Quantification of cAMP antagonist action in vitro and in living cells

European Journal of Cell Biology
Frank GesellchenFriedrich W Herberg

Abstract

cAMP-dependent protein kinase (PKA) plays a key role in intracellular signalling. cAMP antagonists, acting as suppressors of PKA activity by preventing PKA-holoenzyme dissociation, have received increasing attention because of their potential use in diagnostics as well as for therapeutic purposes. A large number of cAMP analogs have been described over the last three decades and methodology has been established to monitor cAMP agonists action by either following enzymatic activity or holoenzyme dissociation. This is not the case for cAMP antagonists, where only a few substances have been demonstrated to exhibit effects in the low micromolar range, for example, Rp-8-Br-cAMPS. A main drawback in the development of new compounds is the lack of technologies to assess antagonist action in an in vitro situation as well as in living cells. Here we quantify the effect of several cAMP analogs applying three different biochemical/biophysical assay setups and one in-cell assay. This includes two methods monitoring subunit dissociation in a test tube, namely AlphaScreen, a bead-based proximity assay, and surface plasmon resonance, determining the association and dissociation patterns of the two PKA subunits in real time in response to anta...Continue Reading

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Citations

May 20, 2009·Proceedings of the National Academy of Sciences of the United States of America·Mohamed R MohamedGrant McFadden
Jan 25, 2011·Pharmacology & Therapeutics·Richard Eglen, Terry Reisine
Dec 13, 2007·Journal of Molecular Recognition : JMR·Rebecca L Rich, David G Myszka
Jul 7, 2007·Cellular Signalling·Mandy DiskarFriedrich W Herberg
Sep 5, 2006·The Biochemical Journal·Anne J StokkaKjetil Taskén

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