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, PROTACs work 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 SERP 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)
Biochemical potency	Evidence of binding to target and E3-ligase (CRBN, VHL, etc.) or other non-E3 degradation effector complexes (<100 nM)
Selectivity	Evidence of in-cell target selectivity e.g. degradation profile measured by mass spectrometry Evidence of wider in vitro profiling, especially within protein class ( >30-fold within family) Broader profiling outside target family is desirable
Target engagement or modulation in cells	Evidence and quantification of target engagement and degradation DC₅₀ and D_max values determined (<1μM, >80%) Time course for degradation defined Evidence of E3, ubiquitin and proteasome-dependence; or dependence on other effector pathways relevant to degradation mechanism
Inactive Controls	A compound that is inactive against the target, and a second non-binding to E3 ligase (or effector complex, >100 fold weaker on target)
Compound properties	Consider solubility, stability, potential assay interference or promiscuous motifs
Cytotoxicity	Absence of compound toxicity in cells ( >10 μM 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

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.
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.
I.V. Hartung et al. (2023) J Med Chem (DOI: 10.1021/acs.jmedchem.3c00550)