CAMP is a universal second messenger. Its signalling is mediated by protein kinase A, Epac and certain types of ion channels in mammalians. cAMP signalling is involved in many physiological processes ranging from vision to the control of insulin secretion, pacemaker activity and gene transcription and therefore selective pharmacological interference is of medical interest. Whereas selective inhibitors of PKA and selective activators of Epac are well established, no inhibitors of Epac were available until recently. Here the action of four of the novel Epac inhibitors was analysed by biophysical means. ESI-05 is confirmed as a selective inhibitor of Epac2. No direct action of Brefeldin A on Epac could be demonstrated. ESI-09 and HJC0197 were found to act as chemicals with general protein denaturing properties and do not act on Epac selectively.

The second messenger cAMP is known to augment glucose-induced insulin secretion. However, its downstream targets in pancreatic β-cells have not been unequivocally determined. Therefore, we designed cAMP analogues by a structure-guided approach that act as Epac2-selective agonists both in vitro and in vivo. These analogues activate Epac2 about two orders of magnitude more potently than cAMP. The high potency arises from increased affinity as well as increased maximal activation. Crystallographic studies demonstrate that this is due to unique interactions. At least one of the Epac2-specific agonists, Sp-8-BnT-cAMPS (S-220), enhances glucose-induced insulin secretion in human pancreatic cells. Selective targeting of Epac2 is thus proven possible and may be an option in diabetes treatment. cAMP is a small molecule produced by cells that activates proteins involved in a wide range of biological processes, including olfaction, pacemaker activity, regulation of gene expression, insulin secretion, and many others. In the case of insulin secretion, cAMP seems to impinge on different stages of the signalling cascade to regulate secretory activity in pancreatic β-cells. Here we have developed a chemically modified version of cAMP that specifically only activates Epac2, one of the cAMP-responsive proteins in this cascade. Furthermore, our cAMP analogue activates Epac2 more potently than cAMP itself does. We have determined several crystal structures of Epac2 in complex with cAMP analogues to help us explain the molecular basis of the observed selectivity and the strong activation potential. In addition, we were able to show that the analogue is able to potentiate glucose-induced secretion of insulin from human pancreatic islets. The principal challenge during this study was identifying and understanding small differences in the cAMP-binding domains of cAMP-regulated proteins and matching these differences with suitable modifications of the cAMP molecule.

Rap1 and Rap2 are closely related proteins of the Ras family of small G-proteins. Rap1 is well known to regulate cell-cell adhesion. Here, we have analysed the effect of Rap-mediated signalling on endothelial permeability using electrical impedance measurements of HUVEC monolayers and subsequent determination of the barrier resistance, which is a measure for the ease with which ions can pass cell junctions. In line with its well-established effect on cell-cell junctions, depletion of Rap1 decreases, whereas activation of Rap1 increases barrier resistance. Despite its high sequence homology with Rap1, depletion of Rap2 has an opposite, enhancing, effect on barrier resistance. This effect can be mimicked by depletion of the Rap2 specific activator RasGEF1C and the Rap2 effector MAP4K4, establishing Rap2 signalling as an independent pathway controlling barrier resistance. As simultaneous depletion or activation of both Rap1 and Rap2 results in a barrier resistance comparable to control cells, Rap1 and Rap2 control barrier resistance in a reciprocal manner. This Rap1-antagonizing effect of Rap2 is established independent of junctional actin formation. These data establish that endothelial barrier resistance is determined by the combined antagonistic actions of Rap1 and Rap2.