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 Biol. 2015; 11, 536-541.
• Blagg J, Workman P. Choose and use your chemical probe wisely to explore cancer biology. Cancer Cell. 2017; 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.
|
|
