Synthesis of 5'-branched derivatives of cyclic ADP-carbocyclic-ribose, a potent Ca2+-mobilizing agent: The first antagonists modified at the N1-ribose moiety

The 5'-branched cyclic ADP-carbocyclic-ribose derivatives were designed and synthesized. These target compounds were identified as the first antagonists of cADPR without a substituent at the adenine 8-position, and were shown to be stable due to the N1-carbocyclic-ribosyl structure.     

Synthesis of cyclic ADP-carbocylcic-xylose and its 3"-O-methyl analogue as stable and potent Ca2+ -mobilizing agents

We previously showed that 3"-deoxy-cyclic ADP-carbocyclic-ribose (3"-deoxy-cADPcR, 3) is a stable and highly potent analogue of cyclic ADP-ribose (cADPR, 1), a Ca2+ -mobilizing second messenger. From these results, we newly designed another 3"-modified analogues of cADPcR and identified the N1-"xylo"-type carbocyclic analogue, i.e., cADPcX (4), as one of the most potent cADPR-related compounds reported so far.     

The discovery and synthesis of highly potent, A2a receptor agonists

A series of N6,2-disubstituted adenosine analogues have been synthesized and their functional activity measured against A2a and A1 receptors. Examples of compounds with both a lipophilic N6-substituent and amino-functionalized 2-position were highly active at the A2a receptor on the human neutrophil.     

The synthesis of highly potent, selective, and water-soluble agonists at the human adenosine A3 receptor

Using a combination of parallel and directed synthesis, the discovery of a highly potent and selective series of adenosine A3 agonists was achieved. High aqueous solubility, required for the intended parenteral route of administration, was achieved by the presence of one or two basic amine functional groups.     

Structural determination of a cyclic metabolite of NAD+ with intracellular Ca2+-mobilizing activity

Incubation of NAD+ with extracts from sea urchin eggs resulted in production of a metabolite which could mobilize intracellular Ca2+ stores of the eggs. In this study we present structural evidence indicating that the metabolite is a cyclized ADP-ribose having an N-glycosyl linkage between the anomeric carbon of the terminal ribose unit and the N6-amino group of the adenine moiety. In view of this structure we propose cyclic ADP-ribose as the common name for the metabolite. The purification procedure for the metabolite consisted of deproteinizing the incubated egg extracts and sequentially chromatographing the extracts through three different high pressure liquid chromatography (HPLC) columns. The homogeneity of the purified metabolite was further verified by HPLC on a Partisil 5 SAX column. Using radioactive precursor NAD+ with label at various positions it was demonstrated that the metabolite was indeed derived from NAD+ and that the adenine ring as well as the adenylate alpha-phosphate were retained in the metabolite whereas the nicotinamide group was removed. This was confirmed by 1H NMR and two-dimensional COSY experiments, which also allowed the identification of all 12 protons on the two ribosyl units as well as the two protons on the adenine ring. From the chemical shifts of the two anomeric protons it was concluded that the C-1 carbons of both ribosyl units were still bonded to nitrogen. The positive and negative ion fast atom bombardment mass spectra showed (M + Na)+, (M - H + 2Na)+, (M - H)-, and (M - 2H + Na)- peaks at m/z 564, 586, 540, and 562, respectively. Exact mass measurements indicated a molecular weight of 540.0526 for (M - H)-. This together with the constraints imposed by the results from NMR, radioactive labeling, and total phosphate determination uniquely specified a molecular composition of C15H21N5O13P2. Analysis by 1H NMR and mass spectroscopy of the only major breakdown product of the metabolite after prolonged incubation at room temperature established that it was ADP-ribose, thus providing strong support for the cyclic structure.     

Tumor necrosis factor as an activator of human granulocytes. Potentiation of the metabolisms triggered by the Ca2+-mobilizing agonists

TNF stimulated superoxide (O2-) release directly in human granulocytes in a dose-dependent manner (1 to 1000 U/ml), although its potency was weak. TNF-induced O2- release was inhibited by cAMP agonists or ionomycin, and was not accompanied with an increase in cytoplasmic free Ca2+ [( Ca2+]i) and membrane potential changes (depolarization). These findings indicate that neither Ca2+ mobilization nor membrane depolarization is required for TNF-receptor-mediated cell activation. The pretreatment of human granulocytes with TNF enhanced O2- release and membrane depolarization in parallel stimulated by the receptor-mediated Ca2+-mobilizing agonists (FMLP, Con A, and wheat germ agglutinin) or the Ca2+ ionophore ionomycin, but not by PMA, a direct activator of protein kinase C. The optimal effect was obtained by pretreatment of cells with 100 U/ml TNF for 5 to 10 min at 37 degrees C, although the magnitude of enhancement varied according to the agonists used as subsequent stimuli. TNF did not affect an increase in [Ca2+]i stimulated by the Ca2+-mobilizing agonists, except Con A. Con A-induced increase in [Ca2+]i was enhanced by TNF in a dose-dependent manner. These diverse effects of TNF could be partly explained by the exclusive potentiation by TNF of the metabolic events triggered by an increase in [Ca2+]i.     

Cyclic ADP-ribose, a putative Ca2+-mobilizing second messenger, operates in submucosal gland acinar cells

Cyclic ADP-ribose (cADPR), a putative Ca(2+)-mobilizing second messenger, has been reported to operate in several mammalian cells. To investigate whether cADPR is involved in electrolyte secretion from airway glands, we used a patch-clamp technique, the measurement of microsomal Ca(2+) release, quantification of cellular cADPR, and RT-PCR for CD38 mRNA in human and feline tracheal glands. cADPR (>6 microM), infused into the cell via the patch pipette, caused ionic currents dependent on cellular Ca(2+). Infusions of lower concentrations (2-4 microM) of cADPR or inositol 1,4,5-trisphosphate (IP(3)) alone were without effect on the baseline current, but a combined application of cADPR and IP(3) mimicked the cellular response to low concentrations of acetylcholine (ACh). Microsomes derived from the isolated glands released Ca(2+) in response to both IP(3) and cADPR. cADPR released Ca(2+) from microsomes desensitized to IP(3) or those treated with heparin. The mRNA for CD38, an enzyme protein involved in cADPR metabolism, was detected in human tissues, including tracheal glands, and the cellular content of cADPR was increased with physiologically relevant concentrations of ACh. We conclude that cADPR, in concert with IP(3), operates in airway gland acinar cells to mobilize Ca(2+), resulting in Cl(-) secretion.     

Intracellular pH-regulatory mechanisms in pancreatic acinar cells. II. Regulation of H+ and HCO3- transporters by Ca2(+)-mobilizing agonists

Pancreatic acini loaded with the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein were used to examine the effect of Ca2(+)-mobilizing agonists on the activity of acid-base transporters in these cells. In the accompanying article (Muallen, S., and Loessberg, P. A. (1990) J. Biol. Chem. 265, 12813-12819) we showed that in 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES)-buffered medium the main pHi regulatory mechanism is the Na+/H+ exchanger, a while in HCO3(-)-buffered medium pHi is determined by the combined activities of a Na+/H+ exchanger, a Na(+)-HCO3- cotransporter and a Cl-/HCO3- exchanger. In this study we found that stimulation of acini with Ca2(+)-mobilizing agonists in HEPES or HCO3(-)-buffered media is followed by an initial acidification which is independent of any identified plasma membrane-located acid-base transporting mechanism, and thus may represent intracellularly produced acid. In HEPES-buffered medium there was a subsequent large alkalinization to pHi above that in resting cells, which could be attributed to the Na+/H+ exchanger. Measurements of the rate of recovery from acid load indicated that the Na+/H+ exchanger was stimulated by the agonists. In HCO3(-)-buffered medium the alkalinization observed after the initial acidification was greatly attenuated. Examination of the activity of each acid-base transporting mechanism in stimulated acini showed that in HCO3(-)-buffered medium: (a) recovery from acid load in the presence of H2-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (H2DIDS) (Na+/H+ exchange) was stimulated similar to that found in HEPES-buffered medium; (b) recovery from acid load in the presence of amiloride and acidification due to removal of external Na+ in the presence of amiloride (HCO3- influx and efflux, respectively, by Na(+)-HCO3- cotransport) were inhibited; and (c) HCO3- influx and efflux due to Cl-/HCO3- exchange, which was measured by changing the Cl- or HCO3- gradients across the plasma membrane, were stimulated. Furthermore, the rate of Cl-/HCO3- exchange in stimulated acini was higher than the sum of H+ efflux due to Na+/H+ exchange and HCO3- influx due to Na(+)-HCO3- cotransport. Use of H2DIDS showed that the latter accounted for the attenuated changes in pHi in HCO3(-)-buffered medium, as much as treating the acini with H2DIDS resulted in similar agonist-mediated pHi changes in HEPES- and HCO3(-)-buffered media. The effect of agonists on the various acid-base transporting mechanisms is discussed in terms of their possible role in transcellular NaCl transport, cell volume regulation, and cell proliferation in pancreatic acini.     

Solubilization of receptors for the novel Ca2+-mobilizing messenger,nicotinic acid adenine dinucleotide phosphate

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca(2+) mobilizing agent in a variety of broken and intact cell preparations. In sea urchin egg homogenates, NAADP releases Ca(2+) independently of inositol trisphosphate or ryanodine receptor activation. Little, however, is known concerning the molecular target for NAADP. Here we report for the first time solubilization of NAADP receptors from sea urchin egg homogenates. Supernatant fractions, prepared following Triton X-100 treatment, bound [(32)P]NAADP with similar affinity and selectivity as membrane preparations. Furthermore, the unusual non-dissociating nature of NAADP binding to its receptor was preserved upon solubilization. NAADP receptors could also be released into supernatant fractions upon detergent treatment of membranes prelabeled with [(32)P]NAADP. Tagged receptors prepared in this way, were readily resolved by native gel electrophoresis as a single protein target. Gel filtration and sucrose density gradient centrifugation analysis indicates that NAADP receptors are substantially smaller than inositol trisphosphate or ryanodine receptors, providing further biochemical evidence that NAADP activates a novel intracellular Ca(2+) release channel.     

Molecular recognition of adenophostin, a very potent Ca2+ inducer, at the D-myo-inositol 1,4,5-trisphosphate receptor.

The recognition mode of adenophostin A at the D-myo-inositol 1,4, 5-trisphosphate [Ins(1,4,5)P(3)] receptor was investigated. Comparison of conformations of Ins(1,4,5)P(3) and adenophostin A by using the combination of NMR and molecular mechanics (MM) calculations demonstrated that adenophostin A adopted a moderately extended conformation regarding the distance between the 2'-phosphoryl group and the 3' ',4' '-bisphosphate motif, as suggested previously [Wilcox, R. A. et al. (1995) Mol. Pharmacol. 47, 1204-1211]. Based on the nuclear Overhauser effect (NOE) observed between 3'-H and 1' '-H and on MM calculations, the molecular shape of adenophostin A proved to be an extended form at least in solution, in contrast to Wilcox's compactly folded, preliminary hairpin model. GlcdR(2,3',4')P(3), an adenophostin analogue without adenine moiety, was found to be less potent than adenophostin A and almost equipotent to Ins(1,4,5)P(3). We propose the possibility that (i) the optimal spatial arrangement of the three phosphoryl groups and/or (ii) the interaction of the adenine moiety of adenophostin A with the putative binding site, if it exists in the vicinity of the Ins(1,4,5)P(3)-binding site, might account for the exceptional potency of adenophostin A.     

Formyl-peptide receptor like 1: a potent mediator of the Ca2+ release-activated Ca2+ current ICRAC

In electrically non-excitable cells, one major source of Ca²⁺ influx is through the store-operated (or Ca²⁺ release-activated Ca²⁺) channel by which the process of emptying the intracellular Ca²⁺ stores results in the activation of Ca²⁺ channels in the plasma membrane. Using both whole-cell patch-clamp and Ca²⁺ imaging technique, we describe the electrophysiology mechanism underlying formyl-peptide receptor like 1 (FPRL1) linked to intracellular Ca²⁺ mobilization. The FPRL1 agonists induced Ca²⁺ release from the endoplasmic reticulum and subsequently evoked I_CRAC-like currents displaying fast inactivation in K562 erythroleukemia cells which expresses FPRL1, but had almost no effect in K562 cells treated with FPRL1 RNA-interference and HEK293 cells which showed no FPRL1 expression. The currents were impaired after either complete store depletion by the sarco/endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin, or after inhibition of PLC by U73122. Our results present the first evidence that FPRL1 is a potent mediator in the activation of CRAC channels.     

EPIC3, a novel Ca2+ indicator located at the cell cortex and in microridges,detects high Ca2+ subdomains during Ca2+ influx and phagocytosis

The construction of a low affinity Ca²⁺-probe that locates to the cell cortex and cell surface wrinkles, is described called. EPIC3 (ezrin-protein indicator of Ca²⁺). The novel probe is a fusion of CEPIA3 with ezrin, and is used in combination with a Ca²⁺-insensitive probe, ezrin-mCherry, both of which locate at the cell cortex. EPIC3 was used to monitor the effect of Ca²⁺ influx on intra-wrinkle Ca²⁺ in the macrophage cell line, RAW 264.7. During experimentally–induced Ca²⁺influx, EPIC3 reported Ca²⁺ concentrations at the cell cortex in the region of 30−50 μM, with peak locations towards the tips of wrinkles reaching 80 μM. These concentrations were associated with cleavage of ezrin (a substrate for the Ca²⁺ activated protease calpain-1) and released the C-terminal fluors. The cortical Ca²⁺ levels, restricted to near the site of phagocytic cup formation and pseudopodia extension during phagocytosis also reached high levels (50−80 μM) during phagocytosis. As phagocytosis was completed, hotspots of Ca²⁺ near the phagosome were also observed.     

2-difluoromethyloestrone 3-O-sulphamate, a highly potent steroid sulphatase inhibitor

Steroid sulphatase is a target enzyme of growing therapeutic importance. The synthesis and in vitro biological evaluation of three novel 2-substituted analogues of oestrone 3-O-sulphamate (EMATE), an established steroid sulphatase inhibitor, are described. One inhibitor, 2-difluoromethyloestrone 3-O-sulphamate (6), was found to have an IC50 of 100 pM and be some 90-fold more potent than EMATE in inhibiting steroid sulphatase activity in a placental microsomal preparation, rendering this agent the most potent steroidal STS inhibitor in vitro reported to date. Lowering of the pKa value of the leaving parent steroid phenol by the 2-difluoromethyl group during irreversible enzyme sulphamoylation most likely facilitates the potent inactivation of steroid sulphatase by (6). However, our preliminary molecular docking studies using the X-ray crystal structure of steroid sulphatase suggest that F.......H interactions between the 2-difluoromethyl group of (6) and hydrogen bond donor residues lining the catalytic site of STS might also contribute to the high potency observed for (6).     

Synthesis of N6-substituted 3'-ureidoadenosine derivatives as highly potent agonists at the mutant A3 adenosine receptor

Several N6-substituted 3 '-ureidoadenosine derivatives were efficiently synthesized starting from D-glucose for the development of H272E mutant A3 adenosine receptor (AR) agonists. Among compounds tested, 3 '-ureido-N6-(3-iodobenzyl)adenosine (2c) exhibited the highest binding affinity (Ki = 0.22 micro M) at the H272E mutant A3 AR without binding to the natural A3AR.     

D-4'-thioadenosine derivatives as highly potent and selective agonists at the human A3 adenosine receptor

4'-Thionucleoside derivatives as potent and selective A3 adenosaine receptor agonists were synthesized, starting from D-gulono-gamma-lactone via D-thioribosyl acetate as a key intermediate, among which the 2-chloro-N6-methyladenosine-5-methyluronamide showed the most potent and selective binding affinity (Ki = 0.28 +/- 0.09 nM) at the human A3 adenosine receptor.     

A novel class of Na+ and Ca2+ channel dual blockers with highly potent anti-ischemic effects

A series of novel arylpiperidines (4a-d) which have highly potent blocking effects for both neuronal Na+ and T-type Ca2+ channels with extremely low affinity for dopamine D2 receptors were synthesized. Among these compounds, 1-(2-hydroxy-3-phenoxy)propyl-4-(4-phenoxyphenyl)-piperidine hydrochloride (4c; SUN N5030) exhibited remarkable neuroprotective activity in a transient middle cerebral artery occlusion (MCAO) model.     

The influx of Ca2+ induced by the administration of glucagon and Ca2+-mobilizing agents to the perfused rat liver could involve at least two separate pathways.

The Ca2+-mobilizing actions of ADP, ATP and epidermal growth factor (EGF) and their interaction with glucagon were studied in a perfused liver system incorporating a Ca2+-selective electrode. ADP (1-100 microM), ATP (1-100 microM) and EGF (10-50 nM) all induced a net efflux, followed by a net uptake of Ca2+ in the intact liver. The co-administration of glucagon (or of cyclic AMP) with these agents resulted in a synergistic potentiation of the Ca2+ uptake response in a way which resembles the synergism observed when glucagon is administered with phenylephrine, vasopressin or angiotensin [Altin & Bygrave (1986) Biochem J. 238, 653-661]. The inability of diltiazem, verapamil and nifedipine to inhibit the Ca2+-influx response suggests that the stimulation of Ca2+ influx does not occur through voltage-sensitive Ca2+ channels. By contrast, the synergistic effects of glucagon in the stimulation of Ca2+ influx are inhibited by 10 mM-neomycin, and a lowering of the extracellular pH to 6.8. Simultaneous measurements of perfusate Ca2+ and pH changes suggest that the Ca2+ influx response is not mediated by a Ca2+/H+ exchange. The inability of neomycin and low extracellular pH to inhibit the refilling of the hormone-sensitive pool of Ca2+, after the administration of Ca2+-mobilizing agents alone, provides evidence for the existence in liver of at least two Ca2+-influx pathways, or mechanisms for regulating Ca2+ influx.     

Synergistic stimulation of Ca2+ uptake by glucagon and Ca2+-mobilizing hormones in the perfused rat liver. A role for mitochondria in long-term Ca2+ homoeostasis.

A perfused liver system incorporating a Ca2+-sensitive electrode was used to study the long-term effects of glucagon and cyclic AMP on the mobilization of Ca2+ induced by phenylephrine, vasopressin and angiotensin. At 1.3 mM extracellular Ca2+ the co-administration of glucagon (10 nM) or cyclic AMP (0.2 mM) and a Ca2+-mobilizing hormone led to a synergistic potentiation of Ca2+ uptake by the liver, to a degree which was dependent on the order of hormone administration. A maximum net amount of Ca2+ influx, corresponding to approx. 3800 nmol/g of liver (the maximum rate of influx was 400 nmol/min per g of liver), was induced when cyclic AMP or glucagon was administered about 4 min before vasopressin and angiotensin. These changes are over an order of magnitude greater than those induced by Ca2+-mobilizing hormones alone [Altin & Bygrave (1985) Biochem. J. 232, 911-917]. For a maximal response the influx of Ca2+ was transient and was essentially complete after about 20 min. Removal of the hormones was followed by a gradual efflux of Ca2+ from the liver over a period of 30-50 min; thereafter, a similar response could be obtained by a second administration of hormones. Dose-response measurements indicate that the potentiation of Ca2+ influx by glucagon occurs even at low (physiological) concentrations of the hormone. By comparison with phenylephrine, the stimulation of Ca2+ influx by vasopressin and angiotensin is more sensitive to low concentrations of glucagon and cyclic AMP, and can be correlated with a 20-50-fold increase in the calcium content of mitochondria. The reversible uptake of such large quantities of Ca2+ implicates the mitochondria in long-term cellular Ca2+ regulation.     

Switching between agonists and antagonists at CRTh2 in a series of highly potent and selective biaryl phenoxyacetic acids

A novel series of biaryl phenoxyacetic acids was discovered as potent, selective antagonists of the chemoattractant receptor-homologous expressed on Th2 lymphocytes receptor (CRTh2 or DP2). A hit compound 4 was discovered from high throughput screening. Modulation of multiple aryl substituents afforded both agonists and antagonists, with small changes often reversing the mode of action. Understanding the complex SAR allowed design of potent antagonists such as potential candidate 34.     

Substitution of a single residue, Asp575, renders the NCKX2 K+-dependent Na+/Ca2+ exchanger independent of K+

The Na(+)/Ca(2+)-K(+) exchanger (NCKX) is a polytopic membrane protein that uses both the inward Na(+) gradient and the outward K(+) gradient to drive Ca(2+) extrusion across the plasma membrane. NCKX1 is found in retinal rod photoreceptors, while NCKX2 is found in retinal cone photoreceptors and is also widely expressed in the brain. Here, we have identified a single residue (out of >100 tested) for which substitution removed the K(+) dependence of NCKX-mediated Ca(2+) transport. Charge-removing replacement of Asp(575) by either asparagine or cysteine rendered the mutant NCKX2 proteins independent of K(+), whereas the charge-conservative substitution of Asp(575) to glutamate resulted in a nonfunctional mutant NCKX2 protein, accentuating the critical nature of this residue. Asp(575) is conserved in the NCKX1-5 genes, while an asparagine is found in this position in the three NCX genes, coding for the K(+)-independent Na(+)/Ca(2+) exchanger.