8‐pCPT‐2′‐O‐Me‐cAMP‐AM: An Improved Epac‐Selective cAMP Analogue

Cyclic adenosine monophosphate (cAMP) is a common second messenger involved in the regulation of many different cellular processes through the activation of protein kinase A (PKA), exchange protein directly activated by cAMP (Epac) and cyclicnucleotide-regulated ion channels. Adenylyl cyclases are ACHTUNGTRENNUNGresponsible for catalysing the formation of cAMP from ATP. Levels of cAMP can be raised in cells in response to a large variety of extracellular stimuli, which act via receptors coupled to heterotrimeric G proteins, which stimulate the activity of adenylyl cyclase. In addition, cAMP levels are controlled by phosphodiesterases (PDE), which catalyse the degradation of cAMP to AMP. In cells, cAMP levels can be artificially elevated by forskolin, which activates adenylyl cyclase directly. Furthermore, cAMP levels can be raised by inhibiting PDEs. These approaches are commonly used in tissue culture experiments, but, by generating cAMP, they do not discriminate between the various target proteins that are activated. Alternatively, membrane-permeable cAMP analogues, which selectively interact with particular receptor proteins, can be applied. For example, signalling pathways activated by Epac and PKA can be ACHTUNGTRENNUNGdistinguished by using 8-pCPT-2’-O-Me-cAMP and 6-Bnz-cAMP, respectively. Epac is a guanine nucleotide exchange factor for the small G protein Rap. Rap cycles between a signalling-inactive GDPbound state and a signalling-active GTP-bound state. cAMP-activated Epac catalyses the exchange of Rap-bound GDP for GTP. Epac and Rap function in a number of different cellular processes including insulin secretion, inhibition of cell scattering, neurotransmitter release and cAMP-induced barrier function in endothelial cells. Even though 8-pCPT-2’-O-Me-cAMP has become a widely used tool in Epac-related research, its biological application is limited by its low membrane permeability, caused by the negatively charged phosphate. However, the negatively charged singly bonded oxygen on the phosphate group can be masked by labile esters. Such a precursor is expected to enter the cell efficiently, where the ester is hydrolysed either directly by water or by cellular esterases to liberate the active compound. We therefore synthesised 8-pCPT-2’-O-Me-cAMP-AM from 8pCPT-2’-O-Me-cAMP, whereby acetoxymethyl bromide was used as a donor for the AM group. The product that was obtained had a purity exceeding 97% and consisted of a mixture of the equatorial and the axial isomers of the ester (Figure S1 in the Supporting Information, Scheme 1). Even though the isomers could be resolved by repetitive analytical HPLC runs, efficient separation on a preparative scale was not possible. Orange peel acetylesterase and esterase from porcine liver cleaved the equatorial isomer about five times more efficiently than the axial isomer within minutes (data not shown). The pharmacokinetics of both isomers are thus expected to be similar, justifying the application of a mixture of both isomers to cells. In any case, the isomeric ratio of an individual synthesis can be easily quality controlled by P NMR (Figure S1). To compare the efficiency of 8-pCPT-2’-O-Me-cAMP-AM and 8-pCPT-2’-O-Me-cAMP in activating Epac1 in vivo, an Epac1based fluorescence resonance energy transfer (FRET) probe was used. In this assay, activation of Epac1 by the binding of cAMP to the Epac1-FRET probe is measured as a reduction in the FRET signal. A431 cells transfected with the FRET probe were stimulated with 8-pCPT-2’-O-Me-cAMP-AM or 8-pCPT-2’O-Me-cAMP (Figure 1). Stimulation of cells with 100 mm 8pCPT-2’-O-Me-cAMP resulted in a decrease of the FRET signal that was approximately one order of magnitude slower than the decrease obtained upon stimulation with 1 mm 8-pCPT-2’O-Me-cAMP-AM. Furthermore, activation of Epac1 following stimulation with 100 mm 8-pCPT-2’-O-Me-cAMP could be further enhanced by the addition of forskolin, whereas 1 mm 8pCPT-2’-O-Me-cAMP-AM induced maximal activity of Epac1 under the given conditions. The activation of Epac by 8-pCPT2’-O-Me-cAMP-AM occurs within one minute after application. This is comparable with the kinetics of forskolin-induced Epac activation, and thus 8-pCPT-2’-O-Me-cAMP-AM mimics the “natural” response time of the signalling pathway. The activity of endogenous Epac can be monitored by isolating selectively Rap·GTP from cell lysates. Primary human umbilical vein endothelial cells (HUVEC) were stimulated with different concentrations of 8-pCPT-2’-O-Me-cAMP and 8-pCPT-2’-OMe-cAMP-AM (Figure 2A). Partial activation of Rap was induced by 10 mm 8-pCPT-2’-O-Me-cAMP, and full activation of the G protein was stimulated by 100 mm 8-pCPT-2’-O-Me-cAMP. In contrast, treatment of the cells with just 0.1 mm 8-pCPT-2’-OMe-cAMP-AM was sufficient to induce full Rap activation. [a] M. J. Vliem, W. -J. Pannekoek, Dr. J. Riedl, M. R. H. Kooistra, Prof. Dr. J. L. Bos, Dr. H. Rehmann Department of Physiological Chemistry Centre for Biomedical Genetics and Cancer Genomics Centre University Medical Center Utrecht Universiteitsweg 100, 3584CG Utrecht (The Netherlands) Fax: (+31)88-75-68101 E-mail : j.l. bos@umcutrecht.nl h.rehmann@umcutrecht.nl [b] B. Ponsioen, Dr. K. Jalink Division of Cell Biology, The Netherlands Cancer Institute Amsterdam (The Netherlands) [c] Dr. F. Schwede, Dr. H. -G. Genieser BIOLOG Life Science Institute Flughafendamm 9a, 28071 Bremen (Germany) [] These authors contribute equally to this work. Supporting information for this article is available on the WWW under http://www.chembiochem. org or from the author.