What is a Chemical Probe?

A chemical probe is simply a reagent—a selective small-molecule modulator of a protein’s function—that allows the user to ask mechanistic and phenotypic questions about its molecular target in cell-based or animal studies.

What is the difference between a chemical probe and a drug?

Although there are major synergies between basic chemical biology and drug discovery research, small-molecule drugs and chemical probes can be very different. From a drug discovery perspective, chemical probes can be key players in the validation of new molecular targets for a therapeutic indication. They can reduce the technical or biological risk of pursuing a particular pathway or target. However, small-molecule drugs and chemical probes are typically distinct molecules tailored to distinct purposes. For example, drugs need not have a highly selective activity profile; indeed, many medicines manifest their clinical effects through polypharmacology. Chemical probes do not need to meet the same requirements in terms of pharmacokinetics, pharmacodynamics, and bioavailability as a drug, but they do need to be potent and selective for their target and engage their target in the applicable biological system.

Why should I use chemical probes?

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. Thus, they can be used to discover new biology relating to that 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.

What is a PAINs molecule?

A PAINs molecule is a Pan-Assay Interference compound, or a compound that is promiscuously active in high-throughput screening assays for any number of reasons including that it induces aggregate formation, reacts non-specifically with proteins or directly interferes with the screening assay.

What is an orthogonal probe?

For any given protein target, scientists would ideally design experiments using two structurally distinct chemical probes (orthogonal probes), as well as their inactive derivatives. The application of orthogonal probes together decreases the probability that a researcher will attribute the off-target activity of a probe to the protein of interest because two probes with distinct structure are not likely to have the same profiles of off-target activity.

Why do you recommend more than one chemical probe as well as a negative control for the same target?

Data for chemical probes is inherently incomplete. A compound that has been shown to modulate protein X without affecting any other proteins surveyed 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 an orthogonal off-target profile; if the results of the two unrelated compounds are similar, there is increased confidence that the effects are from interaction at the target protein. Secondly, we strongly recommend the use of negative control that is structurally related to the known active molecule. For negative control, the concordance between loss of activity against the suspected target in vitro and the loss of a target-related phenotype elicited by the probe in a biological system (cells/organisms) increases confidence that the activity of the probe is indeed related to activity at the designated molecular target.

What is target engagement?

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. 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. Absent 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. Was the target invalid, or did the probe fail to engage the target? 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.

How do I use a chemical probe in cells? How do I know what concentration I should use for my experiment?

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 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.

How does the Portal determine recommended concentrations for each probe?

For each probe, the submitter has the opportunity to recommended concentrations for use. During review by our SAB, we ask our advisors whether they agree with the recommended concentrations or to provide alternate concentrations. In cases where there is disagreement, the Portal typically recommends the lower concentrations as the application of chemical probes at high concentrations, concentrations that over run a probe’s selectivity window, is a common and concerning problem. 

How do I use a chemical probe in vivo? How do I know what concentration I should use for my experiment?

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 avoid 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, potentially some way towards 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 animals 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.

Can I submit a probe I have published for consideration by the Chemical Probes Portal?

Yes. Please submit your probe using our online submission form. You must register for a Portal user account to submit a probe here.

Can I submit my peptide for consideration as a chemical probe?

We are not currently considering bio-active peptides in the portal.

I have data that have not been published that further validate a chemical probe. How can I submit the data to the portal?

At this time, the portal cannot accept data that are not already published in a journal. We aim to add data-hosting capabilities in the near future. We will update this site as soon as this changes.

Why was my chemical probe submission triaged by portal staff rather than considered by the SAB?

There are a lot of ‘chemical probes’ reported in the scientific and patent literature or available through chemical vendors, and scientists use the term chemical probe to mean different things. For consideration by the portal SAB, 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
  • The chemical probe has been deposited to PubChem
  • Data supporting the probe’s validation include 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 processes.

If I disagree with the decision or recommendation of the portal or the portal’s SAB, how can I appeal the decision?

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.

I would like to join the portal’s SAB. What should I do?

Please submit your application here.

I would like more information about the Portal. 

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