Membranes prime the RapGEF EPAC1 to transduce cAMP signaling.

EPAC1, a cAMP-activated GEF for Rap GTPases, is a major transducer of cAMP signaling and a therapeutic target in cardiac diseases. The recent discovery that cAMP is compartmentalized in membrane-proximal nanodomains challenged the current model of EPAC1 activation in the cytosol. Here, we discover that anionic membranes are a major component of EPAC1 activation. We find that anionic membranes activate EPAC1 independently of cAMP, increase its affinity for cAMP by two orders of magnitude, and synergize with cAMP to yield maximal GEF activity. In the cell cytosol, where cAMP concentration is low, EPAC1 must thus be primed by membranes to bind cAMP. Examination of the cell-active chemical CE3F4 in this framework further reveals that it targets only fully activated EPAC1. Together, our findings reformulate previous concepts of cAMP signaling through EPAC proteins, with important implications for drug discovery.

Stability, pharmacokinetics, and biodistribution in mice of the EPAC1 inhibitor (R)-CE3F4 entrapped in liposomes and lipid nanocapsules.

(R)-CE3F4, a specific inhibitor of EPAC1 (exchange protein directly activated by cAMP type 1), has been demonstrated in vitro and in vivo to reduce hypertrophic signaling contributing to heart failure or to control arrhythmia and has shown promise as a drug candidate. However, (R)-CE3F4 exhibits poor solubility in aqueous media and has shown sensitivity to enzyme hydrolysis in plasma. To overcome these issues, the drug was entrapped in liposomes and lipid nanocapsules. Both systems considerably increased the drug apparent solubility in aqueous media. Among these nanocarriers, lipid nanocapsules offered significant protection in vitro against enzymatic degradation by increasing the (R)-CE3F4 apparent half-life from around 40 min to 6 h. Pharmacokinetics and biodistribution of (R)-CE3F4 radiolabeled or not were studied in healthy C57BL/6 mice. The non-encapsulated 3H-CE3F4 showed a very rapid distribution outside the blood compartment. Similar results were observed when using nanocarriers together with a fast dissociation of 3H-CE3F4 from nanocapsules simultaneously labeled with 14C. Thus, essential preclinical information on CE3F4 fate has been obtained, as well as the impact of its formulation using lipid-based nanocarriers.

Interspecies comparison of plasma metabolism and sample stabilization for quantitative bioanalyses: Application to (R)-CE3F4 in preclinical development, including metabolite identification by high-resolution mass spectrometry.

The CE3F4 is an inhibitor of the type 1 exchange protein directly activated by cAMP (EPAC1), which is involved in numerous signaling pathways. The inhibition of EPAC1 shows promising results in vitro and in vivo in different cardiac pathological situations like hypertrophic signaling, contributing to heart failure, or arrhythmia. An HPLC-UV method with a simple and fast sample treatment allowed the quantification of (R)-CE3F4. Sample treatment consisted of simple protein precipitation with 50 µL of ethanol and 150 µL of acetonitrile for a 50 µL biological sample. Two wavelengths were used according to the origin of plasma (220 or 250 nm for human samples and 250 nm for murine samples). Accuracy profile was evaluated for both wavelengths, and the method was in agreement with the criteria given by the EMA in the guideline for bioanalytical method validation for human and mouse plasma samples. The run time was 12 min allowing the detection of the (R)-CE3F4 and a metabolite. This study further permitted understanding the behavior of CE3F4 in plasma by highlighting an important difference between humans and rodents on plasma metabolism and may impact future in vivo studies related to this molecule and translation of results between animal models and humans. Using paraoxon as a metabolism inhibitor was crucial for the stabilization of (R)-CE3F4 in murine samples. HPLC-UV and HPLC-MS/MS studies were conducted to confirm metabolite structure and consequently, the main metabolic pathway in murine plasma.

Synthesis, evaluation and absolute configuration assignment of novel dihydropyrimidin-2-ones as picomolar sodium iodide symporter inhibitors.

A small library of dihydropyrimidin-2-ones (DHPMs) was synthesized and evaluated for their potency to block iodide entrapment in rat thyroid cells. Synthesis was achieved using the multicomponent Biginelli reaction. Twelve compounds were tested for the inhibition of sodium iodide symporter (NIS) in a cell-based assay. One newly synthesized derivative exhibited a remarkably strong activity, with a half-maximum inhibitory concentration value (IC50) of 65 pM. Three DHPMs were further resolved from racemates using chiral HPLC and absolute configurations were assigned using circular dichroism spectroscopy. Biological evaluation showed that most of the activity against NIS resides in one enantiomer. This study provides new insights for the development of anti-thyroid drugs, as well as for the synthesis of novel pharmacological tools designed to investigate iodide transport mechanisms at cellular and molecular levels.

Synthesis and evaluation of 3,4-dihydropyrimidin-2(1H)-ones as sodium iodide symporter inhibitors.

The sodium iodide symporter (NIS) is responsible for the accumulation of iodide in the thyroid gland. This transport process is involved in numerous thyroid dysfunctions and is the basis for human contamination in the case of exposure to radioactive iodine species. 4-Aryl-3,4-dihydropyrimidin-2(1H)-ones were recently discovered by high-throughput screening as the first NIS inhibitors. Described herein are the synthesis and evaluation of 115 derivatives with structural modifications at five key positions on the pyrimidone core. This study provides extensive structure-activity relationships for this new class of inhibitors that will serve as a basis for further development of compounds with in vivo efficacy and adequate pharmacokinetic properties. In addition, the SAR investigation provided a more potent compound, which exhibits an IC(50) value of 3.2 nM in a rat thyroid cell line (FRTL5).

Direct interaction of RNA polymerase II and mediator required for transcription in vivo.

Gene transcription is highly regulated. Altered transcription can lead to cancer or developmental diseases. Mediator, a multisubunit complex conserved among eukaryotes, is generally required for RNA polymerase II (Pol II) transcription. An interaction between the two complexes is known, but its molecular nature and physiological role are unclear. We identify a direct physical interaction between the Rpb3 Pol II subunit of Saccharomyces cerevisiae and the essential Mediator subunit, Med17. Furthermore, we demonstrate a functional element in the Mediator-Pol II interface that is important for genome-wide Pol II recruitment in vivo. Our findings suggest that a direct interaction between Mediator and Pol II is generally required for transcription of class II genes in eukaryotes.

A nonradioactive iodide uptake assay for sodium iodide symporter function.

The standard assay for sodium iodide symporter (NIS) function is based on the measurement of radioiodide uptake ((125)I) in NIS-expressing cells. However, cost and safety issues have limited the method from being used widely. Here we describe a simple spectrophotometric assay for the determination of iodide accumulation in rat thyroid-derived cells (FRTL5) based on the catalytic effect of iodide on the reduction of yellow cerium(IV) to colorless cerium(III) in the presence of arsenious acid (Sandell-Kolthoff reaction). The assay is fast and highly reproducible with a Z' factor of 0.70. This procedure allows the screening of more than 800 samples per day and can easily be adapted to robotic systems for high-throughput screening of NIS function modulators. Using this method, the potency of several known inhibitors of NIS function was evaluated in a single day with high accuracy and reliability. Measured IC(50) values were essentially identical to those determined using Na(125)I.

Synthesis and evaluation of imidazo[2,1-b]thiazoles as iodide efflux inhibitors in thyrocytes.

The Na(+)/I(-) symporter (NIS) mediates iodide uptake in the thyroid gland as well as in other NIS-expressing cells. This transport is the basis for an emerging approach to selective cancer cell destruction by using radioiodide after targeted NIS gene transfer. Therapeutic efficacy requires that radioiodide retention be maximized in tumor cells. A first generation of forty imidazo[2,1-b]thiazole derivatives as iodide efflux inhibitors is described along with the evaluation of their biological properties. Structure-activity relationship studies by using radioiodide uptake in rat thyroid-derived cells (FRTL5) revealed that the 5,6-dihydroimidazo[2,1-b]thiazole heterocycle is required for activity. Introduction of electron-donor substituents on the 3-biphenyl moiety led to the discovery of novel potent compounds. A compound was identified with enhanced potency compared to reference 1. These molecules give the possibility to increase the cellular retention of radioiodide in NIS-expressing tumors, leading to higher absorbed doses and killing efficacy.

Synthesis and evaluation of photoreactive probes to elucidate iodide efflux in thyrocytes.

Four photoreactive analogues of 3-biphenyl-4'-yl-5,6-dihydroimidazo[2,1-b]thiazole were prepared and evaluated as iodide sequestering agents in sodium iodide symporter-expressing cells. One of these new photoactivatable compounds retained biological activity and was further radiolabeled with tritium. This compound may provide a useful tool for labeling, purification, and identification of target protein responsible for iodide efflux in thyrocytes.

Characterization of small-molecule inhibitors of the sodium iodide symporter.

The sodium/iodide symporter (NIS) mediates the active transport of iodide from the bloodstream into thyrocytes. NIS function is strategic for the diagnosis and treatment of various thyroid diseases. In addition, a promising anti-cancer strategy based on targeted NIS gene transfer in non-thyroidal cells is currently developed. However, only little information is available concerning the molecular mechanism of NIS-mediated iodide translocation. Ten small molecules have recently been identified using a high-throughput screening method for their inhibitory effect on iodide uptake of NIS-expressing mammalian cells. In the present study, we analyzed these compounds for their rapid and reversible effects on the iodide-induced current in NIS-expressing Xenopus oocytes. Four molecules almost completely inhibited the iodide-induced current; for three of them the effect was irreversible, for one compound the initial current could be fully re-established after washout. Three molecules showed a rapid inhibitory effect of about 75%, half of which was reversible. Another three compounds inhibited the iodide-induced current from 10 to 50%. Some molecules altered the membrane conductance by themselves, i.e. in the absence of iodide. For one of these molecules the observed effect was also found in water-injected oocytes whereas for some others the iodide-independent effect was associated with NIS expression. The tested molecules show a surprisingly high variability in their possible mode of action, and thus are promising tools for further functional characterization of NIS on a molecular level, and they could be useful for medical applications.

A colorimetric assay for steady-state analyses of iodo- and bromoperoxidase activities.

The standard assay for iodoperoxidase activity is based on the spectrophotometric detection of triiodide formed during the enzymatic reaction. However, chemical instability of I3- has limited the method to high iodide concentrations and acidic conditions. Here we describe a simple spectrophotometric assay for the determination of iodoperoxidase activities of vanadium haloperoxidases based on the halogenation of thymol blue. The relation between color and chemical entities produced by the vHPO/H(2)O(2)/I(-) catalytic system was characterized. The method was extended to bromine and, for the first time, allowed measurement of both iodo- and bromoperoxidase activities using the same assay. The kinetic parameters (K(m) and k(cat)) of bromide and iodide for vanadium bromoperoxidase from Ascophyllum nodosum were determined at pH 8.0 from steady-state kinetic analyses. The results are concordant with an ordered two-substrate mechanism. It is proposed that halide selectivity is guided by the chemical reactivity of peroxovanadium intermediate rather than substrate binding. This method is superior to the standard I3- assay, and we believe that it will find applications for the characterization of other vanadium as well as heme haloperoxidases.

Enhanced iodide sequestration by 3-biphenyl-5,6-dihydroimidazo[2,1-b]thiazole in sodium/iodide symporter (NIS)-expressing cells.

The ability of the sodium/iodide symporter (NIS) to take up iodide has long provided the basis for cytoreductive gene therapy and cancer treatment with radioiodide. One of the major limitations of this approach is that radioiodide retention in NIS-expressing cells is not sufficient for their destruction. We identified and characterized a small organic molecule capable of increasing iodide retention in HEK293 cells permanently transfected with human NIS cDNA (hNIS-HEK293) and in the rat thyroid-derived cell line FRTL-5. In the presence of 3-biphenyl-4'-yl-5,6-dihydroimidazo[2,1-b]thiazole (ISA1), the transmembrane iodide concentration gradient was increased up to 4.5-fold. Our experiments indicate that the imidazothiazole derivative acts either by inhibiting anion efflux mechanisms, or by promoting the relocation of iodide into subcellular compartments. This new compound is not only an attractive chemical tool to investigate the mechanisms of iodide flux at the cellular level, but also opens promising perspectives in the treatment of cancer after NIS gene transfer.

Small-molecule inhibitors of sodium iodide symporter function.

The Na(+)/I(-) symporter (NIS) mediates iodide uptake into thyroid follicular cells. Although NIS has been cloned and thoroughly studied at the molecular level, the biochemical processes involved in post-translational regulation of NIS are still unknown. The purpose of this study was to identify and characterize inhibitors of NIS function. These small organic molecules represent a starting point in the identification of pharmacological tools for the characterization of NIS trafficking and activation mechanisms. The screening of a collection of 17,020 druglike compounds revealed new chemical inhibitors with potencies down to 40 nM. Fluorescence measurement of membrane potential indicates that these inhibitors do not act by disrupting the sodium gradient. They allow immediate and total iodide discharge from preloaded cells in accord with a specific modification of NIS activity, probably through distinct mechanisms.

Microchemical imaging of iodine distribution in the brown alga Laminaria digitata suggests a new mechanism for its accumulation.

Brown algal kelp species are the most efficient iodine accumulators among all living systems, with an average content of 1.0% of dry weight in Laminaria digitata. The iodine distributions in stipe and blade sections from L. digitata were investigated at tissue and subcellular levels. The quantitative tissue mapping of iodine and other trace elements (Cl, K, Ca, Fe, Zn, As and Br) was provided by the proton microprobe with spatial resolutions down to 2 mum. Chemical imaging at a subcellular resolution (below 100 nm) was performed using the secondary ion mass spectrometry microprobe. Sets of samples were prepared by both chemical fixation and cryofixation procedures. The latter prevented the diffusion and the leaching of labile inorganic iodine species, which were estimated at around 95% of the total content by neutron activation analysis. The distribution of iodine clearly shows a huge, decreasing gradient from the meristoderm to the medulla. The contents of iodine reach very high levels in the more external cell layers, up to 191 +/- 5 mg g(-1) of dry weight in stipe sections. The peripheral tissue is consequently the main storage compartment of iodine. At the subcellular level, iodine is mainly stored in the apoplasm and not in an intracellular compartment as previously proposed. This unexpected distribution may provide an abundant and accessible source of labile iodine species which can be easily remobilized for potential chemical defense and antioxidative activities. According to these imaging data, we proposed new hypotheses for the mechanism of iodine storage in L. digitata tissues.

A 96-well automated radioiodide uptake assay for sodium/iodide symporter inhibitors.

A high-throughput screening method based on radioiodide uptake in human embryonic kidney 293 cells expressing the human sodium/iodide symporter was developed. Central to assay development was a homogeneous cell culture in the 96-well microplate coupled with the use of scintillation proximity technology. The assay is fast and highly reproducible with a Z' greater than 0.8. The automated procedure allows the screening of 4,000 compounds per day. Using this methodology, several known substrates of the sodium/iodide symporter were evaluated in a single day. Inhibition of iodide uptake was shown to follow the series PF(6)(-) > ClO(4)(-) > BF(4)(-) > SCN(-) >> NO(3)(-) > IO(4)(-) > N(3)(-) >> Br(-), in accord with the literature. This method represents an initial approach to the search for inhibitors of iodide transport mediated by the sodium/iodide symporter.

Iodine transfers in the coastal marine environment: the key role of brown algae and of their vanadium-dependent haloperoxidases.

Brown algal kelp species are the most efficient iodine accumulators among all living systems, with an average content of 1.0% of dry weight in Laminaria digitata, representing a ca. 30,000-fold accumulation of this element from seawater. Like other marine macroalgae, kelps are known to emit volatile short-lived organo-iodines, and molecular iodine which are believed to be a main vector of the iodine biogeochemical cycle as well as having a significant impact on atmospheric chemistry. Therefore, radioactive iodine can potentially accumulate in seaweeds and can participate in the biogeochemical cycling of iodine, thereby impacting human health. From a radioecological viewpoint, iodine-129 (129I, half-life of 1.6 x 10(7) years) is one of the most persistent radionuclide released from nuclear facilities into the environment. In this context, the speciation of iodine by seaweeds is of special importance and there is a need to further understand the mechanisms of iodine uptake and emission by kelps. Recent results on the physiological role and biochemistry of the vanadium haloperoxidases of brown algae emphasize the importance of these enzymes in the control of these processes.

Inhibitors of cell migration that inhibit intracellular paxillin/alpha4 binding: a well-documented use of positional scanning libraries.

Screening combinatorial libraries for inhibition of Paxillin binding to the cytoplasmic tail of the integrin alpha4 provided the first inhibitors of this protein-protein interaction implicated in enhanced rates of cell migration and chronic inflammation. The preparation of substructure analogs of the lead identified features required for activity, those available for modification, and those that may be removed. The most potent lead structure was shown to inhibit alpha(4)beta(1)-mediated human Jurkat T cell migration in a dose-dependent manner, validating the intracellular Paxillin/alpha4 interaction as a useful and unique target for therapeutic intervention. Moreover, the lead structure emerged from a library that was prepared in two formats: (1) a traditional small mixture format composed of 100 mixtures of 10 compounds and (2) a positional scanning library. Their parallel testing provided the rare opportunity to critically compare two approaches.

Highly chemoselective hydrogenolysis of iodoarenes.

The catalytic hydrodehalogenation reaction using molecular hydrogen and Pd/C has been revisited. It is shown that the speed of removal of halogen increases with increasing electronegativity I < Br < Cl. Nevertheless, selective dehydrohalogenation in compounds containing other reducible functions can be achieved only with iodine and not with bromine or chlorine. Selective deiodination of iodobenzophenone could be accomplished without reducing the carbonyl group. Hydrogenolysis of azidoiodoaromatic compounds to the corresponding azido compounds is high yielding. This selectivity was exploited for the labeling of benzophenone- and azido-containing compounds by deuterium and tritium.