Results

The completion of the REPODRUG involved three phases that will be summarized below.

Phase I

In the first phase of the project, the optimization of the HTS on FFAR1 and FFAR4 receptors was performed and successfully completed. In order to do that, we chose CHO line as a suitable cellular system to heterologoulsy express human FFAR1 and FFAR4 receptors. Since both of them are coupled to Gq/11 proteins, we designed the assay to deliver optimum results using intracellular calcium measurement in real time. The HTS conditions were optimized by using two well known synthetic agonists of FFAR1 and FFAR4, as well as endogenous ligands.

As shown in Figure 1 the stimulation of CHO cells expressing FFAR1 receptors with the synthetic agonist TAK-875, led to robust calcium transients that were quantified and expressed as area under the curve (AUC). In cells not expressing the receptor, the effect is absent.

Figure 1 Effect of 10 uM TAK-875 on intracellular calcium in CHO cells expressing human FFAR1 together with a cytosolic calcium-sensitive probe called G5A.

In Figure 2, the stimulation of the human FFAR4 receptor with several dietary lipids indicated that linoleic acid could significantly activate FFAR4 receptor.

Figure 2 Effect of indicated lipids on intracellular calcium in CHO cells expressing human FFAR4 together with a cytosolic calcium-sensitive probe called G5A.

Phase II

In Phase II of the project. the FDA-approved library has been tested on human FFAR1 and FFAR4 receptors for the ability of the individual compounds to activate both receptors. The compounds of the library were tested at a final concentration of 30 uM. Table 1 presents compounds with the capacity to activate cells that heterologously express either human FFAR1 or human FFAR4.

Table 1 Compounds found in HTS with shown ability to increase intracellular calcium in cells heterologously expressing human FFAR1 and human FFAR4.

In the next phase, these compounds will be tested for their specificity and selectivity.

Phase III

Following the identification of the compounds with potential agonistic effects on FFAR1 and FFAR4

we further examined their specificity of action, meaning the analysis of the dependency of the observed cellular effects on the presence of both FFAR1 and FFAR4. unfortunately, the observed effects were not dependent on the receptors, suggesting that endogenous receptors were responsible for the cellular responses.

Based on these conclusions, we further expanded our screening campaign, by including new compounds. As shown in Figure 3, we identified four compounds that specifically activated the human FFAR1 receptor.

Figure 3 Effect of the solvent and of the indicated compounds on the intracellular calcium in CHO cells expressing empty vector (pcDNA3.1) or human FFAR1 receptor. All the substances were tested at 30 uM.

The pharmacological profiling of these compounds demonstrated that they are potent FFAR1 receptor agonists (Figure 4).

Figure 4 Concentration-dependent responses of the identified compounds on heterologously expressed human FFAR1 receptor in CHO cells together with the cytosolic probe, G5A.

The analysis of the selectivity of the identified compounds demonstrated that cpds 1-4 were specific for FFAR1 as they were not active on the other free fatty acids receptors, FFAR2 and FFAR3.

Next, we wanted to see whether these compounds increased glucose-stimulated insulin secretion (GSIS) in an insulinoma cell line, such as Ins-1.

As shown in Figure 5, compounds 1 to 4 (cpds 1-4) were able to significantly increase GSIS in Ins1 cells, an effect that was reduced by the FFAR1 receptor antagonist.

Figure 5 Effect of the indicated compounds tested at 30 uM on GSIS in Ins 1, in the presence (solvent) or the absence of FFAR1 receptor antagonist (GW1100, 30 uM).

Conclusion

The project has been successfully completed with the finding of specific and previously undescribed compounds that have the ability to activate FFAR1 and increase GSIS in an FFAR1 receptor-specific manner. These results have implications in fundamental science as well as in the pharma industry. as two of the identified substances (cpd 1 and 2) have important medical and industrial properties being widely used.