The Unsuitables: compounds that are too toxic; too promiscuous; interfere with biological assays; or imperfect compounds that have been replaced by better ones. The Unsuitables, formerly referred to on the Chemical Probes Portal as the Historical Compounds, are chemicals deemed inappropriate for use as chemical probes in interrogating specific protein targets.

Chemical probes play a crucial role in biological research. However, there are many compounds that are widely used in research but are not suitable to learn about the biology of a specific protein target. Some of these are listed on the Portal as the Unsuitables, a list of 254 compounds which are unsuitable for use as a chemical probe.

Many of these compounds exhibit insufficient selectivity, impacting numerous proteins. For instance staurosporine, initially intended for protein kinase C, inhibits nearly 400 proteins.

Pathfinder compounds, discovered early in drug development, may demonstrate the feasibility of targeting a specific protein but lack sufficient selectivity to be used to test the specific functions of this one protein. Over time, more potent and selective molecules are discovered, but the original pathfinders often remains in use. An example is LY294002, an initially useful but nonselective PI3K inhibitor now classified as an Unsuitable, with more effective inhibitors available on the Portal (e.g. BYL-719, Pictilisib and Leniolisib).

Some compounds directly interfere with biological assays. Mechanisms for this include colloidal aggregation: causing non-specific inhibition due to absorption onto the surface of proteins [1,2,3]. In cellular assays, toxic effects such as phospholipidosis [4] can occur. Compounds engaging in redox chemistry may interfere with detection methods in both biochemical and cellular assays [5]. General toxicity to cells, particularly in cancer research, can confound results, as compounds with generalised toxicity might produce a particular biological phenotype in cells but lack robust evidence against putative protein targets.

Being listed as an Unsuitable does not mean that the compound should not be used at all in biological research. There may be legitimate value in using a compound that is, for example, active against many kinases in the cell. However, it does mean that the compound should not be used as a probe acting on a specific protein of interest.

Understanding the limitations of the compounds is crucial for accurate experimental interpretation. The list of Unsuitables, along with reviewed compounds on the Chemical Probes Portal, aids in assessing compound suitability for biological research.

Users of the Portal have consistently told us that they find the Unsuitables list very useful. The current list is not comprehensive, but is a collection of compounds that are highly problematic or flawed in various ways. We rely on the community to flag up compounds to be included in the list. So do please let us know if you have examples of Unsuitables.

We will continue to add compounds to the list of Unsuitables, as well as to the Portal. Suggestions for chemical probes can be made through the Portal submission form. For Unsuitables, please get in touch to suggest compounds we should add.

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.
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.
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.
Hu H, Tjaden A, Knapp S, Antolin AA, Müller S. A machine learning and live-cell imaging tool kit uncovers small molecules induced phospholipidosis. Cell Chem Biol 2023 Sep 27:S2451-9456(23)00322-7. DOI: 10.1016/j.chembiol.2023.09.003.
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.

Introducing the Unsuitables