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Nuisance compound advisor

Some chemical compounds can bind non-specifically or be broadly reactive with biomolecules, causing promiscuous activity general toxicity in cellular and in vivo assays. Or they can interfere with biochemical or cellular in vitro assays. leading to an apparent biological effect that is in fact an artefact of the assay system. A useful catch-all term for these is ‘nuisance compounds’ and nuisance behaviour should be avoided in chemical probes1. Many such nuisance 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 non-specific activities. If you are a biologist, consider consulting a chemical biologist or medicinal chemist for additional insights on this topic.

Depending on the assay format used, biochemical or cellular, compounds that produce misleading results, such as are described above, have been grouped into those containing toxicophores – meaning substructures or functional groups that often lead to toxicity, commonly due to widespread chemical reactivity – and those which cause interference with a biochemical assay, leading to misleading readouts. Examples of these nuisance compounds include those that undergo colloidal aggregation, causing non-specific inhibition due to absorption onto the surface of proteins2,3,4; those that react with thiols groups in multiple proteins5; compounds that take part in redox chemistry, which can interfere with detection methods in both biochemical and cellular assays6; and, in the case of cellular assays, compounds that exhibit toxic effects not related to a particular protein target, including phospholipidosis7. Any of these types of non-specific behaviour can confound the interpretation and robustness of experimental results using a small molecule to investigate the involvement of a specific biological target. In addition to nuisance effects in in vitro cellular systems, such behaviour can also cause problems such as toxicity in studies in model organisms. Therefore, it is important to be aware of the risk of these behaviours occurring for a particular chemical structure and take action to evaluate it further and/or mitigate the risk1.

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 published toxicophores and substructures shown to cause assay interference, using the canSAR chemical registration pipeline8. We look for markers of potential assay interference using RDKit9, based on the ‘PAINS’ set assembled by Baell and Holloway10 and we use a list of toxicophore substructures assembled by Hughes and colleagues11. When we detect that a probe possesses any of the known substructures of concern, we ‘flag’ them, and the probe is consequently labelled as containing potential assay-interfering and/or toxicophore substructures, which, as mentioned above, we collectively term ‘nuisance compounds’.

It is important to stress that the presence of these 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, such as if a compound is coloured or fluorescent and a spectrophotometric or spectrofluorimetric readout is being used. Other interferences may be related to the concentration of the compound used as precipitation of a substance can cause non-specific effects. Therefore, a flag is simply a calculated alert to consider when assessing whether to use a particular compound in biological experiments.

It is important that users and probe reviewers are aware of the strengths and limitations of such alerts12 when conducting an experiment with a compound flagged with a structural alert. If there is possible concern, it is important to design experiments to mitigate the risk of confounding effects due to non-specific toxicity or interference with the specific assay used, and/or to use orthogonal techniques to validate the results, including the employment of appropriate controls. Simple assays are available to test for various undesirable effects listed here12.

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. Jung Y, Noda N, Takaya J, Abo M, Toh K, Tajiri K, Cui C, Zhou L, Sato S-I, Uesugi M. Discovery of Non-Cysteine-Targeting Covalent Inhibitors by Activity-Based Proteomic Screening with a Cysteine-Reactive Probe. ACS Chem. Biol. 2022;17:340–347. DOI: 10.1021/acschembio.1c00824.
  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. 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.
  8. 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.
  9. RDKit version 2021.03.1 rdkit.org.
  10. 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.
  11. 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.
  12. 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.