Table 1.

Select criteria for consideration of a molecule as a high-quality chemical probe

This list is not inclusive but is intended to provide an overview of the experimental approaches.

Chemical	Test the relationship between compound structure and function in multiple in vitro and cell-based assays Key molecules were examined for metabolic stability, reactivity, solubility, pharmacokinetics and other pharmacological properties Compound is active at the expected concentrations in cellular and animal models
Biochemical	Immobilized compound will “pull down” the target; this interaction is competitive with free compound but not with related controls Compound, but not control, will stabilize the target in vitro (thermal shift assays) and in cells (CETSA) Evidence of inactivity is against closely related family members Compound affects known biomarkers
Genetic	Treatment with compound resembles phenotype of knockdown or mutagenesis of the target Overexpression or knockdown of the target changes the potency of the molecule (short hairpin RNA or CRISPR screens) Resistance to the compound occurs by mutation of the target or target pathway (drug-resistance screening)