# Live Cell Monitoring of Phosphodiesterase Inhibition by Sulfonylurea Drugs

## TL;DR

This study shows that sulfonylurea drugs used for diabetes can increase cAMP levels by inhibiting phosphodiesterases and activating Epac2 proteins in live cells.

## Contribution

The study reveals a new mechanism of sulfonylurea drugs involving direct PDE inhibition and distinct Epac2 activation.

## Key findings

- SUs induce conformational changes in Epac2 but not in Epac1.
- SUs inhibit PDE3 and PDE4 families, increasing local cAMP levels.
- Tolbutamide inhibits PDE activity through an allosteric mechanism.

## Abstract

Sulfonylureas (SUs) are a class of antidiabetic drugs widely used in the management of diabetes mellitus type 2. They promote insulin secretion by inhibiting the ATP-sensitive potassium channel in pancreatic β-cells. Recently, the exchange protein directly activated by cAMP (Epac) was identified as a new class of target proteins of SUs that might contribute to their antidiabetic effect, through the activation of the Ras-like guanosine triphosphatase Rap1, which has been controversially discussed. We used human embryonic kidney (HEK) 293 cells expressing genetic constructs of various Förster resonance energy transfer (FRET)-based biosensors containing different versions of Epac1 and Epac2 isoforms, alone or fused to different phosphodiesterases (PDEs), to monitor SU-induced conformational changes in Epac or direct PDE inhibition in real time. We show that SUs can both induce conformational changes in the Epac2 protein but not in Epac1, and directly inhibit the PDE3 and PDE4 families, thereby increasing cAMP levels in the direct vicinity of these PDEs. Furthermore, we demonstrate that the binding site of SUs in Epac2 is distinct from that of cAMP and is located between the amino acids E443 and E460. Using biochemical assays, we could also show that tolbutamide can inhibit PDE activity through an allosteric mechanism. Therefore, the cAMP-elevating capacity due to allosteric PDE inhibition in addition to direct Epac activation may contribute to the therapeutic effects of SU drugs.