PROTACs

Proteolysis targeting chimeras (PROTACs) are heterobifunctional small molecules composed of two active domains and a linker capable of promoting the degradation of targeted proteins. Rather than acting as conventional enzyme inhibitors, PROTAC works by inducing selective intracellular proteolysis. PROTACs consist of two covalently linked protein-binding molecules: one capable of engaging an E3 ubiquitin ligase, and another that binds to a target protein meant for degradation. Recruitment of the E3 ligase to the target protein results in ubiquitination and subsequent degradation of the target protein by the proteasome. Because PROTACs need only to bind their targets with high selectivity (rather than inhibit the target protein's enzymatic activity), there are currently many efforts to retool previously ineffective inhibitor molecules as PROTACs for next-generation drugs. 

The Chemical Probe Portal now lists several PROTAC molecules that have been evaluated by our SAB for use in cell and in vivo. Here we report the criteria to select the right PROTAC for your experiment: 



Degrader (PROteolysis TArgeting Chimeras, PROTACs)

Activity

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

 Control Compounds

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.

In-cell validation

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

• 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

Key References

• 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. 10.1016/j.ddtec.2019.02.002

• Kostic M, Jones LH. Critical Assessment of Targeted Protein Degradation as a Research Tool and Pharmacological Modality. Trends Pharmacol Sci. 2020; 41, 305-317 10.1016/j.tips.2020.02.006