Based 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 (, 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 (5
). 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 (E
) point to the binding site as a so far unknown allosteric regulatory site. The small molecules interfere with cAMP binding of the regulatory subunits (H
). 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) (49
). 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 (). In order to fully understand the molecular mechanisms underlying the effects of FMP-API-1 on AKAP-PKA interactions and PKA activity, solved structures of complexes of full-length RII subunits with and without compounds (and of AKAP-R subunit complexes) are needed. However, so far, any attempt to obtain three-dimensional structures of full-length RII subunits has not been successful. In addition, such studies may require compounds binding with higher affinity to RII subunits than those currently available. Our attempts to exactly map the binding region of FMP-API-1 using further truncated versions of RIIα were compromised because the required recombinant proteins were insoluble (e.g. RIIα 87–156) or apparently did not fold correctly (e.g. RIIα 1–156, indicated by STD NMR experiments; data not shown). We did not consider knockdown of RII subunits as a further approach to confirm that PKA is the FMP-API-1 target because knockdown would lead to a reduction of experimentally detectable RII-AKAP interactions.
With respect to inhibition of AKAP-PKA interactions, FMP-API-1 and derivatives resemble PKA anchoring disruptor peptides, such as Ht31 and AKAP18δ-derived peptides, which block AKAP-PKA interactions by binding to the D/D domain of regulatory subunits (6
). Several experiments show that FMP-API-1 and the peptides have similar effects on cellular functions. For example, both agents ablate β-adrenoreceptor-induced increases in L-type Ca2+
channel currents and inhibit negative feedback regulation of adenylyl cyclase-dependent cAMP synthesis ( and ) (12
). Thus, blocking of the canonical AKAP-interacting site, namely the D/D domain, either directly with peptides or indirectly through the binding of a small molecule to an allosteric site, inhibits AKAP-RII subunit interactions. Although the efficacy for inhibition of AKAP-RII interactions with the small molecules identified is considerably lower than that with the peptides (IC50
values are μm versus
), a major advantage of small molecules compared with peptides is their wider applicability for cell and animal experiments. Efficient transfer of peptides to disrupt the interactions in cells and living animals requires costly efforts, including viral transfection or generation of cell-penetrating peptides (12
). Thus, the small molecules discovered here pave the way to new approaches for elucidating functions of AKAP-PKA interactions in vivo
In cardiac myocytes, FMP-API-1 enhances β-adrenoreceptor-induced increases in cAMP levels () and thus reveals a novel mechanism terminating β-adrenoreceptor-dependent signaling in the heart. AKAP79/150 tethers PKA to adenylyl cyclases V and VI to phosphorylate it, rapidly terminating cAMP synthesis upon activation of PKA in the brain and in HEK293 cells (34
). Because adenylyl cyclases V and VI are highly relevant cyclases in cardiac myocytes (55
), and FMP-API-1 disrupts the AKAP150-PKA interaction in these cells (), it is conceivable that the compound blunts negative feedback regulation of these cyclases by interference with this interaction. However, other AKAPs may link PKA to adenylyl cyclases in cardiac myocytes. For example, mAKAPβ is associated with adenylyl cyclase V (54
), and targeting of PKA by Yotiao to various other adenylyl cyclases has recently been observed in neurons (56
). Further evidence confirming that FMP-API-1 interferes with AKAP-PKA interactions that control adenylyl cyclase activities stems from the experiments in uterine tissue (C
). The molecule enhances β-adrenoreceptor-induced cAMP production in uterus preparations, presumably by interference with AKAP150-PKA interactions, which play a role in controlling uterine contraction (57
). Thus, attenuation of adenylyl cyclase activity in response to β-adrenergic agonists regularly seems to rely on AKAP-PKA interactions. Our analyses of the effects of FMP-API-1 on prostaglandin E-stimulated adenylyl cyclase activity in cardiac myocytes shows no FMP-API-1-dependent increase in cAMP formation in response to either PGE1
. The cognate eicosanoid E prostaglandin receptors (EPs) couple to the Gs
/adenylyl cyclase system (33
), and EP signaling mainly involves compartmentalized PKA type I (39
). AKAP-PKA interactions have not been found to be involved in EP signaling (40
). This is consistent with our observations that FMP-API-1 has no effect on EP signaling. The negative feedback regulation may involve PKA phosphorylation of PDE4. Inhibition of AKAP-PKA interactions may reduce such phosphorylation and thereby prevent enhanced cAMP degradation, which in turn would contribute to elevation of the cAMP level in the presence of the inhibitor.
The classical function of AKAPs is to concentrate PKA at defined cellular sites. FMP-API-1 displaces PKA from AKAP18δ (), which tethers PKA to PLN (22
), but this does not result in lower levels of PLN phosphorylation. In contrast, PKA-dependent phosphorylation of PLN in cardiac myocytes is increased by FMP-API-1 challenge (). This unexpected observation might be explained by the dual effect of FMP-API-1. The FMP-API-1-induced direct activation of PKA leads to a local rise in the activity of PKA in the vicinity of PLN where PKA is concentrated by AKAP18δ (22
). Activated PKA, in turn, phosphorylates this substrate. This seems feasible when disruption (i.e.
displacement of PKA from AKAP18δ) and activation occur simultaneously. In addition, the FMP-API-1-induced direct activation of PKA at the AKAP18δ-PKA complex is probably enhanced through the FMP-API-1-induced increases in cAMP levels in the presence of adrenergic stimuli. The same reasons are likely to account for the FMP-API-1-induced and PKA-dependent increases in the phosphorylation of c-TnI, which is an AKAP itself (46
). The increased PLN and c-TnI phosphorylation would account for the resulting positive inotropic and lusitropic effects on cultured adult cardiac myocytes and isolated hearts (). Bond and co-workers (13
) and Patel et al.
) had shown that uncoupling of PKA from AKAPs with the PKA anchoring disruptor peptides Ht31 and AKAD in both cultured cardiac myocytes and intact hearts decreases phosphorylation of PLN and c-TnI (52
). The decrease is most likely due to the lack of PKA-activating capabilities of the peptides (23
). Bond and co-workers (13
) observed a positive inotropic effect in response to uncoupling PKA from AKAPs. Thus, either disruption of AKAP-PKA interactions alone or disruption of the interactions in conjunction with PKA activation results in a positive inotropic response. This outcome is potentially highly relevant therapeutically because the development of small molecule disruptors of AKAP-PKA interactions, either with or without the ability to activate PKA, could pave the way for a novel treatment of chronic heart failure. The classical approaches to elicit positive inotropic responses rely on stimulation of β-adrenoceptors, inhibition of cAMP synthesis by blockade of PDE3, or inhibition of Ca2+
channels with heart glycosides. All of these approaches constitute initially effective treatments, but chronic treatments based on these concepts harm the failing heart and may even increase mortality, as in the case of PDE3 inhibitors (by 28%) (58
). One reason for the failure of the approaches that elevate cellular cAMP levels (β-agonists and PDE blockers) may be that PKA is constitutively and globally activated throughout the cells (59
). Local interference with defined cellular signaling processes using AKAP-PKA disruptors may lead to a more effective treatment with fewer side effects because PKA may not randomly phosphorylate its substrates (). Generally, the specificity and diversity of protein-protein interactions permits potentially highly selective pharmacological interference with defined cellular processes and thus disruption of specific protein-protein interactions promises better drugs (6
Several observations underline that the effects of FMP-API-1 and FMP-API-1/27 on cardiac myocytes and intact hearts are not due to off target effects, although the chemical structure of FMP-API-1 may suggest that it has the potential to react with other proteins. (i) The identification of inactive FMP-API-1 derivatives, such as FMP-API-1/26 (B
), indicates that the molecule does not nonspecifically bind to proteins. (ii) FMP-API-1 interferes with β-adrenergic but not with PGE1/2
signaling (see above). (iii) FMP-API-1 did not prevent direct activation of L-type Ca2+
). (iv) FMP-API-1 did not cause random increases of PKA-dependent substrate phosphorylations (). (v) Activities of several kinases (apart from PKA) that are involved in cardiac myocyte control (including ErbB1, MEK1, ERK1 and -2, ROCK1 and -2, PKCα, CaMKIIα, and GSK3β) are affected by FMP-API-1 only to a minor extent (supplemental Fig. 3
). Consistently, antibodies directed against substrate proteins phosphorylated by PKC, CaMKII, or Akt did not detect changes in the phosphorylation pattern of proteins in cardiac myocytes treated with FMP-API-1 for 30 min (100 or 300 μm
; data not shown). (vi) FMP-API-1 did not influence protein phosphatase and phosphodiesterase activities (, D
). Thus, the molecule apparently interferes selectively with compartmentalized cAMP signaling.
In summary, the identification of the small molecules described here lays the groundwork for the development of high affinity small molecules disrupting the interaction of PKA with AKAPs as well as for revealing novel mechanisms underlying this interaction. Such molecules provide an invaluable tool for new approaches to study functions of AKAP-PKA interactions in living cells and animals. Moreover, they have the potential to be developed as highly targeted therapeutics for the treatment of diseases that are associated with altered cAMP signaling but that are not addressed sufficiently by conventional pharmacotherapy, such as chronic heart failure.