Chemical Probe Criteria for classical modulators and bifunctional degraders (e.g. PROTACs)

In an effort to simplify the selection of Chemical Probes and bifunctional degraders (e.g.  PROteolysis TArgeting Chimeras, PROTACs), the Chemical Probes Team has put together a list of criteria to select the best available compounds for your research. We welcome your input, please send us your feedback.




 

‘Classical’ Modulator

Degrader

Activity

Evidence of target binding/activity modulation. (In-vitro IC50/Ki/Kd etc)

Evidence of binding to target and E3-ligase (CRBN, VHL, etc) or other non-E3 degradation effector complexes

Inactive control

Similar structure with similar physicochemistry, non-binding against target

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.

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 orthogonal probe with alternative chemotype, together with biomarker, to demonstrate target dependence)

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

• Assessment of effect on potent off-target(s) identified from in vitro profiling
• Orthogonal probe (active but different chemotype). Desirable
• 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

Demonstrable by steps above

Key references

•  Antolin AA, Workman P, Al-Lazikani B. Public resources for chemical probes: the journey so far and the road ahead. Future Med Chem. 2019
•  Arrowsmith CH, et al. The promise and peril of chemical probes. Nat Chem Biol2015; 11, 536-541.
•  Blagg J, Workman P. Choose and use your chemical probe wisely to explore cancer biology. Cancer Cell2017;  32, 9-25.
•  Bunnage ME, Chekler EL, Jones LH. Target validation using chemical probes. Nat Chem Biol. 2013; 9, 195-199.
• 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.
•  Frye SV. The art of the chemical probe. Nat Chem Biol. 2010; 6, 159-161.
• Kostic M, Jones LH. Critical Assessment of Targeted Protein Degradation as a Research Tool and Pharmacological Modality. Trends Pharmacol Sci. 2020; 41, 305-317.
•  Workman P, Collins I. Probing the probes: fitness factors for small molecule tools. Chem Biol. 2010; 17, 561-577.