonathan; Bennett, Jonathan; Blagg, Julian; Bountra, Chas; Brennan, Paul E.; Brown, Peter J.; Bunnage, Mark E.; Buser-Doepner, Carolyn; Campbell, Robert M.; Carter, Adrian J.; Cohen, Philip; Copeland, Robert A. (August 2015). "The promise and peril of chemical probes". Nature Chemical Biology. 11 (8): 536–541. doi:10.1038/nchembio.1867. ISSN   1552-4469. PMC   4706458 . PMID   26196764.
  3. ↑ Sterling, Jayden; Baker, Jennifer R.; McCluskey, Adam; Munoz, Lenka (2023-06-03). "Systematic literature review reveals suboptimal use of chemical probes in cell-based biomedical research". Nature Communications. 14 (1): 3228. doi:10.1038/s41467-023-38952-1. ISSN   2041-1723. PMC   10239480 . PMID   37270653.
  4. ↑ Blagg, Julian; Workman, Paul (2017-07-10). "Choose and Use Your Chemical Probe Wisely to Explore Cancer Biology". Cancer Cell. 32 (1): 9–25. doi:10.1016/j.ccell.2017.06.005. ISSN   1878-3686. PMC   5511331 . PMID   28697345.
  5. 1 2 3 4 Licciardello, Marco P.; Workman, Paul (2022-12-14). "The era of high-quality chemical probes". RSC medicinal chemistry. 13 (12): 1446–1459. doi:10.1039/d2md00291d. ISSN   2632-8682. PMC   9749956 . PMID   36545432.
  6. 1 2 Kustatscher, Georg; Collins, Tom; Gingras, Anne-Claude; Guo, Tiannan; Hermjakob, Henning; Ideker, Trey; Lilley, Kathryn S.; Lundberg, Emma; Marcotte, Edward M.; Ralser, Markus; Rappsilber, Juri (July 2022). "Understudied proteins: opportunities and challenges for functional proteomics". Nature Methods. 19 (7): 774–779. doi:10.1038/s41592-022-01454-x. ISSN   1548-7105. PMID   35534633.
  7. ↑ Edwards, Aled M.; Isserlin, Ruth; Bader, Gary D.; Frye, Stephen V.; Willson, Timothy M.; Yu, Frank H. (2011-02-10). "Too many roads not taken". Nature. 470 (7333): 163–165. doi:10.1038/470163a. ISSN   1476-4687. PMID   21307913.
  8. 1 2 3 4 Hartung, Ingo V.; Rudolph, Joachim; Mader, Mary M.; Mulder, Monique P. C.; Workman, Paul (2023-07-27). "Expanding Chemical Probe Space: Quality Criteria for Covalent and Degrader Probes". Journal of Medicinal Chemistry. 66 (14): 9297–9312. doi:10.1021/acs.jmedchem.3c00550. ISSN   1520-4804. PMC   10388296 . PMID   37403870.
  9. ↑ Schreiber, Stuart L. (2021-01-07). "The Rise of Molecular Glues". Cell. 184 (1): 3–9. doi:10.1016/j.cell.2020.12.020. ISSN   1097-4172. PMID   33417864.
  10. ↑ Nabet, Behnam; Roberts, Justin M.; Buckley, Dennis L.; Paulk, Joshiawa; Dastjerdi, Shiva; Yang, Annan; Leggett, Alan L.; Erb, Michael A.; Lawlor, Matthew A.; Souza, Amanda; Scott, Thomas G.; Vittori, Sarah; Perry, Jennifer A.; Qi, Jun; Winter, Georg E. (May 2018). "The dTAG system for immediate and target-specific protein degradation". Nature Chemical Biology. 14 (5): 431–441. doi:10.1038/s41589-018-0021-8. ISSN   1552-4469. PMC   6295913 . PMID   29581585.
  11. ↑ Nabet, Behnam; Ferguson, Fleur M.; Seong, Bo Kyung A.; Kuljanin, Miljan; Leggett, Alan L.; Mohardt, Mikaela L.; Robichaud, Amanda; Conway, Amy S.; Buckley, Dennis L.; Mancias, Joseph D.; Bradner, James E.; Stegmaier, Kimberly; Gray, Nathanael S. (2020-09-18). "Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules". Nature Communications. 11 (1): 4687. doi:10.1038/s41467-020-18377-w. ISSN   2041-1723. PMC   7501296 . PMID   32948771.
  12. ↑ Ercoli, Maria Florencia; Ramos, Priscila Zonzini; Jain, Rashmi; Pilotte, Joseph; Dong, Oliver Xiaoou; Thompson, Ty; Wells, Carrow I.; Elkins, Jonathan M.; Edwards, Aled M.; Couñago, Rafael M.; Drewry, David H.; Ronald, Pamela C. (November 2022). "An open source plant kinase chemogenomics set". Plant Direct. 6 (11): e460. doi:10.1002/pld3.460. ISSN   2475-4455. PMC   9694430 . PMID   36447653.
  13. ↑ Müller, Susanne; Ackloo, Suzanne; Al Chawaf, Arij; Al-Lazikani, Bissan; Antolin, Albert; Baell, Jonathan B.; Beck, Hartmut; Beedie, Shaunna; Betz, Ulrich A. K.; Bezerra, Gustavo Arruda; Brennan, Paul E.; Brown, David; Brown, Peter J.; Bullock, Alex N.; Carter, Adrian J. (2022-01-27). "Target 2035 - update on the quest for a probe for every protein". RSC medicinal chemistry. 13 (1): 13–21. doi:10.1039/d1md00228g. ISSN   2632-8682. PMC   8792830 . PMID   35211674.
  14. ↑ Bain, Jenny; Plater, Lorna; Elliott, Matt; Shpiro, Natalia; Hastie, C. James; McLauchlan, Hilary; Klevernic, Iva; Arthur, J. Simon C.; Alessi, Dario R.; Cohen, Philip (2007-12-15). "The selectivity of protein kinase inhibitors: a further update". The Biochemical Journal. 408 (3): 297–315. doi:10.1042/BJ20070797. ISSN   1470-8728. PMC   2267365 . PMID   17850214.
  15. ↑ Workman, Paul; Collins, Ian (2010-06-25). "Probing the probes: fitness factors for small molecule tools". Chemistry & Biology. 17 (6): 561–577. doi:10.1016/j.chembiol.2010.05.013. ISSN   1879-1301. PMC   2905514 . PMID   20609406.
  16. ↑ Mader, Mary M.; Rudolph, Joachim; Hartung, Ingo V.; Uehling, David; Workman, Paul; Zuercher, William (2023-10-05). "Which Small Molecule? Selecting Chemical Probes for Use in Cancer Research and Target Validation". Cancer Discovery. 13 (10): 2150–2165. doi:10.1158/2159-8290.CD-23-0536. ISSN   2159-8290. PMID   37712569.
  17. ↑ Antolin, Albert A.; Workman, Paul; Al-Lazikani, Bissan (April 2021). "Public resources for chemical probes: the journey so far and the road ahead". Future Medicinal Chemistry. 13 (8): 731–747. doi:10.4155/fmc-2019-0231. ISSN   1756-8927. PMID   31778323.
  18. ↑ Sanfelice, Domenico; Antolin, Albert A.; Crisp, Alisa; Chen, Yi; Bellenie, Benjamin; Brennan, Paul E.; Edwards, Aled; Müller, Susanne; Al-Lazikani, Bissan; Workman, Paul (2025-01-06). "The Chemical Probes Portal - 2024: update on this public resource to support best-practice selection and use of small molecules in biomedical research". Nucleic Acids Research. 53 (D1): D1663 –D1669. doi:10.1093/nar/gkae1062. ISSN   1362-4962. PMC   11701680 . PMID   39558166.
  19. ↑ Antolin, Albert A.; Sanfelice, Domenico; Crisp, Alisa; Villasclaras Fernandez, Eloy; Mica, Ioan L.; Chen, Yi; Collins, Ian; Edwards, Aled; Müller, Susanne; Al-Lazikani, Bissan; Workman, Paul (2023-01-06). "The Chemical Probes Portal: an expert review-based public resource to empower chemical probe assessment, selection and use". Nucleic Acids Research. 51 (D1): D1492 –D1502. doi:10.1093/nar/gkac909. ISSN   1362-4962. PMC   9825478 . PMID   36268860.
  20. ↑ Antolin, Albert A.; Tym, Joseph E.; Komianou, Angeliki; Collins, Ian; Workman, Paul; Al-Lazikani, Bissan (2018-02-15). "Objective, Quantitative, Data-Driven Assessment of Chemical Probes". Cell Chemical Biology. 25 (2): 194–205.e5. doi:10.1016/j.chembiol.2017.11.004. ISSN   2451-9448. PMC   5814752 . PMID   29249694.
  21. ↑ Antolin, Albert A.; Aye, Yimon; Bar-Peled, Liron; Vita, Elena De; Dudkina, Natavan; Jewett, Michael C.; Kiely-Collins, Hannah; Mazitschek, Ralph; Zhang, Zhenrun Jerry (2024-09-19). "What is chemical biology?". Cell Chemical Biology. 31 (9): 1562–1565. doi:10.1016/j.chembiol.2024.08.011. ISSN   2451-9456. PMID   39303695.
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