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Toxicophores and PAINS Alerts

Some chemical compounds can be broadly reactive with biomolecules, causing promiscuous activity and general toxicity in cellular and in vivo assays, or can interfere with biological or in vitro assays leading to an apparent biological effect that is in fact an artefact of the assay system.

Many such molecules are enriched in certain chemical groups or substructures, and the presence of these chemical features can be worth taking into consideration as potential alerts for unwanted activities. It can be helpful to distinguish between compounds containing toxicophores – meaning substructures or functional groups often leading to toxicity, commonly due to widespread chemical reactivity – and so-called Pan-Assay Interference compounds (PAINS) – which are compounds that can interfere with biochemical and cell-based assay detection methods leading to false readouts. Compounds that undergo colloidal aggregation can cause non-specific inhibition due to absorption onto the surface of proteins 2,3,4. In cellular assays, non-target related toxic effects can include phospholipidosis 5. Compounds that take part in redox chemistry can interfere with detection methods in both biochemical and cellular assays 6.

Any of these types of non-specific behaviour can confound the interpretation and robustness of experimental results using a small molecule to investigate the effect of a specific biological target. Therefore, it is important to be aware of the risk of these behaviours occurring for a particular chemical structure 1.

Within the Chemical Probes Portal, the chemical structure of each potential chemical probe that is submitted to us is screened virtually against a library of known toxicophores and PAINS substructures using the canSAR chemical registration pipeline 7 . When we detect that a probe possesses any of the known substructures, we ‘flag’ them, and the probe is consequently labelled as containing potential PAINS and/or toxicophore substructures. We look for markers of pan-assay interference using RDKit 8, based on the PAINS set assembled by Baell and Holloway 9 and we use a list of toxicophore substructures assembled by Hughes and colleagues 10. It is important to stress that the presence of the toxicophore or PAINS substructures within the chemical structure of a compound does not necessarily mean that it will be non-specifically active or toxic, or give rise to assay interference. The substructure alerts can sometimes produce false positives. Some assay interferences are associated with particular assay conditions or technologies. Therefore, a toxicophore or PAINS flag is simply an alert to consider.

It is important that users are aware of the strengths and limitations of such alerts 11 when conducting an experiment with a compound flagged with a structural alert. For scientists who are not specialists in chemical biology, talking to an expert may be helpful. Because of the potential for artefactual results, it is even more important to design experiments to mitigate the risk of confounding effects due to non-specific toxicity or assay interference. Simple assays are available to test for the various undesirable effects listed here 11. The use of multiple, structurally distinct chemical probes, negative control probes and varied, orthogonal assay technologies is strongly recommended.

References

  1. Baell J and Walters MA. Chemical con artists foil drug discovery. Nature 2014 513: 481-3. DOI: 10.1038/513481a.
  2. LaPlante SR, Roux V, Shahout F, LaPlante G, Woo S, Denk MM, Larda ST, Ayotte Y. Probing the free-state solution behavior of drugs and their tendencies to self-aggregate into nano-entities. Nature Protocols 2021 16: 5250–5273. DOI: 10.1038/s41596-021-00612-3.
  3. O'Donnell HR, Tummino TA, Bardine B, Craik CS, Shoichet BK. Colloidal Aggregators in Biochemical SARS-CoV-2 Repurposing Screens. J Med Chem 2021 64(23): 17530-17539. DOI: 10.1021/acs.jmedchem.1c01547.
  4. Irwin JJ, Duan D, Torosyan H, Doak AK, Ziebart KT, Sterling T, Tumanian G, Shoichet BK. An Aggregation Advisor for Ligand Discovery. J Med Chem. 58(17): 7076-87. DOI: 10.1021/acs.jmedchem.5b01105.
  5. Tummino TA, Rezelj VV, Fischer B, Fischer A, O'Meara MJ, Monel B, Vallet T, White KM, Zhang Z, Alon A, Schadt H, O'Donnell HR, Lyu J, Rosales R, McGovern BL, Rathnasinghe R, Jangra S, Schotsaert M, Galarneau JR, Krogan NJ, Urban L, Shokat KM, Kruse AC, García-Sastre A, Schwartz O, Moretti F, Vignuzzi M, Pognan F, Shoichet BK. Drug-induced phospholipidosis confounds drug repurposing for SARS-CoV-2. Science 2021 373(6554): 541-547. DOI: 10.1126/science.abi4708.
  6. Johnston PA. Redox cycling compounds generate H2O2 in HTS buffers containing strong reducing reagents – real hits or promiscuous artifacts? Curr Opin Chem Biol 2011 15(1): 174–182. DOI: 10.1016/j.cbpa.2010.10.022.
  7. Mitsopoulos C, Di Micco P, Fernandez EV, Dolciami D, Holt E, Mica IL, Coker EA, Tym JE, Campbell J, Che KH, Ozer B, Kannas C, Antolin AA, Workman P, Al-Lazikani B. canSAR: update to the cancer translational research and drug discovery knowledgebase. Nucleic Acids Res. 2021 49 (D1): D1074-D1082. DOI: 10.1093/nar/gkaa1059 .
  8. RDKit version 2021.03.1 rdkit.org.
  9. Baell JB and Holloway GA. New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays. J Med Chem 2010 53(7): 2719-40. DOI: 10.1021/jm901137j.
  10. Hughes JD, Blagg J, Price DA, Bailey S, Decrescenzo GA, Devraj RV, Ellsworth E, Fobian YM, Gibbs ME, Gilles RW, Greene N, Huang E, Krieger-Burke T, Loesel J, Wager T, Whiteley L, Zhang Y. Physiochemical drug properties associated with in vivo toxicological outcomes. Bioorg Med Chem Lett 2008 18(17): 4872-5. DOI: 10.1016/j.bmcl.2008.07.071.
  11. Aldrich C, Bertozzi C, Georg GI, Kiessling L, Lindsley C, Liotta D, Merz KM Jr., Schepartz A, Wang S. The Ecstasy and Agony of Assay Interference Compounds. J Med Chem 2017 60(6): 2165–68. DOI: 10.1021/acs.jmedchem.7b00229.