Testing Cytokine Production of Small Molecule, Organically Synthesized Drug Candidates

The Problem

A biotechnology startup designed a panel of compounds to activate macrophages, dendritic cells and B cells. They wanted to know how structural changes would affect cell activation, so they turned to Astarte to test cytokine production of cells following culture with their compounds.

The Results

Our initial experiment did not detect any significant cytokine production in B cells, so we replicated the experiment using monocytes. Monocytes are known to produce numerous cytokines including IL-6, TNF-α and MIP-1α, and this experiment was no exception. After overnight culture, the levels of MIP-1α produced were off the charts, beyond the upper limit of the assay. There was little IFNγ, IL-10 or IL-12, but IL-6 and TNF-α gave a useful measure of the activity of the compounds.

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Figure 1. Increasing concentrations of compound 1 stimulate increasing production of IL-6. Compounds 2 and 4 also stimulate IL-6 production but at 30 fold lower concentration than compound 1. Compound 18 is intermediate in its ability to stimulate IL-6.

Figure 1 shows the differences in IL-6 production following culture for four of the compounds tested. Each compound in the panel activated cytokine production by monocytes, but compound 1 was less potent than the others. IL-6 production was highest at the highest concentration, 30 µM. Compounds 2 and 4 were most active at the lowest concentration, and compound 18 had peak activity at concentrations above 1 µM but below 30 µM.

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Figure 2. The metabolic activity of monocytes (ATP levels) decreases in the presence of increasing concentrations of the test compounds.

A sharp-eyed Astarte scientist noticed that the monocytes looked dead at the top concentrations of these compounds, so she used a chemiluminescent reagent to generate a more objective measurement of the ATP levels (Figure 2). CellTiter-Glo® is sold by Promega and is a convenient reagent for measuring cell numbers and metabolic activity. The amount of ATP is directly related to the number of cells present but is also affected by cell metabolism. By adding CellTiter-Glo® to the cultures and measuring the luminescence, the differences in the cultures was obvious. In all cases, the ATP in the cultures decreased as the concentration of the test compounds increased. This confirmed the visual inspection of the cultures but also detected changes that were not visually apparent.

The Conclusion

Our research concluded that all of the compounds in the panel had activity, but the ratio of activity and toxicity was different among the compounds.

Follow-up experiments are needed to determine the optimal concentration for compounds 2 and 4 and to further understand the toxicity or metabolic effects of these compounds. Since this experiment included many other compounds, it informed the synthetic chemistry approach to focus on the eventual drug candidate.

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