Information Centre

Welcome to the Chemical Probes Portal Information Centre, containing information, resources and training materials about chemical probes and the Portal.

Please let us know what else you would like to see here. We’re adding new content all the time.

Probes Criteria

Chemical Probes / Classical Modulators

Chemical probes (classical modulators) are small-molecule ligands targeting specific biomolecular targets (proteins). They allow scientists to ask mechanistic and phenotypic questions about a target in cell-based or animal studies.

Chemical probes play a major role in linking a phenotype to a gene allowing the functional annotation of the human genome and validating new molecular targets. When a phenotype is observed upon treatment with the chemical probe, it is attributed to the protein targeted by the probe hence selectivity and potency, are essential attributes of chemical probes.

The Chemical Probes Team has put together a list of criteria to be used when selecting a probe for your experiment.

'Classical' Modulator
Activity Evidence of target binding/activity modulation. (In-vitro IC50/Ki/Kd, etc.)
Control Compounds Similar structure with similar physicochemistry, non-binding against target
Off-target activity Evidence of wider in vitro profiling, especially within protein class
In-cell validation Evidence and quantification of target engagement
  • Need direct measure of target engagement (e.g. in cell binding or stabilisation) or proximal PD biomarker (e.g. specific phosphosite)
  • Phenotype is target-engagement dependent (use inactive analogue as well as an orthogonal probe with alternative chemotype, together with biomarker, to demonstrate target dependence)
Off-target activity in cells
  • Assessment of effect on potent off-target(s) identified from in vitro profiling
  • Orthogonal probe (active but different chemotype). Desirable
Evidence of cellular permeability Demonstrable by steps above
Key references
  1. Antolin AA, Workman P, Al-Lazikani B. Public resources for chemical probes: the journey so far and the road ahead. Future Med Chem. 2021 Apr;13(8):731-747. doi: 10.4155/fmc-2019-0231.
  2. Arrowsmith CH, et al. The promise and peril of chemical probes. Nat Chem Biol. 2015; 11, 536-541 doi: 10.1038/nchembio.1867.
  3. Blagg J, Workman P. Choose and use your chemical probe wisely to explore cancer biology. Cancer Cell. 2017; 32, 9-25 doi: 10.1016/j.ccell.2017.06.005.
  4. Bunnage ME, Chekler EL, Jones LH. Target validation using chemical probes. Nat Chem Biol. 2013; 9, 195-199 doi: 10.1038/nchembio.1197.
  5. Frye SV. The art of the chemical probe. Nat Chem Biol. 2010; 6, 159-161 doi: 10.1038/nchembio.296.
  6. Workman P, Collins I. Probing the probes: fitness factors for small molecule tools. Chem Biol. 2010; 17, 561-577 doi: 10.1016/j.chembiol.2010.05.013.


Proteolysis targeting chimeras (PROTACs) are heterobifunctional small molecules composed of two active domains and a linker capable of promoting the degradation of targeted proteins. Rather than acting as conventional enzyme inhibitors, PROTACs work by inducing selective intracellular proteolysis. PROTACs consist of two covalently linked protein-binding molecules: one capable of engaging an E3 ubiquitin ligase, and another that binds to a target protein meant for degradation. Recruitment of the E3 ligase to the target protein results in ubiquitination and subsequent degradation of the target protein by the proteasome. Because PROTACs need only to bind their targets with high selectivity (rather than inhibit the target protein's enzymatic activity), there are currently many efforts to retool previously ineffective inhibitor molecules as PROTACs for next-generation drugs.

The Chemical Probe Portal now lists several PROTAC molecules that have been evaluated by our SERP for use in cell and in vivo. Here we report the criteria to select the right PROTAC for your experiment:

Degrader (PROteolysis TArgeting Chimeras, PROTACs)
Activity Evidence of binding to target and E3-ligase (CRBN, VHL, etc.) or other non-E3 degradation effector complexes
Control Compounds def A probe that is inactive against target, and a second non-binding to E3 ligase (or effector complex)
Off-target activity Evidence of wider in vitro profiling, especially within protein class
In-cell validation Evidence and quantification of target engagement and degradation
  • DC50 and Dmax values determined
  • Time course for degradation defined
  • Evidence of E3, ubiquitin and proteasome-dependence; or dependence on other effector pathways relevant to degradation mechanism
  • Phenotype is degradation dependent (comparison to non-degrading target binder)
Off-target activity in cells Evidence of in-cell target selectivity
  • e.g. degradation profile measured by MS / proteomics
  • e.g. measurement of off-target engagement / inhibition / depletion
  • Orthogonal probe (active but different chemotype). Desirable
Evidence of cellular permeability Demonstrable by steps above
Key references
  1. Chopra R, Sadok A, Collins I. A critical evaluation of the approaches to targeted protein degradation for drug discovery. Drug Discov Today Technol. 2019; 31, 5-13 doi: 10.1016/j.ddtec.2019.02.002.
  2. Kostic M, Jones LH. Critical Assessment of Targeted Protein Degradation as a Research Tool and Pharmacological Modality. Trends Pharmacol Sci. 2020; 41, 305-317 doi: 10.1016/

Molecular Glues and Molecular Glue Degraders

Molecular glues are small molecules that exert a biological effect by inducing new protein-protein associations.1 The induced interaction can lead to homo- or hetero-dimerization or oligomerization of the proteins involved. The binding of a small-molecule glue to one protein can induce a new protein-protein interaction through different mechanisms. Binding to a pocket on one protein may create a new protein surface at the pocket, creating complementarity to the surface of a new binding partner. The glue is bound at the interface between the interacting proteins in a ternary or higher-order complex and stabilizes their interaction (Figure 1). Alternatively, binding of the small molecule to one protein may lead to an allosteric conformational change in the protein that creates and stabilizes a new surface for new protein-protein interactions remote from the small molecule binding site. Examples of molecular glues include immune-modulatory compounds such as Cyclosporin A, Rapamycin and other binders of the protein FKBP12, and microtubule-stabilising compounds such as Taxol and discodermalide.1 The criteria for assessing molecular glues as chemical tools parallel those for chemical probes, assessing for binding of both targets 1.

The criteria for assessing molecular glues as chemical tools parallel those for chemical probes, assessing for binding of both targets.

Molecular glue degraders are a subset of molecular glues that induce the interaction of an E3 ubiquitin ligase or other protein degradation-effecting complex with a new protein target 2 . The neo-substrate interaction induced by the molecular glue leads to tagging of the target protein, for example by poly-ubiquitination in the case of an E3 ligase, and triggers degradation of the protein by the cell’s proteasomal or autophagic machinery. (Figure 2). Examples of small molecule glue degraders that recruit new proteins to interact with E3 ligases include thalidomide derivatives and indisulam1,2.

The criteria for assessing molecular glue degraders as chemical tools parallel those suggested for the larger, bifunctional PROTAC inducers of degradation.

Molecular Glues listed on the Chemical Probe Portal can be found here.

1. S. Schreiber (2021) Cell, 184, 3-9. (DOI: 10.1016/j.cell.2020.12.020)
2. R. Chopra, A. Sadok, I. Collins (2019) Drug Discov. Today Technol., 31, 5-13. (DOI: 10.1016/j.ddtec.2019.02.002)

Molecular Glue Degrader
  • Evidence of binding to E3-ligase (CRBN, VHL, etc) or other non-E3 degradation effector complexes
  • Optional: in vitro complex formation between E3 component protein, molecular glue and target protein
Control Compounds def
  • Derivative of the molecular glue that is non-binding to E3 ligase or another effector complex
  • Inhibitor or another non-degrading modulator of the degraded target
Off-target activity
  • Optional: in vitro pharmacology screening against potential off-targets
In-cell validation
  • Evidence and quantification of target engagement and degradation
  • DC50 and Dmax values determined
  • Time course for degradation defined
  • Evidence of E3, ubiquitin and proteasome-dependence; or dependence on other effector pathways relevant to degradation mechanism
  • Phenotype is degradation dependent (comparison to non-degrading derivative)
Off target activity in cells
  • Evidence of in-cell target selectivity
    • e.g. degradation profile measured by MS / proteomics
    • e.g. measurement of off-target engagement / inhibition / depletion
  • Optional: Orthogonal probe (active but different chemotype)
Evidence of cellular permeability Demonstrable by steps above

Activity-Based Probes

Activity-based probes (ABPs) are small molecules that show a tripartite structure. They consist of a reactive group, also called ‘warhead’, a linker or recognition element and a detection group suitable to either enrich the protein(s) of interest or detect them in solution or on a solid carrier (Figure 1).

The warhead often consists of an electrophilic reactive group for covalent binding to specific amino acids on the protein target(s). The linker group acts as a spacer preventing a steric clash between the warhead and the recognition element, but may also contain elements improving the selectivity of a probe. Additionally, the linker positions the reactive group for selective covalent targeting. In some cases, the linker and warhead may not be completely distinguishable. The detection group consist of a tag such as a click handle or biotin allowing high-affinity coupling to a solid support in order to enrich the protein for subsequent mass spectrometric analysis. Also fluorophores are used to visualise ABP-labelled proteins. In addition, radioisotopes may be coupled for gel-based visualisation or in vivo application e.g. in positron emission tomography. The tag may alter the properties of the probe including its cell penetrance or subcellular localisation.

Most ABPs are not designed to bind to a specific protein, but recently several potent and selective ABPs have been described.

The Chemical Probes Portal does not currently evaluate ABPs.


  1. D. Conole, M. Mondal, J.D. Majmudar, E.W. Tate (2019) Front. Chem., DOI: 10.3389/fchem.2019.00876
  2. Benns HJ, Wincott CJ, Tate EW, Child MA. (2021) Curr Opin Chem Biol., DOI: 10.1016/j.cbpa.2020.06.011
  3. S. Heinzlmeier, S. Müller (2021), Drug Discovery Today, DOI: 10.1016/j.drudis.2021.10.021

dTAG system

by Dr Hadley Sheppard, the Institute of Cancer Research, London

Targeted protein degradation systems have emerged which enable pharmacological modelling of protein degradation in the absence of a chemical probe. Once such system is the dTAG system, developed by the Gray lab (formerly of Harvard and now Stanford University). The dTAG system enables degradation of a given target both in vitro and in vivo1,2. The system makes use of highly selective FKBP12F36V directed ligands (known as the dTAG molecule) and expression of a target protein tagged with the degron FKBP12F36V, to create a fusion protein known as the dTAG-protein (Figure 1). Once the dTAG-protein is expressed in a cell, for example by CRISPR-mediated knock in or transgenic expression, the cell-permeable dTAG molecule is added and forms a dual linker between the dTAG-protein and an endogenous E3 ligase. As a result, the dTAG-protein is ubiquitinated and targeted for proteasomal degradation.

dTAG-mediated degradation is extremely rapid and can completely remove a given target in as little as an hour. The system is reversible and washout of the dTAG molecule will enable re-expression of the dTAG-protein. To set up a dTAG system, there are a series of plasmids and sequences available from Addgene, detailed protocols, and the dTAG molecules are commercially available. There are now dTAG molecules that will recruit either the endogenous cereblon or VHL E3 ligases and both are compatible with same dTAG epitope tag on the target protein. It is important to confirm which terminus can accommodate the FKBP12F36V tag without interfering with function as well evaluate toxicity of the dTAG molecules in parental cells prior to performing degradation experiments. Once the system is established, the dTAG is ideal for target validation in the process of drug discovery. The dTAG system combines the precision of genetics with the time-dependent opportunities of small molecules.

Figure 2: Schematic of the dTAG system for degrading protein target T, through fusion with the degron, FKPB12F36V. Addition of the dTAG molecule leads to binding with Cereblon and degradation of the target protein.


  1. Nabet, B. et al. The dTAG system for immediate and target-specific protein degradation. Nat Chem Biol 14, 431-441, DOI:10.1038/s41589-018-0021-8 (2018).
  2. Nabet, B. et al. Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules. Nat Commun 11, 4687, doi:10.1038/s41467-020-18377-w (2020).

Guidelines for characterization of probes to be used in animals

Chemical probes that meet the desired criteria for in vitro cell systems may or may not be suitable for use in animal models, such as rodent species that are widely employed in biomedical research. In particular, it is important that a probe can be administered so as to achieve the appropriate level of exposure that would support modulation of the desired protein target for the required duration.

In order to aid the evaluation of probes in animal models, typically rodents, the Chemical Probes Team recommends a set of measured criteria considered essential for the in vivo pharmacokinetic and pharmacodynamic characterization of chemical probes. These can be applied to guide the choice of probe and rational selection of the dosing schedule of a compound for preclinical in vivo experiments1,2.

Pharmacokinetic data should be provided to inform on the plasma and target organ exposure achieved with the compound at specified doses and dosing route(s). Protein binding data should be included to enable calculation of the unbound (free) concentration of the compound, i.e. the amount of compound not bound by plasma and tissue protein.

Together with the measured in vitro potency of the compound, this information will typically enable estimation of the dose(s) of compound required to achieve unbound concentrations sufficient to interact with the compound’s intended molecular target, and for which duration3. The likelihood of interactions with known off-targets can be similarly quantified.

Where available, inclusion of quantitative data on the modulation of specific biomarkers reporting on the activity of the intended molecular target is encouraged, as evidence of target engagement in vivo, providing a pharmacological audit trail at the measured exposures4.

Guidelines for useful pharmacokinetic data to be provided (rodents 1

Specification of the preclinical model(s) in which pharmacokinetic parameters have been measure
Specification of the preclinical model(s) in which pharmacokinetic parameters have been measure
  • Species used
  • Dosing route and vehicle
  • Dose quantity and schedule
Pharmacokinetic data informing on the plasma exposure of the drug and the plasma free fraction
  • Elimination half-life (T1/2)
  • Systemic clearance (CL)
  • Fraction of the compound that is protein-bound (fb)
  • Maximum plasma concentration after drug administration (Cmax) and time to reach maximum plasma concentration (Tmax) for the given dose
For compounds targeting the CNS
  • Ratio of drug in the brain to that in plasma (B:P)
  • Ratio of drug found free in the brain (Cu,p) defined as Cu,b/Cu,p
  • Any potential impact of drug transporters (found on the rodent blood-brain-barrier) in limiting brain exposure


  1. Kleiman RJ, Ehlers MD. Sci Transl Med 2016 8: 320ps1. doi: 10.1126/scitranslmed.aac9888
  2. Rossanese O, Eccles S, Springer C, Swain A, Raynaud FI, Workman P, Kirkin V. Drug Discovery Today: Disease Models 2016 21: 23-32. doi: 10.1016/j.ddmod.2017.07.002
  3. Smith DA, Di L, Kerns EH. Nat Rev Drug Discov 2010 9: 929-39. doi: 10.1038/nrd3287
  4. Banerji U, Workman P. Semin Oncol 2016 43: 436-45. doi: 10.1053/j.seminoncol.2016.06.001

Toxicophores and PAINS Alerts

Some chemical compounds can be broadly reactive with biomolecules, causing promiscuous activity and general toxicity in cellular and in vivo assays, or can interfere with biological or in vitro assays leading to an apparent biological effect that is in fact an artefact of the assay system.

Many such molecules are enriched in certain chemical groups or substructures, and the presence of these chemical features can be worth taking into consideration as potential alerts for unwanted activities. It can be helpful to distinguish between compounds containing toxicophores – meaning substructures or functional groups often leading to toxicity, commonly due to widespread chemical reactivity – and so-called Pan-Assay Interference compounds (PAINS) – which are compounds that can interfere with biochemical and cell-based assay detection methods leading to false readouts. Compounds that undergo colloidal aggregation can cause non-specific inhibition due to absorption onto the surface of proteins2,3,4. In cellular assays, non-target related toxic effects can include phospholipidosis5. Compounds that take part in redox chemistry can interfere with detection methods in both biochemical and cellular assays6.

Any of these types of non-specific behaviour can confound the interpretation and robustness of experimental results using a small molecule to investigate the effect of a specific biological target. Therefore, it is important to be aware of the risk of these behaviours occurring for a particular chemical structure1.

Within the Chemical Probes Portal, the chemical structure of each potential chemical probe that is submitted to us is screened virtually against a library of known toxicophores and PAINS substructures using the canSAR chemical registration pipeline7. When we detect that a probe possesses any of the known substructures, we ‘flag’ them, and the probe is consequently labelled as containing potential PAINS and/or toxicophore substructures. We look for markers of pan-assay interference using RDKit 8, based on the PAINS set assembled by Baell and Holloway9 and we use a list of toxicophore substructures assembled by Hughes and colleagues 10. It is important to stress that the presence of the toxicophore or PAINS substructures within the chemical structure of a compound does not necessarily mean that it will be non-specifically active or toxic, or give rise to assay interference. The substructure alerts can sometimes produce false positives. Some assay interferences are associated with particular assay conditions or technologies. Therefore, a toxicophore or PAINS flag is simply an alert to consider.

It is important that users are aware of the strengths and limitations of such alerts11 when conducting an experiment with a compound flagged with a structural alert. For scientists who are not specialists in chemical biology, talking to an expert may be helpful. Because of the potential for artefactual results, it is even more important to design experiments to mitigate the risk of confounding effects due to non-specific toxicity or assay interference. Simple assays are available to test for the various undesirable effects listed here11. The use of multiple, structurally distinct chemical probes, negative control probes and varied, orthogonal assay technologies is strongly recommended.

  1. Baell J and Walters MA. Chemical con artists foil drug discovery. Nature 2014 513: 481-3. doi: 10.1038/513481a .
  2. LaPlante SR, Roux V, Shahout F, LaPlante G, Woo S, Denk MM, Larda ST, Ayotte Y. Probing the free-state solution behavior of drugs and their tendencies to self-aggregate into nano-entities. Nature Protocols 2021 16: 5250–5273. doi: 10.1038/s41596-021-00612-3 .
  3. O'Donnell HR, Tummino TA, Bardine B, Craik CS, Shoichet BK. Colloidal Aggregators in Biochemical SARS-CoV-2 Repurposing Screens. J Med Chem 2021 64(23): 17530-17539. doi: 10.1021/acs.jmedchem.1c01547 .
  4. Irwin JJ, Duan D, Torosyan H, Doak AK, Ziebart KT, Sterling T, Tumanian G, Shoichet BK. An Aggregation Advisor for Ligand Discovery. J Med Chem. 58(17): 7076-87. doi: 10.1021/acs.jmedchem.5b01105 .
  5. Tummino TA, Rezelj VV, Fischer B, Fischer A, O'Meara MJ, Monel B, Vallet T, White KM, Zhang Z, Alon A, Schadt H, O'Donnell HR, Lyu J, Rosales R, McGovern BL, Rathnasinghe R, Jangra S, Schotsaert M, Galarneau JR, Krogan NJ, Urban L, Shokat KM, Kruse AC, García-Sastre A, Schwartz O, Moretti F, Vignuzzi M, Pognan F, Shoichet BK. Drug-induced phospholipidosis confounds drug repurposing for SARS-CoV-2. Science 2021 373(6554): 541-547. doi: 10.1126/science.abi4708 .
  6. Johnston PA. Redox cycling compounds generate H2O2 in HTS buffers containing strong reducing reagents – real hits or promiscuous artifacts? Curr Opin Chem Biol 2011 15(1): 174–182. doi: 10.1016/j.cbpa.2010.10.022 .
  7. Mitsopoulos C, Di Micco P, Fernandez EV, Dolciami D, Holt E, Mica IL, Coker EA, Tym JE, Campbell J, Che KH, Ozer B, Kannas C, Antolin AA, Workman P, Al-Lazikani B. canSAR: update to the cancer translational research and drug discovery knowledgebase. Nucleic Acids Res. 2021 49 (D1): D1074-D1082. doi: 10.1093/nar/gkaa1059 .
  8. RDKit version 2021.03.1 .
  9. Baell JB and Holloway GA. New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays. J Med Chem 2010 53(7): 2719-40. doi: 10.1021/jm901137j .
  10. Hughes JD, Blagg J, Price DA, Bailey S, Decrescenzo GA, Devraj RV, Ellsworth E, Fobian YM, Gibbs ME, Gilles RW, Greene N, Huang E, Krieger-Burke T, Loesel J, Wager T, Whiteley L, Zhang Y. Physiochemical drug properties associated with in vivo toxicological outcomes. Bioorg Med Chem Lett 2008 18(17): 4872-5. doi: 10.1016/j.bmcl.2008.07.071 .
  11. Aldrich C, Bertozzi C, Georg GI, Kiessling L, Lindsley C, Liotta D, Merz KM Jr., Schepartz A, Wang S. The Ecstasy and Agony of Assay Interference Compounds. J Med Chem 2017 60(6): 2165–68. doi: 10.1021/acs.jmedchem.7b00229 .

Historical Compounds

The Portal features a class of compounds we termed Historical Compounds. These small molecules are typically non-selective or not sufficiently potent compared with other available chemical probes to merit the probe designation. We opted for the name "Historical Compounds" to provide a category that allows us to distinguish them from chemical probes. The title recognizes that many of these compounds were once valuable, for example if they were the first compound available to study a protein target or target family. We also recognize that some scientists select non-specific compounds for experiments precisely because they are non-specific, as these compounds allow them to impact the activity of many proteins at the same time. Our goal with this category of compounds is not to indicate that they cannot be useful in research. Rather, we hope to discourage the misapplication of these reagents as if they were chemical probes (i.e., specific and selective tools for a particular target).

A table of all Historical Compounds currently catalogued on the portal can be found here. To recommend a historical compound that we do not yet feature - use our probe submission form.

In the Portal, historical compounds can be readily distinguished from chemical probes: they are catalogued without much of the detailed validation information that accompanies a probe, they feature a generic historical compound image, and they are not rated by our SAB. To ensure there is no confusion, each historical compound is also designated by a generic and specific note to clarify the categorization. The generic note simply indicates that it is a historical compound; the specific note provides some context for why it is considered a historical compound. For an example, please visit the staurosporine page.

Rating System

Drawing from their experience and expertise, SERP members review the data for a probe as well as the publication reporting the probe and rate the probe for its in cellular and/or in vivo model systems (e.g., mice). Probes are evaluated for use in these two types of systems independently. This means that a probe can have top marks for cellular use without being suitable for use in model organisms.

While we expect SERP members to consider similar data for each probe (including potency, selectivity, evidence for target engagement, and an understanding of the mechanism by which the probe modulates the target), we have not defined rigid criteria that translate into Portal endorsement. We merely provide guidelines to the SERPs to be considered when evaluating probes. Expert reviewers bring their experience and knowledge of how small molecules interact with protein targets and the unique challenges that may come to bear in different situations. In addition to rating probes, SERP members can comment on each probe, providing guidance to users to ensure readers understand the strengths and weaknesses of a probe, know about control compounds such as orthogonal probes that should be used in tandem with the probe, and are aware of other potential pitfalls before they begin an experiment.

Our SERP members provide a star rating (1-4 stars) for each chemical probe they review. Star ratings for each probe are based on the data and whether they are sufficient to demonstrate that the probe is valid for use in cellular systems or in animal models. The star ratings appearing on the probe pages represent the average of all ratings provided by SERP members, and the number of votes for each probe is indicated. If no stars are shown (i.e., for in animal use of some probes), then the probe was not considered for this application. Please note that not being recommended as a chemical probe for a specific protein target does not mean that a compound may not be useful in other contexts. The star ratings correlate to the following recommendations:

  • 4 stars = Recommended as a probe for this target
  • 3 stars = Best available probe for this target, or a high-quality probe that is a useful orthogonal tool
  • 2 stars = Insufficient validation data to recommend
  • 1 star = Not recommended as a probe for this target

The Chemical Probes Portal only endorses compounds as chemical probes for use as specific and selective modulators of the proposed target if they receive three or more (3-4) stars.


Key Publications

This page will be expanded over time to include more references, but we begin by recommending a small number of outstanding, important papers that cover topics that should be of broad interest to scientists who use chemical probes.

How to cite the Chemical Probes Portal

A manuscript describing the new portal is in preparation, meanwhile please cite the original publication:

  • The promise and peril of chemical probes, Arrowsmith et al Nat Chem Biol 2015 11;536-531. doi: 10.1038/nchembio.1867 ( Nature Chemical Biology)

Please add our URL to your publication: