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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' Modulators
Biochemical potency Evidence of target binding or modulation (biochemical/biophysical). (In-vitro IC50/Ki/Kd, etc. <100 nM )
Selectivity Evidence of wider in vitro profiling (biochemical/biophysical/cellular), especially within protein class (>30-fold selectivity within family)
Broader selectivity profiling outside target family is desirable
Target engagement or modulation in cells Evidence and quantification ( <1μM ) of target engagement
  • Direct measurement of target engagement (e.g. in cell binding or stabilisation) or proximal PD biomarker (e.g. specific phosphosite)
  • Phenotype is target-engagement dependent
Inactive control Similar structure with same reactive group but modifications in other parts of the molecule, non-binding against the target ( >100 fold weaker on target)
Compound properties Consider solubility, stability, potential assay interference or promiscuous motifs
Chemical Stability Use of reactivity assay to rule out promiscuity
Cytotoxicity Absence of compound toxicity in cells (>10-fold window compared to cellular potency, unless target-mediated)
Usage Use at or below the recommended concentration
Use inactive control and orthogonal active compounds where available

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.