The Ecstasy and Agony of Assay Interference Compounds

Alarmingly, up to 80–100% of initial hits from screening can be artifacts if appropriate control experiments are not employed. The source of this artificial behavior has been thoroughly summarized in the literature.9？12 Misleading assay results can arise through a variety of mechanisms including covalent protein reactivity,13 redox activity, interference with assay spectroscopy,14？16 membrane disruption,17 decomposition in buffers,18 and the formation of colloidal aggregates.2,19,20 If not properly controlled, colloidal aggregation is perhaps the most common artifact from high-throughput screening: between 1 and 3% of molecules in many screening libraries will aggregate at relevant concentrations and up to 95% of “hits” identified from a screen can be assigned as aggregates,21 and the colloids that they form inhibit,20？22 or occasionally activate, proteins.23 PAINS molecules can be synthetic in origin or derived from natural products; the latter have been termed Invalid Metabolic PanaceaS, or IMPS.24 Even marketed drugs can aggregate and may also contain PAINS chemotypes. Over 60 FDA-approved and worldwide drugs contain PAINS chemotypes,25 and about the same number have been shown to aggregate.26 Although some drugs can contain PAINS and can aggregate at micromolar concentrations, such examples do not imply that any molecule that acts via a PAINS or aggregation mechanism can become a drug. Hence, noting or “flagging” any PAINS-containing hits and performing detailed follow-up experiments are essential to validate that the function of the molecule is as expected prior to discarding it from further consideration.27 However, it is important to realize that no PAINS-containing drug has ever been developed starting from a protein-reactive PAINS target-based screening hit.2