PaintMe Like One of Your French Cells: The Potential of Real-Time Compound BioactivityAnalysis in Drug Development With theadvent of cell-painting, an experimental technique for quickly andsimultaneously analyzing dozens of cells exposed to unique compounds, theefficient annotation of compounds’ bioactivity and their potential use inmedicine is within reach1.MitchellMann The use of small molecules to elicitcertain effects in diseased cells is an increasingly popular and effectivetreatment option for many illnesses, and with improved techniques, developing newcompounds has become increasingly fast and easy. Unfortunately, the analysis ofthese molecules’ effects on cellular functions and structures has been hinderedby the challenges, time, and costs of their invivo analysis2. However, a recent study by Gerry et al. (2016) presents a potentiallygame-changing approach to this issue and offers the chance to dramaticallyincrease the speed, efficiency, and ease of such analyses.
The product of theirwork has the potential to revolutionize drug development; by offering a quickassessment of compounds within cells and their categorization based on function,rather than just structure, the use of these compounds as new drugs to fight aplethora of diseases will be expedited and more cost-effective. Gerry et al. demonstrated the power of simultaneously profiling severalsynthetic compounds in vivo with anexperimental technique called cell painting3, allowing them to testfor the compounds’ effects on cell processes1. The authors marriedthis technique to the creation of libraries in which chemical compounds are thensorted based on their functional patterns within cells1. Thesensitivity of the assay is profound, capable of delineating the unique effectsof compounds with significant structural similarities in a manner not possibleby more traditional analyses1. Accordingly, it sets the stage for determiningthe compounds’ mechanisms of action (MoAs), how the molecules affect cells interms of specific cell processes and structures, and offers the chance tocategorize molecules based on MoA to better group new potential drugs1,4-6.
The researcher group first synthesizedten compounds containing pyrroles, five-membered ring structures consisting offour carbons and a nitrogen, and transformed them to develop two uniqueproducts from each1,7-10. These products were either aziridines,containing a three-membered ring with a nitrogen and two carbons, or secondaryimines, containing a carbon-nitrogen double-bond where the nitrogen is also boundto a second carbon1. Each pyrrole and its subsequent aziridine andimine are constitutional isomers, meaning that they have identical atomiccompositions but a different arrangement of those atoms1. Thisallows for the structure of the atoms forming the compounds’ backbones to potentiallyremain consistent (although significant differences can still occur), while thestructures of the outlying ones are more diverse, offering for a wide range ofgeometric possibilities to be explored11.
When using cell painting to profilethe function of each molecule, the authors simultaneously applied one newcompound (at multiple concentrations) to a collection of bone cancer-derivedcells, known as U2OS cells, placed in separate wells for 24 hours1,12.After staining the cells with a series of dyes that each highlight specific cellularstructures, the researchers imaged them via fluorescence microscopy (Fig. 1)1,12.
This allowed for their morphological features to be visualized andcompared to control cells that were not exposed to the compounds1,12.Each compound was given an activity score to quantify its effect on the targetcell, a value derived from the difference between test and control cells1.Small molecules that killed cells, inhibited their reproduction, or alteredtheir morphology were the most active. Furthermore, pairwise comparisons of themolecules’ effects allowed for insights towards the MoAs of the most bioactivecompounds1. A group of nitrile-containing molecules showed thestrongest bioactivity, a trait seen in other nitriles14, which the researcherspresume is due to their ability to, depending on their surroundings, serve asboth nucleophiles and donate electrons, and as electrophiles, with a tendencyto attract electrons1,14. By sorting new compounds in libraries basedon bioactivity and comparing such activity to known MoAs of other molecules,the determination of appropriate drugs for a given ailment will be expedited.
As well, comparing known MoAs to the bioactivities of test molecules will aidin determining the MoAs of such test compounds.In all, Gerry et al.established the effectiveness of cell painting in offering an efficient, powerful,and inexpensive method to characterize the bioactivity of unique compounds.
Theirmethods can be applied to all small molecules and in a wide range of cell types12,offering an opportunity to study the bioactivity of compounds of diversestructural features to delineate their MoAs1,15,16. Ultimately,these methods will be useful for generating large libraries of syntheticmolecules, organized based on function, rather than just structure, and offersthe opportunity to improve the speed and efficiency of drug discovery.Figure 1. Cell painting image of U2OS cells. Cells were incubatedfor 24 h in DMSO solution (negative control) or with compounds 5c or 10b,stained with five dyes, and imaged at wavelengths specific to each viafluorescence microscopy to discern changes in cell structure and populationlevels. Figure from Gerry et al.