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Covalent Inhibitors
In the past decade, covalent small molecules have seen a surge, both in clinical drugs as well as in research tools to study protein functions. However, similar to reversible small-molecule probes, the quality of covalent molecular probes is crucial for their value in cell biology studies. Rigorous evaluation of covalent molecules is necessary to state with confidence that they are specific modulators of a protein target. In particular, the time-dependency of covalent inhibitors needs to be taken into consideration. Based on the publication by Hartung et al, the Chemical Probes Team proposes the following criteria for covalent chemical probes.
| Covalent Probes | |
|---|---|
| Biochemical potency |
Evidence of in vitro target binding/activity
modulation. Provide kinact/Ki (rate of inactivation/potency to the target) which is more informative than Ki/Kd, etc. |
| Selectivity |
Evidence of proteome-wide selectivity Alternatively, evidence of wider in vitro profiling (>30-fold within protein family) and broader selectivity profiling outside target family is desirable |
| Target engagement or modulation in cells |
Evidence and quantification ( <1μM ) of
target engagement
|
| Inactive Controls | Similar structure with same reactive group but modifications in other parts of the molecule, non-binding against the target (>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 |
- 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)