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Review
. 2021 Mar 18;28(3):356-370.
doi: 10.1016/j.chembiol.2021.01.021. Epub 2021 Feb 15.

Nuisance compounds in cellular assays

Jayme L Dahlin  1 , Douglas S Auld  2 , Ina Rothenaigner  3 , Steve Haney  4 , Jonathan Z Sexton  5 , J Willem M Nissink  6 , Jarrod Walsh  7 , Jonathan A Lee  8 , John M Strelow  9 , Francis S Willard  9 , Lori Ferrins  10 , Jonathan B Baell  11 , Michael A Walters  12 , Bruce K Hua  13 , Kamyar Hadian  3 , Bridget K Wagner  14
Affiliations
Review

Nuisance compounds in cellular assays

Jayme L Dahlin et al. Cell Chem Biol. .

Abstract

Compounds that exhibit assay interference or undesirable mechanisms of bioactivity ("nuisance compounds") are routinely encountered in cellular assays, including phenotypic and high-content screening assays. Much is known regarding compound-dependent assay interferences in cell-free assays. However, despite the essential role of cellular assays in chemical biology and drug discovery, there is considerably less known about nuisance compounds in more complex cell-based assays. In our view, a major obstacle to realizing the full potential of chemical biology will not just be difficult-to-drug targets or even the sheer number of targets, but rather nuisance compounds, due to their ability to waste significant resources and erode scientific trust. In this review, we summarize our collective academic, government, and industry experiences regarding cellular nuisance compounds. We describe assay design strategies to mitigate the impact of nuisance compounds and suggest best practices to efficiently address these compounds in complex biological settings.

Keywords: artifacts; chemical biology; drug discovery; high-content screening; high-throughput screening; interference; nuisance compounds; phenotypic drug discovery; phenotypic screening.

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Conflict of interest statement

Declaration of interests B.K.W. is an editor of Cell Chemical Biology.

Figures

Figure 1.
Figure 1.. Misconceptions and nomenclature regarding cellular nuisance compounds.
Figure 2.
Figure 2.. Cellular nuisance compound framework.
Interferences can be broadly divided into technology-related (“artifacts”) and non-technology-related categories (undesirable, nonspecific activity), and can overlap. Most non-technology-related interference mechanisms can lead to cytotoxicity. High-quality hits can still have interference such as auto-fluorescence or selective cytotoxicity.
Figure 3.
Figure 3.. Common pitfalls of cellular nuisance compounds.
(A) Pfizer model for conceptualizing cellular assay readouts (Vincent et al., 2015). (B) KAT3 inhibitors A-485 and C646 both decrease cellular H3K27ac levels as expected, but through specific and nonspecific target engagement, respectively (Dahlin et al., 2017; Lasko et al., 2017; Shrimp et al., 2015). (C) Active cell painting morphologies are enriched in compounds that decrease final cell number (Bray et al., 2017). (D) Compounds can fluoresce and interfere with the interpretation of readouts in microscopy-based cellular assays. Scale: 50 μm. (E) Nuisance compounds from phenotypic assays can appear “active” in subsequent target-based assays and are at risk for confirmation bias.
Figure 4.
Figure 4.. Proactively addressing cellular nuisance compounds during assay development.
Figure 5.
Figure 5.. Approaches to identify cellular nuisance compounds.
Figure 6.
Figure 6.. Decision aid for nuisance compound triage.
Figure 7.
Figure 7.. Framework for proposed nuisance compound informer set.
See also Supplemental File 1.
See this image and copyright information in PMC

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