Small Molecule AKAP-Protein Kinase A (PKA) Interaction Disruptors That Activate PKA Interfere with Compartmentalized cAMP Signaling in Cardiac Myocytes

Abstract

A-kinase anchoring proteins (AKAPs) tether protein kinase A (PKA) and other signaling proteins to defined intracellular sites, thereby establishing compartmentalized cAMP signaling. AKAP-PKA interactions play key roles in various cellular processes, including the regulation of cardiac myocyte contractility. We discovered small molecules, 3,3′-diamino-4,4′-dihydroxydiphenylmethane (FMP-API-1) and its derivatives, which inhibit AKAP-PKA interactions in vitro and in cultured cardiac myocytes. The molecules bind to an allosteric site of regulatory subunits of PKA identifying a hitherto unrecognized region that controls AKAP-PKA interactions. FMP-API-1 also activates PKA. The net effect of FMP-API-1 is a selective interference with compartmentalized cAMP signaling. In cardiac myocytes, FMP-API-1 reveals a novel mechanism involved in terminating β-adrenoreceptor-induced cAMP synthesis. In addition, FMP-API-1 leads to an increase in contractility of cultured rat cardiac myocytes and intact hearts. Thus, FMP-API-1 represents not only a novel means to study compartmentalized cAMP/PKA signaling but, due to its effects on cardiac myocytes and intact hearts, provides the basis for a new concept in the treatment of chronic heart failure. A-kinase anchoring proteins (AKAPs) tether protein kinase A (PKA) and other signaling proteins to defined intracellular sites, thereby establishing compartmentalized cAMP signaling. AKAP-PKA interactions play key roles in various cellular processes, including the regulation of cardiac myocyte contractility. We discovered small molecules, 3,3′-diamino-4,4′-dihydroxydiphenylmethane (FMP-API-1) and its derivatives, which inhibit AKAP-PKA interactions in vitro and in cultured cardiac myocytes. The molecules bind to an allosteric site of regulatory subunits of PKA identifying a hitherto unrecognized region that controls AKAP-PKA interactions. FMP-API-1 also activates PKA. The net effect of FMP-API-1 is a selective interference with compartmentalized cAMP signaling. In cardiac myocytes, FMP-API-1 reveals a novel mechanism involved in terminating β-adrenoreceptor-induced cAMP synthesis. In addition, FMP-API-1 leads to an increase in contractility of cultured rat cardiac myocytes and intact hearts. Thus, FMP-API-1 represents not only a novel means to study compartmentalized cAMP/PKA signaling but, due to its effects on cardiac myocytes and intact hearts, provides the basis for a new concept in the treatment of chronic heart failure. IntroductionCyclic AMP (cAMP)-dependent protein kinase (protein kinase A; PKA) 3The abbreviations used are: PKA, protein kinase A; AKAP, A-kinase anchoring protein; RII, type II regulatory subunit of PKA; STD, saturation transfer difference; FMP-API-1, 3,3′-diamino-4,4′-dihydroxydiphenylmethane; D/D, dimerization and docking; PDE, phosphodiesterase; SPR, surface plasmon resonance; CNGC, cyclic nucleotide-gated channel(s); 8-AHA-cAMP, 8-(6-aminohexylamino)adenosine-3′,5′-cyclic monophosphate; ISO, isoproterenol; PLN, phospholamban; c-TnI, cardiac troponin I; PGE, prostaglandin E; EP, eicosanoid E prostaglandin receptor. is a ubiquitous serine/threonine kinase controlling a variety of cellular functions. PKA holoenzyme consists of a dimer of regulatory subunits (RIα, RIβ, RIIα, or RIIβ) and two catalytic subunits (Cα, Cβ, or Cγ), each bound to an R subunit. RI-containing holoenzyme is termed PKA type I, whereas RII-containing PKA is termed PKA type II. Binding of cAMP to the R subunits induces a conformational change, causing the release and thus activation of the catalytic subunits, which then phosphorylate various substrates (1Taylor S.S. Kim C. Cheng C.Y. Brown S.H. Wu J. Kannan N. Biochim. Biophys. Acta. 2008; 1784: 16-26Crossref PubMed Scopus (173) Google Scholar). Specificity of PKA action is achieved by controlling its cellular localization through a family of A-kinase anchoring proteins (AKAPs).AKAPs bind PKA through an amphipathic α-helical structure consisting of 14–18 amino acids (RII-binding domain), which interacts with the hydrophobic groove formed by the N-terminal dimerization and docking (D/D) domain of regulatory subunit dimers (2Newlon M.G. Roy M. Morikis D. Hausken Z.E. Coghlan V. Scott J.D. Jennings P.A. Nat. Struct. Biol. 1999; 6: 222-227Crossref PubMed Scopus (183) Google Scholar, 3Kinderman F.S. Kim C. von Daake S. Ma Y. Pham B.Q. Spraggon G. Xuong N.H. Jennings P.A. Taylor S.S. Mol. Cell. 2006; 24: 397-408Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 4Gold M.G. Lygren B. Dokurno P. Hoshi N. McConnachie G. Taskén K. Carlson C.R. Scott J.D. Barford D. Mol. Cell. 2006; 24: 383-395Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 5Sarma G.N. Kinderman F.S. Kim C. von Daake S. Chen L. Wang B.C. Taylor S.S. Structure. 2010; 18: 155-166Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Besides PKA, AKAPs can directly bind various signaling proteins, such as other protein kinases, protein phosphatases, cAMP phosphodiesterases (PDEs), GTP-binding proteins, adaptor proteins, and substrate proteins of PKA. Thus, AKAPs coordinate multiprotein signaling complexes, thereby establishing compartmentalized signaling (6Hundsrucker C. Klussmann E. Handb. Exp. Pharmacol. 2008; 186: 483-503Crossref Scopus (26) Google Scholar, 7McCahill A.C. Huston E. Li X. Houslay M.D. Handb. Exp. Pharmacol. 2008; 186: 125-166Crossref Scopus (38) Google Scholar, 8Carnegie G.K. Means C.K. Scott J.D. IUBMB Life. 2009; 61: 394-406Crossref PubMed Scopus (140) Google Scholar, 9Skroblin P. Grossmann S. Schäfer G. Rosenthal W. Klussmann E. Int. Rev. Cell Mol. Biol. 2010; 283: 235-330Crossref PubMed Scopus (138) Google Scholar).The functional roles of AKAP-PKA interactions have often been uncovered in in vitro and in cell-based studies by disruption of the interactions using peptides derived from the RII-binding domains of AKAPs. Several such peptides have been developed (6Hundsrucker C. Klussmann E. Handb. Exp. Pharmacol. 2008; 186: 483-503Crossref Scopus (26) Google Scholar). For example, peptide Ht31 was derived from the RII-binding domain of AKAP-Lbc (10Carr D.W. Hausken Z.E. Fraser I.D. Stofko-Hahn R.E. Scott J.D. J. Biol. Chem. 1992; 267: 13376-13382Abstract Full Text PDF PubMed Google Scholar), AKAP in silico (AKAPIS) was derived from a bioinformatics approach (11Alto N.M. Soderling S.H. Hoshi N. Langeberg L.K. Fayos R. Jennings P.A. Scott J.D. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 4445-4450Crossref PubMed Scopus (146) Google Scholar), superAKAPIS was derived from AKAPIS (4Gold M.G. Lygren B. Dokurno P. Hoshi N. McConnachie G. Taskén K. Carlson C.R. Scott J.D. Barford D. Mol. Cell. 2006; 24: 383-395Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar), and others were derived from the RII-binding domain of AKAP18δ (12Hundsrucker C. Krause G. Beyermann M. Prinz A. Zimmermann B. Diekmann O. Lorenz D. Stefan E. Nedvetsky P. Dathe M. Christian F. McSorley T. Krause E. McConnachie G. Herberg F.W. Scott J.D. Rosenthal W. Klussmann E. Biochem. J. 2006; 396: 297-306Crossref PubMed Scopus (50) Google Scholar). Peptides like these have, for instance, been used to uncouple PKA from AKAPs in cardiac myocytes and thereby to demonstrate that AKAP-PKA interactions facilitate β-adrenoreceptor-induced increases in cardiac myocyte contractility (13Mauban J.R. O'Donnell M. Warrier S. Manni S. Bond M. Physiology. 2009; 24: 78-87Crossref PubMed Scopus (61) Google Scholar).Although peptides have proven invaluable for such purposes, their restricted membrane permeability and poor oral availability limit their use for therapeutic purposes and in animal studies. These drawbacks may be overcome with small molecules. Various examples show that disruption of protein-protein interactions with small molecules is feasible. Both small molecules interfering with interactions by association with the interacting surfaces or by allosteric binding have been identified (14Wells J.A. McClendon C.L. Nature. 2007; 450: 1001-1009Crossref PubMed Scopus (1549) Google Scholar, 15Yin H. Hamilton A.D. Angew Chem. Int. Ed. Engl. 2005; 44: 4130-4163Crossref PubMed Scopus (418) Google Scholar, 16Buchwald P. IUBMB Life. 2010; 62: 724-731Crossref PubMed Scopus (89) Google Scholar). The specificity and diversity of protein-protein interactions permits highly selective pharmacological interference. Thus, targeting protein-protein interactions with small molecules opens new avenues for the study of molecular mechanisms. In addition, the development of small molecules targeting disease-relevant protein-protein interactions may lead to novel therapeutic strategies, which, potentially, result in higher specificity and fewer side effects.Here we report the discovery of small molecules that have a dual effect. FMP-API-1 and its derivatives inhibit AKAP-PKA associations and also activate PKA. Using cardiac myocytes, we show that these molecules provide a new means to analyze functions of compartmentalized cAMP/PKA signaling. Moreover, we show that the approach of targeting scaffolding proteins with small molecules may pave the way to a novel concept for the treatment of chronic heart failure.DISCUSSIONBased on high throughput screening and subsequent chemical modification, we have identified small molecules, namely FMP-API-1 and its derivative FMP-API-1/27, that disrupt interactions between AKAPs and PKA. The D/D domain at the N terminus of the RII subunits is the interface at which AKAPs interact through their RII-binding domains. Together, NMR, Biacore, and in vitro kinase assays show that the molecules reversibly bind to RII subunits C-terminal of the D/D domain (Fig. 1, F–H, and supplemental Fig. 1). To date, the D/D domain has been the only portion of the RII subunits implicated in either direct binding or regulation of binding to AKAPs (5Sarma G.N. Kinderman F.S. Kim C. von Daake S. Chen L. Wang B.C. Taylor S.S. Structure. 2010; 18: 155-166Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 9Skroblin P. Grossmann S. Schäfer G. Rosenthal W. Klussmann E. Int. Rev. Cell Mol. Biol. 2010; 283: 235-330Crossref PubMed Scopus (138) Google Scholar). The fact that the new small molecule disruptors so markedly affect the binding of AKAPs to regulatory subunits without interaction with the core AKAP-regulatory subunit interface and the fact that they act additively with Ht31 peptides (Fig. 1E) point to the binding site as a so far unknown allosteric regulatory site. The small molecules interfere with cAMP binding of the regulatory subunits (Figs. 1H and 2B). It is currently not known whether this occurs through blockade of the cyclic nucleotide-binding domain or binding of the molecule outside the cyclic nucleotide-binding domain, which might lead to a conformational change inhibiting the interaction with cAMP. The region following N-terminally the D/D domain contains an autoinhibitory site (amino acids 92–102 in human RIIα) (49Taylor S.S. Buechler J.A. Yonemoto W. Annu. Rev. Biochem. 1990; 59: 971-1005Crossref PubMed Scopus (953) Google Scholar). This inhibitory site binds to the active site cleft of the catalytic subunit in the holoenzyme. The interaction of a small molecule within this region could prevent the inhibitory functioning of this part of the protein, thereby leading to the dissociation, and hence activation, of catalytic subunits. Taken together, not only does the D/D domain seem to interact with RII-binding domains of AKAPs, but additional regions of the regulatory PKA subunits are involved in the interaction.Interference of the small molecules with the autoinhibitory site may underpin the observed increase in catalytic activity (Fig. 3). In order to fully understand the molecular the effects of FMP-API-1 on AKAP-PKA interactions and PKA of of RII subunits with and without of subunit are so to of RII subunits has not been In addition, such studies may binding with higher to RII subunits currently to the binding region of FMP-API-1 using of were the proteins were or not by not We not of RII subunits as a approach to that PKA is the FMP-API-1 lead to a of to of AKAP-PKA FMP-API-1 and derivatives PKA anchoring such as Ht31 and which AKAP-PKA interactions by binding to the D/D domain of regulatory subunits (6Hundsrucker C. Klussmann E. Handb. Exp. Pharmacol. 2008; 186: 483-503Crossref Scopus (26) Google Scholar, D.W. Hausken Z.E. Fraser I.D. Stofko-Hahn R.E. Scott J.D. J. Biol. Chem. 1992; 267: 13376-13382Abstract Full Text PDF PubMed Google Scholar, C. Krause G. Beyermann M. Prinz A. Zimmermann B. Diekmann O. Lorenz D. Stefan E. Nedvetsky P. Dathe M. Christian F. McSorley T. Krause E. McConnachie G. Herberg F.W. Scott J.D. Rosenthal W. Klussmann E. Biochem. J. 2006; 396: 297-306Crossref PubMed Scopus (50) Google Scholar, Y. A. B. J. J. N. C. G. P.A. Taylor S.S. J. Biol. Chem. 2010; Full Text Full Text PDF PubMed Scopus Google Scholar). Several show that FMP-API-1 and the peptides have effects on cellular functions. For example, β-adrenoreceptor-induced increases in and and inhibit regulation of cAMP (12Hundsrucker C. Krause G. Beyermann M. Prinz A. Zimmermann B. Diekmann O. Lorenz D. Stefan E. Nedvetsky P. Dathe M. Christian F. McSorley T. Krause E. McConnachie G. Herberg F.W. Scott J.D. Rosenthal W. Klussmann E. Biochem. J. 2006; 396: 297-306Crossref PubMed Scopus (50) Google Scholar, D. W. J. Scott J.D. J. 2006; PubMed Scopus Google Scholar, L. J. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, R.E. T. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: PubMed Scopus Google Scholar). Thus, of the namely the D/D domain, either directly with peptides or through the binding of a small molecule to an allosteric subunit interactions. the for of interactions with the small molecules identified is that with the peptides are a of small molecules with peptides is their for and animal transfer of peptides to disrupt the interactions in and including or of peptides (12Hundsrucker C. Krause G. Beyermann M. Prinz A. Zimmermann B. Diekmann O. Lorenz D. Stefan E. Nedvetsky P. Dathe M. Christian F. McSorley T. Krause E. McConnachie G. Herberg F.W. Scott J.D. Rosenthal W. Klussmann E. Biochem. J. 2006; 396: 297-306Crossref PubMed Scopus (50) Google Scholar, Y. A. B. J. J. N. C. G. P.A. Taylor S.S. J. Biol. Chem. 2010; Full Text Full Text PDF PubMed Scopus Google Scholar, J.A. C. Bond M. PubMed Scopus Google Scholar, N. K. J. F. R. M. Bond M. J. Biol. Chem. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, the small molecules discovered pave the way to new for functions of AKAP-PKA interactions in cardiac myocytes, FMP-API-1 β-adrenoreceptor-induced increases in cAMP (Fig. and thus reveals a novel mechanism terminating signaling in the PKA to and to phosphorylate terminating cAMP activation of PKA in the and in J. D. G.K. W. Hoshi N. Langeberg L.K. Scott J.D. Mol. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, Mol. Pharmacol. 2009; PubMed Scopus (183) Google Scholar). and are highly in cardiac myocytes S. T. G. K. Nat. Rev. 2009; PubMed Scopus Google Scholar), and FMP-API-1 the interaction in these (Fig. is that the regulation of these by interference with this other AKAPs may PKA to in cardiac myocytes. For example, is with Mol. Pharmacol. 2009; PubMed Scopus (183) Google Scholar), and targeting of PKA by to various other has been observed in Scott J.D. Proc. Natl. Acad. Sci. U.S.A. 2008; PubMed Scopus Google Scholar). that FMP-API-1 with AKAP-PKA interactions that from the in (Fig. The molecule β-adrenoreceptor-induced cAMP in by interference with which play a in controlling D.W. 1999; PubMed Scopus Google Scholar). Thus, of activity in to to on AKAP-PKA interactions. of the effects of FMP-API-1 on prostaglandin activity in cardiac myocytes increase in cAMP in to either or The eicosanoid E prostaglandin to the Y. S. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar), and signaling compartmentalized PKA type G. A. V. A. Li X. Houslay M.D. M. 2008; PubMed Scopus Google Scholar). AKAP-PKA interactions have not been to be involved in signaling J. Biol. Chem. Full Text PDF PubMed Google Scholar, M. J. Cell Biol. 2006; PubMed Scopus Google Scholar). This is with that FMP-API-1 has effect on signaling. The regulation may PKA of of AKAP-PKA interactions may such and thereby prevent cAMP which in to of the cAMP in the of the of AKAPs is to PKA at defined cellular FMP-API-1 PKA from AKAP18δ (Fig. which PKA to B. Carlson C.R. K. V. McSorley T. J. Lorenz D. B. Rosenthal W. M. Taskén K. Klussmann E. 2007; PubMed Scopus Google Scholar), but this does not result in of In of in cardiac myocytes is by FMP-API-1 (Fig. This might be by the dual effect of The direct activation of PKA leads to a in the activity of PKA in the of PKA is by AKAP18δ B. Carlson C.R. K. V. McSorley T. J. Lorenz D. B. Rosenthal W. M. Taskén K. Klussmann E. 2007; PubMed Scopus Google Scholar). PKA, in this This disruption of PKA from and activation In addition, the direct activation of PKA at the is through the increases in cAMP in the of The are to for the and increases in the of c-TnI, which is an AKAP J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The and for the and effects on cultured cardiac myocytes and (Fig. Bond and (13Mauban J.R. O'Donnell M. Warrier S. Manni S. Bond M. Physiology. 2009; 24: 78-87Crossref PubMed Scopus (61) Google and Y. A. B. J. J. N. C. G. P.A. Taylor S.S. J. Biol. Chem. 2010; Full Text Full Text PDF PubMed Scopus Google that of PKA from AKAPs with the PKA anchoring peptides Ht31 and in cultured cardiac myocytes and intact of and J.A. C. Bond M. PubMed Scopus Google Scholar, N. K. J. F. R. M. Bond M. J. Biol. Chem. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). The is due to the of of the peptides E. K. B. Beyermann M. Rosenthal W. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). Bond and (13Mauban J.R. O'Donnell M. Warrier S. Manni S. Bond M. Physiology. 2009; 24: 78-87Crossref PubMed Scopus (61) Google Scholar, N. K. J. F. R. M. Bond M. J. Biol. Chem. 2009; Full Text Full Text PDF PubMed Scopus Google observed a effect in to PKA from AKAPs. Thus, either disruption of AKAP-PKA interactions or disruption of the interactions in with PKA activation in a This is highly the development of small molecule disruptors of AKAP-PKA either with or without the to activate PKA, could pave the way for a novel treatment of chronic heart failure. The to on of of cAMP by blockade of or of with heart of these but chronic on these the heart and may increase as in the of M. J.R. R. Engl. J. PubMed Scopus Google Scholar). for the of the that cellular cAMP and may be that PKA is and the C.L. N. S. M. J.A. PubMed Scopus Google Scholar). interference with defined cellular signaling using AKAP-PKA disruptors may lead to a treatment with fewer side effects PKA may not phosphorylate its substrates (Fig. the specificity and diversity of protein-protein interactions permits highly selective pharmacological interference with defined cellular and thus disruption of protein-protein interactions (6Hundsrucker C. Klussmann E. Handb. Exp. Pharmacol. 2008; 186: 483-503Crossref Scopus (26) Google that the effects of FMP-API-1 and on cardiac myocytes and intact are not due to the chemical structure of FMP-API-1 may that has the to with other The of FMP-API-1 derivatives, such as (Fig. that the molecule does not bind to FMP-API-1 with but not with signaling FMP-API-1 not prevent direct activation of (Fig. FMP-API-1 not increases of substrate (Fig. of from PKA) that are involved in cardiac myocyte and and and are by FMP-API-1 only to a Fig. 3). substrate proteins by or not in the of proteins in cardiac myocytes with FMP-API-1 for or not FMP-API-1 not protein and (Figs. and Thus, the molecule with compartmentalized cAMP the of the small molecules the for the development of high small molecules the interaction of PKA with AKAPs as as for novel this molecules provide an invaluable for new to study functions of AKAP-PKA interactions in and Moreover, they have the to be developed as highly for the treatment of that are with cAMP signaling but that are not by such as chronic heart failure. IntroductionCyclic AMP (cAMP)-dependent protein kinase (protein kinase A; PKA) 3The abbreviations used are: PKA, protein kinase A; AKAP, A-kinase anchoring protein; RII, type II regulatory subunit of PKA; STD, saturation transfer difference; FMP-API-1, 3,3′-diamino-4,4′-dihydroxydiphenylmethane; D/D, dimerization and docking; PDE, phosphodiesterase; SPR, surface plasmon resonance; CNGC, cyclic nucleotide-gated channel(s); 8-AHA-cAMP, 8-(6-aminohexylamino)adenosine-3′,5′-cyclic monophosphate; ISO, isoproterenol; PLN, phospholamban; c-TnI, cardiac troponin I; PGE, prostaglandin E; EP, eicosanoid E prostaglandin receptor. is a ubiquitous serine/threonine kinase controlling a variety of cellular functions. PKA holoenzyme consists of a dimer of regulatory subunits (RIα, RIβ, RIIα, or RIIβ) and two catalytic subunits (Cα, Cβ, or Cγ), each bound to an R subunit. RI-containing holoenzyme is termed PKA type I, whereas RII-containing PKA is termed PKA type II. Binding of cAMP to the R subunits induces a conformational change, causing the release and thus activation of the catalytic subunits, which then phosphorylate various substrates (1Taylor S.S. Kim C. Cheng C.Y. Brown S.H. Wu J. Kannan N. Biochim. Biophys. Acta. 2008; 1784: 16-26Crossref PubMed Scopus (173) Google Scholar). Specificity of PKA action is achieved by controlling its cellular localization through a family of A-kinase anchoring proteins (AKAPs).AKAPs bind PKA through an amphipathic α-helical structure consisting of 14–18 amino acids (RII-binding domain), which interacts with the hydrophobic groove formed by the N-terminal dimerization and docking (D/D) domain of regulatory subunit dimers (2Newlon M.G. Roy M. Morikis D. Hausken Z.E. Coghlan V. Scott J.D. Jennings P.A. Nat. Struct. Biol. 1999; 6: 222-227Crossref PubMed Scopus (183) Google Scholar, 3Kinderman F.S. Kim C. von Daake S. Ma Y. Pham B.Q. Spraggon G. Xuong N.H. Jennings P.A. Taylor S.S. Mol. Cell. 2006; 24: 397-408Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 4Gold M.G. Lygren B. Dokurno P. Hoshi N. McConnachie G. Taskén K. Carlson C.R. Scott J.D. Barford D. Mol. Cell. 2006; 24: 383-395Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 5Sarma G.N. Kinderman F.S. Kim C. von Daake S. Chen L. Wang B.C. Taylor S.S. Structure. 2010; 18: 155-166Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Besides PKA, AKAPs can directly bind various signaling proteins, such as other protein kinases, protein phosphatases, cAMP phosphodiesterases (PDEs), GTP-binding proteins, adaptor proteins, and substrate proteins of PKA. Thus, AKAPs coordinate multiprotein signaling complexes, thereby establishing compartmentalized signaling (6Hundsrucker C. Klussmann E. Handb. Exp. Pharmacol. 2008; 186: 483-503Crossref Scopus (26) Google Scholar, 7McCahill A.C. Huston E. Li X. Houslay M.D. Handb. Exp. Pharmacol. 2008; 186: 125-166Crossref Scopus (38) Google Scholar, 8Carnegie G.K. Means C.K. Scott J.D. IUBMB Life. 2009; 61: 394-406Crossref PubMed Scopus (140) Google Scholar, 9Skroblin P. Grossmann S. Schäfer G. Rosenthal W. Klussmann E. Int. Rev. Cell Mol. Biol. 2010; 283: 235-330Crossref PubMed Scopus (138) Google Scholar).The functional roles of AKAP-PKA interactions have often been uncovered in in vitro and in cell-based studies by disruption of the interactions using peptides derived from the RII-binding domains of AKAPs. Several such peptides have been developed (6Hundsrucker C. Klussmann E. Handb. Exp. Pharmacol. 2008; 186: 483-503Crossref Scopus (26) Google Scholar). For example, peptide Ht31 was derived from the RII-binding domain of AKAP-Lbc (10Carr D.W. Hausken Z.E. Fraser I.D. Stofko-Hahn R.E. Scott J.D. J. Biol. Chem. 1992; 267: 13376-13382Abstract Full Text PDF PubMed Google Scholar), AKAP in silico (AKAPIS) was derived from a bioinformatics approach (11Alto N.M. Soderling S.H. Hoshi N. Langeberg L.K. Fayos R. Jennings P.A. Scott J.D. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 4445-4450Crossref PubMed Scopus (146) Google Scholar), superAKAPIS was derived from AKAPIS (4Gold M.G. Lygren B. Dokurno P. Hoshi N. McConnachie G. Taskén K. Carlson C.R. Scott J.D. Barford D. Mol. Cell. 2006; 24: 383-395Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar), and others were derived from the RII-binding domain of AKAP18δ (12Hundsrucker C. Krause G. Beyermann M. Prinz A. Zimmermann B. Diekmann O. Lorenz D. Stefan E. Nedvetsky P. Dathe M. Christian F. McSorley T. Krause E. McConnachie G. Herberg F.W. Scott J.D. Rosenthal W. Klussmann E. Biochem. J. 2006; 396: 297-306Crossref PubMed Scopus (50) Google Scholar). Peptides like these have, for instance, been used to uncouple PKA from AKAPs in cardiac myocytes and thereby to demonstrate that AKAP-PKA interactions facilitate β-adrenoreceptor-induced increases in cardiac myocyte contractility (13Mauban J.R. O'Donnell M. Warrier S. Manni S. Bond M. Physiology. 2009; 24: 78-87Crossref PubMed Scopus (61) Google Scholar).Although peptides have proven invaluable for such purposes, their restricted membrane permeability and poor oral availability limit their use for therapeutic purposes and in animal studies. These drawbacks may be overcome with small molecules. Various examples show that disruption of protein-protein interactions with small molecules is feasible. Both small molecules interfering with interactions by association with the interacting surfaces or by allosteric binding have been identified (14Wells J.A. McClendon C.L. Nature. 2007; 450: 1001-1009Crossref PubMed Scopus (1549) Google Scholar, 15Yin H. Hamilton A.D. Angew Chem. Int. Ed. Engl. 2005; 44: 4130-4163Crossref PubMed Scopus (418) Google Scholar, 16Buchwald P. IUBMB Life. 2010; 62: 724-731Crossref PubMed Scopus (89) Google Scholar). The specificity and diversity of protein-protein interactions permits highly selective pharmacological interference. Thus, targeting protein-protein interactions with small molecules opens new avenues for the study of molecular mechanisms. In addition, the development of small molecules targeting disease-relevant protein-protein interactions may lead to novel therapeutic strategies, which, potentially, result in higher specificity and fewer side effects.Here we report the discovery of small molecules that have a dual effect. FMP-API-1 and its derivatives inhibit AKAP-PKA associations and also activate PKA. Using cardiac myocytes, we show that these molecules provide a new means to analyze functions of compartmentalized cAMP/PKA signaling. Moreover, we show that the approach of targeting scaffolding proteins with small molecules may pave the way to a novel concept for the treatment of chronic heart failure.