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
- Desirable: Orthogonal probe (active
but different chemotype)
| -
Evidence of in-cell target selectivity
-
e.g. degradation profile measured by MS /
proteomics
-
e.g. measurement of off-target engagement /
inhibition / depletion
- Desirable: Orthogonal probe (active
but different chemotype)
|
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. 2021
Apr;13(8):731-747. doi:
10.4155/fmc-2019-0231.
-
Arrowsmith CH, et al. The promise and peril of
chemical probes. Nat Chem Biol. 2015; 11, 536-541
doi:
10.1038/nchembio.1867.
-
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.
-
Bunnage ME, Chekler EL, Jones LH. Target validation
using chemical probes. Nat Chem Biol. 2013; 9,
195-199 doi:
10.1038/nchembio.1197.
-
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 doi:
10.1016/j.ddtec.2019.02.002.
-
Frye SV. The art of the chemical probe. Nat Chem
Biol. 2010; 6, 159-161 doi:
10.1038/nchembio.296.
-
Kostic M, Jones LH. Critical Assessment of Targeted
Protein Degradation as a Research Tool and
Pharmacological Modality. Trends Pharmacol Sci.
2020; 41, 305-317 doi:
10.1016/j.tips.2020.02.006.
-
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.
|