Me Like One of Your French Cells: The Potential of Real-Time Compound Bioactivity
Analysis in Drug Development


With the
advent of cell-painting, an experimental technique for quickly and
simultaneously analyzing dozens of cells exposed to unique compounds, the
efficient annotation of compounds’ bioactivity and their potential use in
medicine is within reach1.

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            The use of small molecules to elicit
certain effects in diseased cells is an increasingly popular and effective
treatment option for many illnesses, and with improved techniques, developing new
compounds has become increasingly fast and easy. Unfortunately, the analysis of
these molecules’ effects on cellular functions and structures has been hindered
by the challenges, time, and costs of their in
vivo analysis2. However, a recent study by Gerry et al. (2016) presents a potentially
game-changing approach to this issue and offers the chance to dramatically
increase the speed, efficiency, and ease of such analyses. The product of their
work has the potential to revolutionize drug development; by offering a quick
assessment of compounds within cells and their categorization based on function,
rather than just structure, the use of these compounds as new drugs to fight a
plethora of diseases will be expedited and more cost-effective.

            Gerry et al. demonstrated the power of simultaneously profiling several
synthetic compounds in vivo with an
experimental technique called cell painting3, allowing them to test
for the compounds’ effects on cell processes1. The authors married
this technique to the creation of libraries in which chemical compounds are then
sorted based on their functional patterns within cells1. The
sensitivity of the assay is profound, capable of delineating the unique effects
of compounds with significant structural similarities in a manner not possible
by more traditional analyses1. Accordingly, it sets the stage for determining
the compounds’ mechanisms of action (MoAs), how the molecules affect cells in
terms of specific cell processes and structures, and offers the chance to
categorize molecules based on MoA to better group new potential drugs1,4-6.

            The researcher group first synthesized
ten compounds containing pyrroles, five-membered ring structures consisting of
four carbons and a nitrogen, and transformed them to develop two unique
products from each1,7-10. These products were either aziridines,
containing a three-membered ring with a nitrogen and two carbons, or secondary
imines, containing a carbon-nitrogen double-bond where the nitrogen is also bound
to a second carbon1. Each pyrrole and its subsequent aziridine and
imine are constitutional isomers, meaning that they have identical atomic
compositions but a different arrangement of those atoms1. This
allows for the structure of the atoms forming the compounds’ backbones to potentially
remain consistent (although significant differences can still occur), while the
structures of the outlying ones are more diverse, offering for a wide range of
geometric possibilities to be explored11.

            When using cell painting to profile
the function of each molecule, the authors simultaneously applied one new
compound (at multiple concentrations) to a collection of bone cancer-derived
cells, 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 cellular
structures, the researchers imaged them via fluorescence microscopy (Fig. 1)
1,12. This allowed for their morphological features to be visualized and
compared to control cells that were not exposed to the compounds1,12.
Each compound was given an activity score to quantify its effect on the target
cell, a value derived from the difference between test and control cells1.
Small molecules that killed cells, inhibited their reproduction, or altered
their morphology were the most active. Furthermore, pairwise comparisons of the
molecules’ effects allowed for insights towards the MoAs of the most bioactive
compounds1. A group of nitrile-containing molecules showed the
strongest bioactivity, a trait seen in other nitriles14, which the researchers
presume is due to their ability to, depending on their surroundings, serve as
both nucleophiles and donate electrons, and as electrophiles, with a tendency
to attract electrons1,14. By sorting new compounds in libraries based
on 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 aid
in 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. Their
methods 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 diverse
structural features to delineate their MoAs1,15,16. Ultimately,
these methods will be useful for generating large libraries of synthetic
molecules, organized based on function, rather than just structure, and offers
the opportunity to improve the speed and efficiency of drug discovery.

Figure 1. Cell painting image of U2OS cells. Cells were incubated
for 24 h in DMSO solution (negative control) or with compounds 5c or 10b,
stained with five dyes, and imaged at wavelengths specific to each via
fluorescence microscopy to discern changes in cell structure and population
levels. Figure from Gerry et al. (2016).