A chemical probe is a selective small-molecule modulator – usually an inhibitor – of a protein’s function, which allows the user to ask mechanistic and phenotypic questions about its molecular target in cell-based or animal research.
Chemical probes are selective small-molecule modulators that enable the study of a specific protein. Drugs, in contrast, do not need to be selective but should be efficacious for a certain disease in vivo. Therefore, despite the existing synergies between basic chemical biology and drug discovery, small-molecule drugs and chemical probes are conceptually different.
Chemical probes can be key players in the validation of new molecular targets for a therapeutic indication but do not need to meet the same requirements as drugs in terms of pharmacokinetics, pharmacodynamics, and bioavailability.
Some drugs that can be used as chemical probes, but others are effective precisely because of their lack of selectivity and are therefore not suitable to be used as chemical probes to study a specific protein.
Chemical probes can be used to help establish the relationship between a molecular target and the broader biological consequences of modulating that target in cells or organisms. They are also highly synergistic to biological or genetic tools that can be used to study protein functions.
Chemical probes facilitate the fine tuning of protein inhibition through concentration-response experiments and the possibility to easily time the modulation of the protein. Thus, chemical probes can be used to discover new biology relating to a specific target, to clarify the relationship between the target and a phenotype, and to validate that a particular target is a suitable intervention point to impact the progression or outcome of a disease.
Unsuitables (previously termed as “Historical Compounds”) are small molecules that are not fit to be used as chemical probes. They are often non-selective or not sufficiently potent compared with other available chemical probes. The name “Unsuitables” was created by the Chemical Probes Team 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 PAINS compound is a Pan-Assay INterference compound, or a compound that produces frequent artefacts in biological assays.
These compounds often interfere with the detection method of the screening assay and are therefore regularly reported as being active across a multitude of assays/targets. They can also be compounds that are promiscuous because they aggregate, react non-specifically with proteins or are redox active.
PAINS compounds are generally not suitable as chemical probes to study specific targets. Computational filters are available that provide alerts for substructures or functional groups that can result in PAINS behaviour and also for ‘Toxicophore’ substructures leading to toxicity, often again due to widespread chemical reactivity. In addition, simple laboratory assays are available to test for the above behaviours.
Search PAINS for more information and how we screen for, and report on, known Toxicophore and PAINS substructures within compounds submitted to the portal as potential chemical probes.
For any given protein target, scientists should ideally design experiments using two structurally distinct chemical probes (orthogonal probes), as well as their inactive derivatives. The use of orthogonal probes decreases the probability that a researcher will attribute an off-target activity of a probe to the protein of interest because two probes of distinct structure are not likely to interact with similar off-targets.
Data for chemical probes is inherently incomplete. A compound that has been shown to modulate a protein without affecting other tested proteins may still modulate other proteins against which it has not been evaluated. We try to address this in two ways. First, a second, structurally distinct chemical probe is likely (but not guaranteed) to have a different off-target profile; if the results of the two unrelated compounds are the same, there is increased confidence that the phenotypic effects are from interaction with the target protein. Secondly, we strongly recommend the use of a negative control that is structurally related to the chemical probe whenever possible, but considerably less active on the main target. Negative controls are likely (but not guaranteed) to share the same off-targets with the chemical probe as they are structurally similar. Suggested read: The Promise and Peril of Chemical Probe Negative Control, ACS Chem. Biol. 2021, 10.1021/acschembio.1c00036
Validation of a chemical probe’s activity in vitro, in cells, and in vivo requires verification that the chemical probe engages – that is binds to – its intended target in the model system used. Assays to detect target engagement can be proximal or distal to the target. The more proximal the assay to the probe-target interaction (i.e., direct binding assay), the less likely unanticipated activity of the probe will mislead the researcher into believing a phenotype is due to on-target activity when it is not.
Biomarkers that are reliable surrogates for target engagement enable researchers to directly correlate target engagement with specific phenotypes. If, for instance, full target occupancy is confirmed for a chemical probe in cells or in vivo and the probe fails to produce an expected phenotype, then the target and mechanism were properly tested and invalidated. Without measurements of target engagement, it can be very difficult to discern the basis for lack of activity or for researchers to have confidence they have tested their hypothesis.
An ideal target-engagement assay measures (i) the extent of target engagement, which can help to determine doses or concentrations that produce the phenotype while limiting side effects, and (ii) the potential for interactions with off-target proteins.
For more information, see Determining target engagement in living systems.
The Chemical Probes Portal provides guidance on the maximum concentration that a probe should be used in cells.
However, when applying a chemical probe that was validated in one cellular system to another, it is important to consider several factors. Is the target expressed in the new system at levels comparable to those in the original? If not, the concentration of the probe that you will need to impact the activity of the target will differ.
Similarly, are likely off-target proteins expressed at the same levels? It is important to balance the amount of the probe that you use in cells with its selectivity, and the best concentration may need to be determined empirically by assessing on- and off-target activity profiles.
Similarly, we recommend validating that the chemical probe engages its intended target when moving to a new cellular system. Proteins can adopt different conformations and participate in distinct complexes in different cells; thus, it is essential to demonstrate that the protein target is accessible by the probe in the new system before proceeding with new experiments.
The requirements for a chemical probe that is fit for use in an in vivo animal model (e.g., in the mouse or rat) are more stringent than for chemical probes that will be used only in vitro, for example in cell culture. In particular, the chemical probe must be capable of achieving concentrations in plasma and tissue that are sufficient to provide meaningful modulation of the intended target, as defined initially by cellular potency from in vitro experiments.
Thus, over and above the characteristics that are key in vitro, the pharmacokinetic properties of the candidate probe are particularly important. The probe must also not display rapid metabolic and other forms of clearance; for example, the candidate probe should be stable in microsomes with no major CYP450 inhibition.
The candidate probe must also be sufficiently well tolerated for the period of experimentation such that the necessary dose can be administered to achieve the required levels of pharmacokinetic exposures in terms of concentration and time.
It is important to demonstrate target engagement using appropriate pharmacodynamic biomarkers in the in vivo model and ideally to establish a pharmacokinetic-pharmacodynamic-effect relationship. To obtain these properties it is commonly necessary to invest substantial medicinal chemistry resources, in some way similar to those required in a lead optimization program that aims to achieve a preclinical drug candidate.
Importantly, when a chemical probe is to be used in vivo in animal models such as mice, rats or higher species, the quantity of the compound that is required will be much greater than those that are sufficient for in vitro use.
For more details, see Probing the probes: Fitness factors for small molecule tools and Data gaps limit the potential of preclinical research.
FAQ for Chemical Probes
The ChemicalProbes.org database can be searched by Protein Target or Chemical Probe name. After the search, you will be presented with the results of your query summarising key properties of the chemical probes that match your search with the best probe to use shown first. Upon clicking on each probe, a page with more detailed information can be accessed.
The probe page will also recommend the use of suitable control compounds and/or orthogonal probes, which will provide you with more confidence in the observed results. Alternatively, it is also possible to browse ChemicalProbes.org and use filters to reduce the number of results.
Search results are sorted based on SERP star rating (please check here for more details). The higher the star rating the better the probe is according to expert assessment.
We recommend that to validate your experiments you use more than one Probe (see Orthogonal Compounds). We aim to have at least two probes for each protein target assessed by experts in The Chemical Probes Portal. In addition, our SERP members, provide useful comments explaining the best way to use a specific probe at which concentrations to use the compounds and potential pitfalls.
Yes. Please submit your probe using our online submission form. You must register for a Portal user account to submit a probe.
We are not currently considering bio-active peptides in the portal.
Although we recognise the scientific relevance of Activity-based probes the Chemical Probes Portal will not currently host such probes as their intent is different from classical modulators.
In general, the portal cannot accept data that are not already published, and peer reviewed by a journal. Recently, we have started adding probes that don’t have a specific publication but have been peer reviewed through external committees, and the data are made available to the scientific community (e.g. SGC Chemical Probes and Donated Chemical Probes). We aim to add data-hosting capabilities in the near future. We will update this site as soon as the situation changes.
After a period of inactivity, from 2020 ChemicalProbes.org is active again. The Portal is manually curated and maintained by experts in various scientific fields. Probes activity data are either submitted or extracted from scientific literature and assessed against our criteria for suitable chemical probes. We try our best to keep the database as up to date and accurate as possible, but we rely on the scientific community to highlight any flaws that are in our database or of new data becoming available for a specific compound changing its profile.
If you want to get in touch with the team regarding the Portal content, please contact us.
Yes. You can use the Download feature from the browse page to download all data about chemical probes on the Portal. Use the filters feature to select a subset of the data by protein family, star rating or date added. Include historical compounds to download the full database. There is also a download data button on each individual probe page which allows you to download the data for that compound.
There are many potential ‘chemical probes’ reported in the scientific and patent literature or available through chemical vendors, and scientists use the term chemical probe to refer to slightly different things. For consideration by the portal SERP, portal staff review all chemical probe submissions to ensure they meet our standards for review. These standards* include:
- The chemical probe and data supporting its validation are published in a peer-reviewed scientific journal
- Data supporting the probe’s validation includes reasonable evidence that the probe is acting selectively on the target of interest in cells
Please note, assays that demonstrate a chemical probe impacts cell proliferation and/or cell death are rarely specific enough to provide the type of validation for selectivity that we seek.
*Note these standards are subject to change over time, we will update this page and announce any changes before we incorporate them into our decision processes.
Please send a letter of appeal, including your name, the name of your probe and a scientific explanation for why you believe the portal’s decision was incorrect, to the Chemical Probe Team.
Please get in touch with the team.
If you are searching for a specific chemical probe, and it does not appear on the Portal, it might be because it is not a high-quality chemical probe or because it has not yet been included. The Portal is a manually curated and expert-assessed resource so it can take time for all potential probes to be reviewed.
If you are searching for a specific protein, try using the gene name, as this is the key information stored in the database.
If you want to use a compound not yet included on the Portal, check our chemical probe criteria to determine whether a compound is potent and selective enough for use in research.
Suggest new compounds
We are continually adding new compounds and protein targets to the Portal, but you can help us with this effort. If you think we are missing a protein target that has chemical probes, please suggest a compound or protein.
If you still need to find the best chemical probe for your target of interest as soon as possible, you can consider using alternative resources.
Probe Miner, is a data-driven resource that provides objective assessment of chemical probes in public large-scale, publicly available, medicinal chemistry databases. Probe Miner can be searched by target and the best probes according to their computed scores will appear on top. However, be aware that the compounds on top of the list might not be sufficiently potent, selective or characterised in cells to study your target of interest. Please check our criteria for a good chemical probe.
In addition, you can also explore the sets of chemogenomic compounds assembled by the EUbOPEN Project. Chemogenomic compounds might not be as selective as probes, and sufficient controls are needed to make sure that the phenotype you observe is due to the modulation of the target you are interested in.
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: www.chemicalprobes.org.
Support the Portal by submitting new compounds or suggesting new compounds or targets for us to include.
If you are a team leader or equivalent, please consider supporting the Portal by joining our Scientific Expert Review Panel and reviewing probes.
If you would like to donate to the Portal, please get in touch with the team.