Chemical Probes logo

< back to information centre home

Chemical Probes (Classical Modulators) Criteria

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.