Conformational States of Exchange Protein Directly Activated by cAMP (EPAC1) Revealed by Ensemble Modeling and Integrative Structural Biology.

Exchange proteins directly activated by cAMP (EPAC1 and EPAC2) are important allosteric regulators of cAMP-mediated signal transduction pathways. To understand the molecular mechanism of EPAC activation, we performed detailed Small-Angle X-ray Scattering (SAXS) analysis of EPAC1 in its apo (inactive), cAMP-bound, and effector (Rap1b)-bound states. Our study demonstrates that we can model the solution structures of EPAC1 in each state using ensemble analysis and homology models derived from the crystal structures of EPAC2. The N-terminal domain of EPAC1, which is not conserved between EPAC1 and EPAC2, appears folded and interacts specifically with another component of EPAC1 in each state. The apo-EPAC1 state is a dynamic mixture of a compact (Rg = 32.9 Å, 86%) and a more extended (Rg = 38.5 Å, 13%) conformation. The cAMP-bound form of EPAC1 in the absence of Rap1 forms a dimer in solution; but its molecular structure is still compatible with the active EPAC1 conformation of the ternary complex model with cAMP and Rap1. Herein, we show that SAXS can elucidate the conformational states of EPAC1 activation as it proceeds from the compact, inactive apo conformation through a previously unknown intermediate-state, to the extended cAMP-bound form, and then binds to its effector (Rap1b) in a ternary complex.

Stimulation of proglucagon gene expression by human GPR119 in enteroendocrine L-cell line GLUTag.

GPR119 is a G protein-coupled receptor expressed on enteroendocrine L-cells that synthesize and secrete the incretin hormone glucagon-like peptide-1 (GLP-1). Although GPR119 agonists stimulate L-cell GLP-1 secretion, there is uncertainty concerning whether GLP-1 biosynthesis is under the control of GPR119. Here we report that GPR119 is functionally coupled to increased proglucagon (PG) gene expression that constitutes an essential first step in GLP-1 biosynthesis. Using a mouse L-cell line (GLUTag) that expresses endogenous GPR119, we demonstrate that PG gene promoter activity is stimulated by GPR119 agonist AS1269574. Surprisingly, transfection of GLUTag cells with recombinant human GPR119 (hGPR119) results in a constitutive and apparently ligand-independent increase of PG gene promoter activity and PG mRNA content. These constitutive actions of hGPR119 are mediated by cAMP-dependent protein kinase (PKA) but not cAMP sensor Epac2. Thus, the constitutive action of hGPR119 to stimulate PG gene promoter activity is diminished by: 1) a dominant-negative Gαs protein, 2) a dominant-negative PKA regulatory subunit, and 3) a dominant-negative A-CREB. Interestingly, PG gene promoter activity is stimulated by 6-Bn-cAMP-AM, a cAMP analog that selectively activates α and ß isoforms of type II, but not type I PKA regulatory subunits expressed in GLUTag cells. Finally, our analysis reveals that a specific inhibitor of Epac2 activation (ESI-05) fails to block the stimulatory action of 6-Bn-cAMP-AM at the PG gene promoter, nor is PG gene promoter activity stimulated by: 1) a constitutively active Epac2, or 2) cAMP analogs that selectively activate Epac proteins. Such findings are discussed within the context of ongoing controversies concerning the relative contributions of PKA and Epac2 to the control of PG gene expression.

Identification and characterization of small molecules as potent and specific EPAC2 antagonists.

EPAC1 and EPAC2, two isoforms of exchange proteins directly activated by cAMP (EPAC), respond to the second messenger cAMP and regulate a wide variety of intracellular processes under physiological and pathophysiological circumstances. Herein, we report the chemical design, synthesis, and pharmacological characterization of three different scaffolds (diarylsulfones, N,N-diarylamines, and arylsulfonamides) as highly potent and selective antagonists of EPAC2. Several selective EPAC2 antagonists have been identified including 20i (HJC0350), which has an apparent IC(50) of 0.3 µM for competing with 8-NBD-cAMP binding of EPAC2 and is about 133-fold more potent than cAMP. Compounds 1 (ESI-05), 14c (HJC0338), and 20i, selected from each series, have exhibited no inhibition of EPAC1-mediated Rap1-GDP exchange activity at 25 µM, indicating that they are EPAC2-specific antagonists. Moreover, live-cell imaging studies using EPAC1, EPAC2, or PKA FRET sensor also demonstrate that 20i functions as an EPAC2 specific antagonist.

A novel EPAC-specific inhibitor suppresses pancreatic cancer cell migration and invasion.

Exchange protein directly activated by cAMP (EPAC) and cAMP-dependent protein kinase (PKA) are two intracellular receptors that mediate the effects of the prototypic second messenger cAMP. Identifying pharmacological probes for selectively modulating EPAC activity represents a significant unmet need within the research field. Herein, we report the identification and characterization of 3-(5-tert-butyl-isoxazol-3-yl)-2-[(3-chloro-phenyl)-hydrazono]-3-oxo-propionitrile (ESI-09), a novel noncyclic nucleotide EPAC antagonist that is capable of specifically blocking intracellular EPAC-mediated Rap1 activation and Akt phosphorylation, as well as EPAC-mediated insulin secretion in pancreatic ß cells. Using this novel EPAC-specific inhibitor, we have probed the functional roles of overexpression of EPAC1 in pancreatic cancer cells. Our studies show that EPAC1 plays an important role in pancreatic cancer cell migration and invasion, and thus represents a potential target for developing novel therapeutic strategies for pancreatic cancer.

Structural analyses of a constitutively active mutant of exchange protein directly activated by cAMP.

Exchange proteins directly activated by cAMP (EPACs) are important allosteric regulators of cAMP-mediated signal transduction pathways. To understand the molecular mechanism of EPAC activation, we have combined site-directed mutagenesis, X-ray crystallography, and peptide amide hydrogen/deuterium exchange mass spectrometry (DXMS) to probe the structural and conformational dynamics of EPAC2-F435G, a constitutively active EPAC2 mutant. Our study demonstrates that conformational dynamics plays a critical role in cAMP-induced EPAC activation. A glycine mutation at 435 position shifts the equilibrium of conformational dynamics towards the extended active conformation.

Isoform-specific antagonists of exchange proteins directly activated by cAMP.

The major physiological effects of cAMP in mammalian cells are transduced by two ubiquitously expressed intracellular cAMP receptors, protein kinase A (PKA) and exchange protein directly activated by cAMP (EPAC), as well as cyclic nucleotide-gated ion channels in certain tissues. Although a large number of PKA inhibitors are available, there are no reported EPAC-specific antagonists, despite extensive research efforts. Here we report the identification and characterization of noncyclic nucleotide EPAC antagonists that are exclusively specific for the EPAC2 isoform. These EAPC2-specific antagonists, designated as ESI-05 and ESI-07, inhibit Rap1 activation mediated by EAPC2, but not EPAC1, with high potency in vitro. Moreover, ESI-05 and ESI-07 are capable of suppressing the cAMP-mediated activation of EPAC2, but not EPAC1 and PKA, as monitored in living cells through the use of EPAC- and PKA-based FRET reporters, or by the use of Rap1-GTP pull-down assays. Deuterium exchange mass spectroscopy analysis further reveals that EPAC2-specific inhibitors exert their isoform selectivity through a unique mechanism by binding to a previously undescribed allosteric site: the interface of the two cAMP binding domains, which is not present in the EPAC1 isoform. Isoform-specific EPAC pharmacological probes are highly desired and will be valuable tools for dissecting the biological functions of EPAC proteins and their roles in various disease states.

5-Cyano-6-oxo-1,6-dihydro-pyrimidines as potent antagonists targeting exchange proteins directly activated by cAMP.

Exchange proteins directly activated by cAMP (Epac) are a family of guanine nucleotide exchange factors that regulate a wide variety of intracellular processes in response to second messenger cAMP. To explore the structural determinants for Epac antagonist properties of high throughput screening (HTS) hit ESI-08, pyrimidine 1, a series of 5-cyano-6-oxo-1,6-dihydro-pyrimidine analogues have been synthesized and evaluated for their activities for Epac inhibition. Structure-activity relationship (SAR) analysis led to the identification of three more potent Epac antagonists (6b, 6g, and 6h). These inhibitors may serve as valuable pharmacological probes for further elucidation of the physiological functions and mechanisms of Epac regulation. Our SAR results and molecular docking studies have also revealed that further optimization of the moieties at the C-6 position of pyrimidine scaffold may allow us to discover more potent Epac-specific antagonists.

A fluorescence-based high-throughput assay for the discovery of exchange protein directly activated by cyclic AMP (EPAC) antagonists.

BACKGROUND: The discovery, more than ten years ago, of exchange proteins directly activated by cAMP (EPAC) as a new family of intracellular cAMP receptors revolutionized the cAMP signaling research field. Extensive studies have revealed that the cAMP signaling network is much more complex and dynamic as many cAMP-related cellular processes, previously thought to be controlled by protein kinase A, are found to be also mediated by EPAC proteins. Although there have been many important discoveries in the roles of EPACs greater understanding of their physiological function in cAMP-mediated signaling is impeded by the absence of EPAC-specific antagonist. METHODOLOGY/PRINCIPAL FINDINGS: To overcome this deficit, we have developed a fluorescence-based high throughput assay for screening EPAC specific antagonists. Our assay is highly reproducible and simple to perform using the "mix and measure" format. A pilot screening using the NCI-DTP diversity set library led to the identification of small chemical compounds capable of specifically inhibiting cAMP-induced EPAC activation while not affecting PKA activity. CONCLUSIONS/SIGNIFICANCE: Our study establishes a robust high throughput screening assay that can be effectively applied for the discovery of EPAC-specific antagonists, which may provide valuable pharmacological tools for elucidating the biological functions of EPAC and for promoting an understanding of disease mechanisms related to EPAC/cAMP signaling.

Mechanism of intracellular cAMP sensor Epac2 activation: cAMP-induced conformational changes identified by amide hydrogen/deuterium exchange mass spectrometry (DXMS).

Epac2, a guanine nucleotide exchange factor, regulates a wide variety of intracellular processes in response to second messenger cAMP. In this study, we have used peptide amide hydrogen/deuterium exchange mass spectrometry to probe the solution structural and conformational dynamics of full-length Epac2 in the presence and absence of cAMP. The results support a mechanism in which cAMP-induced Epac2 activation is mediated by a major hinge motion centered on the C terminus of the second cAMP binding domain. This conformational change realigns the regulatory components of Epac2 away from the catalytic core, making the later available for effector binding. Furthermore, the interface between the first and second cAMP binding domains is highly dynamic, providing an explanation of how cAMP gains access to the ligand binding sites that, in the crystal structure, are seen to be mutually occluded by the other cAMP binding domain. Moreover, cAMP also induces conformational changes at the ionic latch/hairpin structure, which is directly involved in RAP1 binding. These results suggest that in addition to relieving the steric hindrance imposed upon the catalytic lobe by the regulatory lobe, cAMP may also be an allosteric modulator directly affecting the interaction between Epac2 and RAP1. Finally, cAMP binding also induces significant conformational changes in the dishevelled/Egl/pleckstrin (DEP) domain, a conserved structural motif that, although missing from the active Epac2 crystal structure, is important for Epac subcellular targeting and in vivo functions.

Exchange protein directly activated by cyclic AMP isoform 2 is not a direct target of sulfonylurea drugs.

It has been reported by Zhang et al. that antidiabetic sulfonylurea drugs promote insulin secretion by directly binding to exchange protein directly activated by cyclic AMP isoform 2 (Epac2) and activating its down-stream effector Rap1. However, a critical link for an unambiguous validation of a direct interaction between Epac2 and sulfonylurea using purified individual components is missing. Our in vitro analyses using purified full-length Epac2 and Rap1 suggest that sulfonylureas are not able to directly bind to Epac2, nor are they capable of triggering Epac2-dependent Rap1 activation.

Mechanism of Epac activation: structural and functional analyses of Epac2 hinge mutants with constitutive and reduced activities.

Epac2 is a member of the family of exchange proteins directly activated by cAMP (Epac). Our previous studies suggest a model of Epac activation in which cAMP binding to the enzyme induces a localized "hinge" motion that reorients the regulatory lobe relative to the catalytic lobe without inducing large conformational changes within individual lobes. In this study, we identified the location of the major hinge in Epac2 by normal mode motion correlation and structural alignment analyses. Targeted mutagenesis was then performed to test the functional importance of hinge bending for Epac activation. We show that substitution of the conserved residue phenylalanine 435 with glycine (F435G) facilitates the hinge bending and leads to a constitutively active Epac2 capable of stimulating nucleotide exchange in the absence of cAMP. In contrast, substitution of the same residue with a bulkier side chain, tryptophan (F435W), impedes the hinge motion and results in a dramatic decrease in Epac2 catalytic activity. Structural parameters determined by small angle x-ray scattering further reveal that whereas the F435G mutant assumes a more extended conformation in the absence of cAMP, the F435W mutant is incapable of adopting the fully extended and active conformation in the presence of cAMP. These findings demonstrate the importance of hinge motion in Epac activation. Our study also suggests that phenylalanine at position 435 is the optimal size side chain to keep Epac closed and inactive in the absence of cAMP while still allowing the proper hinge motion for full Epac extension and activation in the presence of cAMP.

Epac and PKA: a tale of two intracellular cAMP receptors.

cAMP-mediated signaling pathways regulate a multitude of important biological processes under both physiological and pathological conditions, including diabetes, heart failure and cancer. In eukaryotic cells, the effects of cAMP are mediated by two ubiquitously expressed intracellular cAMP receptors, the classic protein kinase A (PKA)/cAMP-dependent protein kinase and the recently discovered exchange protein directly activated by camp (Epac)/cAMP-regulated guanine nucleotide exchange factors. Like PKA, Epac contains an evolutionally conserved cAMP binding domain that acts as a molecular switch for sensing intracellular second messenger cAMP levels to control diverse biological functions. The existence of two families of cAMP effectors provides a mechanism for a more precise and integrated control of the cAMP signaling pathways in a spatial and temporal manner. Depending upon the specific cellular environments as well as their relative abundance, distribution and localization, Epac and PKA may act independently, converge synergistically or oppose each other in regulating a specific cellular function.

Effect of glutathione on homo- and heterotropic cooperativity in cytochrome P450 3A4.

Glutathione (GSH) exerted a profound effect on the oxidation of 7-benzyloxy-4-(trifluoromethyl)coumarin (BFC) and 7-benzyloxyquinoline (BQ) by human liver microsomes as well as by CYP3A4-containing insect cell microsomes (Baculosomes). The cooperativity in O-debenzylation of both substrates is eliminated in the presence of 1-4mM GSH. Addition of GSH also increased the amplitude of the 1-PB induced spin shift with purified CYP3A4 and abolished the cooperativity of 1-PB or BFC binding. Changes in fluorescence of 6-bromoacetyl-2-dimethylaminonaphthalene attached to the cysteine-depleted mutant CYP3A4(C58,C64) suggest a GSH-induced conformational changes in proximity of alpha-helix A. Importantly, the K(S) value for formation of the GSH complex and the concentrations in which GSH decreases CYP3A4 cooperativity are consistent with the physiological concentrations of GSH in hepatocytes. Therefore, the allosteric effect of GSH on CYP3A4 may play an important role in regulation of microsomal monooxygenase activity in vivo.

Mechanism of interactions of alpha-naphthoflavone with cytochrome P450 3A4 explored with an engineered enzyme bearing a fluorescent probe.

Design of a partially cysteine-depleted C98S/C239S/C377S/C468A cytochrome P450 3A4 mutant designated CYP3A4(C58,C64) allowed site-directed incorporation of thiol-reactive fluorescent probes into alpha-helix A. The site of modification was identified as Cys-64 with the help of CYP3A4(C58) and CYP3A4(C64), each bearing only one accessible cysteine. Changes in the fluorescence of CYP3A4(C58,C64) labeled with 6-(bromoacetyl)-2-(dimethylamino)naphthalene (BADAN), 7-(diethylamino)-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM), or monobromobimane (mBBr) were used to study the interactions with bromocriptine (BCT), 1-pyrenebutanol (1-PB), testosterone (TST), and alpha-naphthoflavone (ANF). Of these substrates only ANF has a specific effect, causing a considerable decrease in fluorescence intensity of BADAN and CPM and increasing the fluorescence of mBBr. This ANF-binding event in the case of the BADAN-modified enzyme is characterized by an S50 of 18.2 +/- 0.7, compared with the value of 2.2 +/- 0.3 for the ANF-induced spin transition, thus revealing an additional low-affinity binding site. Studies of the effect of TST, 1-PB, and BCT on the interactions of ANF monitored by changes in fluorescence of CYP3A4(C58,C64)-BADAN or by the ANF-induced spin transition revealed no competition by these substrates. Investigation of the kinetics of fluorescence increase upon H2O2-dependent heme depletion suggests that labeled CYP3A4(C58,C64) is represented by two conformers, one of which has the fluorescence of the BADAN and CPM labels completely quenched, presumably by photoinduced electron transfer from the neighboring Trp-72 and/or Tyr-68 residues. The binding of ANF to the newly discovered binding site appears to affect the interactions of the label with the above residue(s), thus modulating the fraction of the fluorescent conformer.