The conformation of nascent polylysine and polyphenylalanine peptides on ribosomes

Polypeptide synthesis using either phenylalanine or lysine was initiated on Escherichia coli ribosomes; then the position and conformation of the nascent peptide were monitored by fluorescence techniques. To this end, fluorophores had been attached to the amino terminus of each nascent peptide, and major differences were observed as chain extension occurred. Polyphenylalanine appeared to build up as a hydrophobic mass adjacent to the peptidyl transferase center while polylysine apparently was extended directly from the ribosome into the surrounding solution. An explanation for these differences may be provided by the physical and chemical properties of each polypeptide. These properties may be responsible for the route by which each peptide exits the peptidyl transferase center as demonstrated by the different sensitivity of each to inhibition by erythromycin

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 th

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

Dose-dependent response of selected cAMP analogs of known activity.

<p>Active compounds: cAMP and 8-Cl-cAMP; inactive compounds: 2′-deoxy-cAMP and cXMP. Data are from three independent experiments with error bars representing standard deviations.</p

Summary of assay statistic parameters^* of independent experiments.

<p>*S: mean signal; σ_S: standard deviation of signal; CV: coefficient of variation; B: mean background (control); σ_B: standard deviation of background; S/B: signal-to-background ratio; S/N: signal-to-noise ratio; Z′: Z′ score as determined by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030441#pone.0030441-Zhang1" target="_blank">[21]</a>.</p

Relative potency of identified EPAC antagonists.

<p>Dose-dependent competition of EPAC antagonists with 8-NBD-cAMP in binding to EPAC2: open circles, cAMP control; open squares, 25 µM NSC45576; open diamonds, 25 µM NSC119911; and open triangles up, 25 µM NSC686365. Similar results were obtained from three independent experiments.</p

Structural Analyses of a Constitutively Active Mutant of Exchange Protein Directly Activated by cAMP

<div><p>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.</p> </div

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, <i>N</i>,<i>N</i>-diarylamines, and arylsulfonamides) as highly potent and selective antagonists of EPAC2. Several selective EPAC2 antagonists have been identified including <b>20i</b> (HJC0350), which has an apparent IC₅₀ of 0.3 μM for competing with 8-NBD-cAMP binding of EPAC2 and is about 133-fold more potent than cAMP. Compounds <b>1</b> (ESI-05), <b>14c</b> (HJC0338), and <b>20i</b>, 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 <b>20i</b> functions as an EPAC2 specific antagonist