Multiple Subtypes of Excitatory Amino Acid Receptors Coupled to the Hydrolysis of Phosphoinositides in Rat Brain

Abstract: The excitatory amino acid (EAA) analogues quisqualate, ibotenate, and trans-(±)-1-amino-1, 3-cyclopentanedicarboxylate (trans-ACPD) activate the metabotropic EAA receptors that are coupled to the hydrolysis of Phosphoinositides (PI). Previous studies of hippocampal cross sections demonstrated that PI hydrolysis stimulated by these agonists can be inhibited by either L-aspartate-β- hydroxamate (L-AβHA) or DL-2-amino-3-phosphonopropionate (DL-AP3). The goal of the present studies was to determine if all metabotropic EAA receptors are sensitive to L-AβHA and DL-AP3. Two approaches were used. In the first, using cerebellar cross sections, the effects of these agonists and inhibitors were examined. The EC₅₀ values (the concentrations required to evoke half-maximal stimulation) of quisqualate, ibotenate, and trans-ACPD in cerebellum were similar to the EC₅₀ values that we observed previously in hippocampus, but neither L-AβHA nor DL- AP3 blocked PI hydrolysis. The EC₅₀ values were 0.65 ± 0.17 μM for quisqualate, 12.8 ± 2.5 μM for ibotenate, and 18.1 ± 3.1 μM for trans-ACPD. All data were best fit to theoretical curves that had Hill slopes of 1. In the second approach, another EAA analogue, D-aspartate, was identified as an agonist that stimulates PI hydrolysis. The EC₅₀for PI hydrolysis stimulated by D-aspartate was 470 ± 90 μM in hippocampus. Neither L-AβSHA nor DL-AP3 blocked PI hydrolysis stimulated by D-aspartate in hippocampus. Furthermore, antagonists of ionotropic EAA receptors, antagonists of other receptor systems coupled to PI hydrolysis, and inhibitors of the Na⁺-dependent L-glutamate transport process also did not block PI hydrolysis stimulated by D-aspartate. These data support the presence of three pharmacologically distinct metabotropic EAA receptor subtypes.     

Signal transduction and pharmacological characteristics of a metabotropic glutamate receptor, mGluR1, in transfected CHO cells.

The signal transduction and pharmacological properties of a metabotropic glutamate receptor, mGluR1, were studied in CHO cells permanently expressing the cloned receptor. mGluR1 stimulated phosphatidylinositol (PI) hydrolysis in the potency rank order of quisqualate greater than L-glutamate greater than or equal to ibotenate greater than L-homocysteine sulfinate greater than or equal to trans-ACPD. This receptor also evoked the stimulation of cAMP formation and arachidonic acid release with comparable agonist potencies. DL-AP3 and L-AP4, the effective antagonists reported for glutamate-stimulated PI hydrolysis in brain slices, showed no appreciable effects on mGluR1, suggesting the existence of an additional subtype of this receptor family. Pertussis toxin and phorbol ester produced distinct effects on the three transduction cascades, implying that mGluR1 independently links to the multiple transduction pathways probably through different G proteins.     

L-[3H]Glutamate Binding to Hippocampal Synaptic Membranes: Two Binding Sites Discriminated by Their Differing Affinities for Quisqualate

The excitatory glutamate analogs quisqualate and ibotenate were employed to distinguish multiple binding sites for L-[3H]glutamate on freshly prepared hippocampal synaptic membranes. The fraction of bound radioligand that was displaceable by 5 microM quisqualate was termed GLU A binding. That which persisted in the presence of 5 microM quisqualate, but was displaceable by 100 microM ibotenate, was termed GLU B binding. GLU A binding equilibrated within 5 min and remained unchanged for up to 80 min. GLU B binding appeared to equilibrate at least as rapidly, but incubation with ligand unmasked latent binding sites. Saturation binding curves were best fitted by single exponentials, which yielded KD values of about 200 nM (GLU A) and 1 microM (GLU B). On the average, GLU B binding sites were about twice as abundant in these membranes as were GLU A sites. Rapid freezing of the membranes, followed by storage at -26 degrees C and rapid thawing markedly diminished GLU A binding, but nearly tripled GLU B binding. Both site bound L-glutamate with 10-30 times the affinity of D-glutamate. The GLU A site also bound L-glutamate with about 10 times the affinity of L-aspartate and discriminated poorly between L- and D-aspartate. In contrast, the GLU B site bound L-aspartate with an affinity similar to that for L-glutamate, and with an order-of-magnitude greater affinity than D-aspartate. The structural specificities of the GLU A and GLU B binding sites suggest that these sites may correspond to receptors on hippocampal pyramidal cell dendrites that are activated by iontophoretically applied L-glutamate.     

Presynaptic Glutamate/Quisqualate Receptors: Effects on Synaptosomal Free Calcium Concentrations

Intracellular free [Ca2+]i was measured using fura-2 in synaptosomes prepared from cerebral cortices of adult male rats (12 weeks). L-(+)-Glutamate, D-(-)-glutamate, and quisqualate produced similar dose-dependent increases in [Ca2+]i, with EC50 values of 0.38 microM, 0.74 microM, and 0.1 microM, respectively, and maximum increases of approximately 40%. Ibotenate showed less affinity (EC50 4.4 microM) but had a greater maximum effect (57%). N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) did not increase [Ca2+]i. The increases in [Ca2+]i induced by quisqualate and ibotenate were not diminished in the absence of extrasynaptosomal Ca2+. L-2-Amino-4-phosphonobutyrate (L-AP4) (1 microM) completely blocked the changes in [Ca2+]i induced by L-(+)-glutamate, D-(-)-glutamate, quisqualate, or ibotenate. The effects of quisqualate and ibotenate on [Ca2+]i were also blocked by coincubation of synaptosomes with L-(+)-serine-O-phosphate (L-SP) (1 mM) (which, like L-AP4, blocks the effects of quisqualate and ibotenate on inositol phospholipid metabolism). 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) had no effect on agonist-mediated increases in [Ca2+]i when coincubated with either quisqualate or ibotenate. These data are consistent with the existence of presynaptic glutamate receptors (of the excitatory amino acid metabotropic type) which activate phospholipase C leading to the elevation of inositol 1,4,5-trisphosphate and release of Ca2+ from intracellular stores.     

Excitatory amino acid receptor agonists stimulate membrane inositol phospholipid hydrolysis and increase cytoplasmic free Ca2+ in primary cultures of retinal neurons

A variety of neurotransmitters are believed to elicit effects through receptor-stimulated inositol phospholipid metabolism. It appears that most major types of retinal neurons receive a direct glutamatergic input. The aim of the present studies was to characterize excitatory amino acid (EAA) receptor-mediated breakdown of inositol phospholipids and changes in Ca2+ homeostasis in primary avian retinal cell cultures. Cell monolayers, prepared from 8-day-old chick embryo neural retina, were labelled with [3H]inositol for 48 h, and used after 7 days in vitro. Kainic acid stimulated the accumulation of inositol phosphates in a time- and dose-dependent manner (ED50 = 30 microM). The EAA receptor agonists glutamate, N-methyl-D-aspartate (NMDA), ibotenate and quisqualate were all active, with the rank order: glutamate greater than kainate greater than NMDA much greater than ibotenate approximately quisqualate. External Ca2+ was required for these effects. Agonist actions were inhibited by type-specific antagonists, and also Mg2+ in the case of glutamate and NMDA. Glutamate, NMDA and kainate also elevated cytosolic free Ca2+ in individual retinal cells loaded with the Ca2(+)-sensitive dye Fura-2, as assessed by digital fluorescence ratio imaging microscopy. The agonist-induced increases in [Ca2+]i were largely dependent on extracellular Ca2+, independent of membrane depolarization and were blocked by Mg2+ for glutamate and NMDA. These results demonstrate that vertebrate retinal cells possess EAA receptors coupled to intracellular signal transduction pathways.     

Pretreatment of Cerebellar Granule Cells with Concanavalin A Potentiates Quisqualate-Stimulated Phosphoinositide Hydrolysis

The hydrolysis of phosphoinositides (PI) elicited in cerebellar granule cell cultures by agonists of metabolotropic glutamate receptors, glutmate and quisqualate, was enhanced when the cells were pretreated with concanavalin A (Con-A). A similar effect was produced by wheat germ agglutinin, but not by several other lectins tested. Con-A produced a dose-dependent effect (EC50 = 3 microM) and increased the efficacy but not the potency of the agonists. In contrast, Con-A failed to enhance PI hydrolysis evoked by N-methyl-D-aspartate, kainate, carbachol, the calcium ionophore A23187, or 50 mM K+. The Con-A stimulatory effect was prevented by simultaneous pretreatment with the agonists of ionotropic quisqualate receptors quisqualate, kainate, and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, but not by the antagonist 6-cyano-7-nitroquioxaline-2,3-dione (CNQX). CNQX, which did not inhibit quisqualate-stimulated PI hydrolysis in untreated cells, abolished the component of quisqualate response enhanced by Con-A pretreatment. The pretreatment with Con-A also increased the influx of 45Ca2+ in granule cells stimulated by quisqualate. This increase was inhibited by CNQX. Moreover, the potentiation of PI hydrolysis by Con-A, but not the response to quisqualate alone, was abolished in the absence of Ca2+ and Na+. Pretreatment of granule cells with pertussis toxin inhibited PI hydrolysis stimulated by the metabolotropic quisqualate receptor and the Con-A-potentiated response by the same percentage, but Ca2+ influx induced by quisqualate was not affected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of Excitatory Amino Acid-Stimulated Phosphoinositide Hydrolysis in the Neonatal Rat Hippocampus by 2-Amino-3-Phosphonopropionate

The effects of excitatory amino acid agonists and alpha-amino-omega-phosphonocarboxylic acid antagonists on phosphoinositide hydrolysis in hippocampal slices of the 7-day neonatal rat were examined. Significant stimulation of [3H]inositol monophosphate formation was observed with ibotenate, quisqualate, L-glutamate, L-aspartate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, L-homocysteate, and kainate. N-Methyl-D-aspartate had no effect. Of these agonists, ibotenate and quisqualate were the most potent and efficacious. Stimulations by ibotenate and quisqualate were partially inhibited by L-2-amino-4-phosphonobutyrate (10(-3) M), but this antagonist had no effect on L-glutamate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, or kainate. At 10(-3) M, D,L-2-amino-3-phosphonopropionate completely inhibited ibotenate and quisqualate stimulations, partially inhibited L-glutamate stimulation, and had no effect on alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-, kainate-, or carbachol-induced [3H]inositol monophosphate formation. Concentration-effect experiments showed D,L-2-amino-3-phosphonopropionate to be five times more potent as an antagonist of ibotenate-stimulated phosphoinositide hydrolysis than L-2-amino-4-phosphonobutyrate. Thus in the neonatal rat hippocampus, like in the adult rat brain, D,L-2-amino-3-phosphonopropionate is a selective and relatively potent inhibitor of excitatory amino acid-stimulated phosphoinositide hydrolysis. Because this glutamate receptor is uniquely sensitive to D,L-2-amino-3-phosphonopropionate, these studies provide further pharmacological evidence for the existence of a novel excitatory amino acid receptor subtype that is coupled to phosphoinositide hydrolysis in brain.     

Excitatory amino acids inhibit stimulated phosphoinositide hydrolysis in the rat prefrontal cortex

In rat prefrontal cortical slices, the excitatory amino acids N-methyl-D-aspartate (NMDA), ibotenate, L-aspartate, quisqualate, kainate and L-glutamate inhibit carbachol-induced phosphoinositide hydrolysis as measured by the accumulation of [3H]inositol-1-phosphate ([3H]IP1). NMDA dose-dependently inhibited the carbachol response (IC50 = 14.4 microM), and this inhibition was blocked by the NMDA receptor antagonist D,L-aminophosphonovaleric acid. Lowering medium Na+ concentration to 10 mM or exposing slices to pertussis toxin alleviated the inhibitory effect of NMDA on carbachol-induced [3H]IP1 formation. Serotonin-induced stimulation of [3H]IP1 was also inhibited by NMDA; in contrast, stimulation by norepinephrine, epinephrine or dopamine was unaffected. The results suggest that excitatory amino acids, besides their traditional role as stimulatory substances, can also act to inhibit the production of 2nd messengers activated by certain neurotransmitters in the brain.     

The Effects of l-Glutamate and trans-(±)-1-Amino-1,3-Cyclopentanedicarboxylate on Phosphoinositide Hydrolysis Can Be Pharmacologically Differentiated

Abstract: The excitatory amino acid analogues l -glutamate ( l -Glu), l -aspartate ( l -Asp), d -Asp, and trans -(±)-1-amino-1,3-cyclopentanedicarboxylate ( trans -ACPD) stimulate the hydrolysis of phosphoinositides (PI). In the present studies, the effects of noncompetitive and competitive inhibitors on PI hydrolysis stimulated by excitatory amino acid analogues were examined. When agonist and inhibitor were added simultaneously to hippocampal tissue, the noncompetitive inhibitor l -2-amino-3-phosphonopropionate ( l -AP3) did not block the effects of l -Glu, l -Asp, or d -Asp at concentrations that block the effects of trans -ACPD by more than 80%. When tissue was pre-incubated with l -AP3, the effects of l -Glu, l -Asp, or d -Asp were blocked (IC₅₀ values between 65 and 210 µ M ). Unlike l -AP3, l -aspartate-β-hydroxamate ( l -AβHA) inhibited PI hydrolysis stimulated by trans -ACPD, l -Glu, l -Asp, or d -Asp when agonist and inhibitor were added simultaneously in hippocampus; its effects were not time-dependent. In cerebellum, both l -AP3 and l -AβHA had agonist activity. Inhibition by the recently identified competitive inhibitor (+)-α-methyl-4-carboxyphenylglycine [(+)-MCPG] of PI hydrolysis was also examined. (+)-MCPG blocked PI hydrolysis stimulated by trans -ACPD, l -Asp, or d -Asp in both hippocampus and cerebellum (IC₅₀ values between 220 and 1,700 µ M ). The effects of (+)-MCPG were consistent with a competitive mechanism of action. (+)-MCPG (up to 3 m M ) blocked PI hydrolysis stimulated by l -Glu by less than 25% in both hippocampus and cerebellum.     

Complex binding of L-[3H]glutamate to hippocampal synaptic membranes in the absence of sodium

Specific binding of L-[3H]glutamate was investigated with a thoroughly washed synaptic membrane preparation from rat hippocampal formation, a region of brain densely innervated by putatively glutamatergic fibers. L-[3H]Glutamate bound rapidly, saturably, and reversibly to these membranes in the absence of Na+. Specific binding was greatest around 38 degrees C and at a slightly acidic pH. Saturation isotherms fit a model of two independent binding sites with dissociation constants of 11 and 570 nM and corresponding densities of 2.5 and 47 pmol/mg protein. All potent amino acid excitants, except N-methyl-D-aspartate and kainate, and several excitatory amino acid antagonists inhibited specific radioligand binding with IC50 values between 10(-7) M and 10(-4) M. In contrast, weak amino acid excitants and an inhibitor of glutamate uptake were nearly inactive. Displacement curves were analyzed with a computer program that assumed the simultaneous contributions of two independent sites at which each compound competitively inhibited the binding of L-[3H]glutamate. According to this analysis, ibotenate and the L- and D-isomers of glutamate and aspartate bind preferentially to the high-affinity site, whereas quisqualate, L-alpha-aminoadipate, and the L- and D-isomers of homocysteate bind preferentially to the low-affinity site. With the notable exception of gamma-D-glutamylglycine, all of the more potent antagonists appear to bind preferentially to the low-affinity site. Both sites exhibit marked stereoselectivity for L-glutamate. D- and L-Homocysteate and most excitatory amino acid antagonists increased specific binding at concentrations below those required to demonstrate inhibition. Some properties of the low-affinity binding site resemble those of junctional glutamate receptors on insect muscle, but neither site appears to correspond to the "N-methyl-D-aspartate receptor" or the "quisqualate receptor."     

Influence of the degree of unsaturation of the acyl side chain upon the interaction of analogues of 1-arachidonoylglycerol with monoacylglycerol lipase and fatty acid amide hydrolase

Little is known as to the structural requirements of the acyl side chain for interaction of acylglycerols with monoacylglycerol lipase (MAGL), the enzyme chiefly responsible for the metabolism of the endocannabinoid 2-arachidonoylglycerol (2-AG) in the brain. In the present study, a series of twelve analogues of 1-AG (the more stable regioisomer of 2-AG) were investigated with respect to their ability to inhibit the metabolism of 2-oleoylglycerol by cytosolic and membrane-bound MAGL. In addition, the ability of the compounds to inhibit the hydrolysis of anandamide by fatty acid amide hydrolase (FAAH) was investigated. For cytosolic MAGL, compounds with 20 carbon atoms in the acyl chain and 2-5 unsaturated bonds inhibited the hydrolysis of 2-oleoylglycerol with similar potencies (IC50 values in the range 5.1-8.2 microM), whereas the two compounds with a single unsaturated bond were less potent (IC50 values 19 and 21 microM). The fully saturated analogue 1-monoarachidin did not inhibit the enzyme, whereas the lower side chain analogues 1-monopalmitin and 1-monomyristin inhibited the enzyme with IC50 values of 12 and 32 microM, respectively. The 22-carbon chain analogue of 1-AG was also potent (IC50 value 4.5 microM). Introduction of an alpha-methyl group for the C20:4, C20:3, and C22:4 compounds did not affect potency in a consistent manner. For the FAAH and the membrane-bound MAGL, there was no obvious relationship between the degree of unsaturation of the acyl side chain and the ability to inhibit the enzymes. It is concluded that increasing the number of unsaturated bonds on the acyl side chain of 1-AG from 1 to 5 has little effect on the affinity of acylglycerols for cytosolic MAGL.     

Metabotropic Excitatory Amino Acid Receptor Activation Stimulates Phospholipase D in Hippocampal Slices

Metabotropic excitatory amino acid (EAA) receptors are coupled to effector systems through G proteins. Because various G protein-coupled receptors stimulate the hydrolysis of phosphatidylcholine by phospholipase D (PLD), we examined the possibility that metabotropic EAA receptors exist that are coupled to the activation of PLD. We found that the selective metabotropic glutamate receptor (mGluR) agonists 1S,3R-amino-1,3-cyclopentanedicarboxylic acid (ACPD) and 1S,3S-ACPD, but not the inactive isomer, 1R,3S-ACPD, induce a concentration-dependent increase in PLD activity in hippocampal slices. Selective ionotropic glutamate receptor (iGluR) antagonists did not block 1S,3R-ACPD-induced PLD stimulation. Furthermore, although selective iGluR agonists did not activate this response, the nonselective mGluR-iGluR agonists, ibotenate and quisqualate, caused significant increases in PLD activity (all in the presence of iGluR antagonists). L-2-Amino-3-phosphonopropionic acid, which blocks the mGluR that is coupled to phosphoinositide hydrolysis in various brain regions, activates PLD to the same extent as the active isomers of ACPD. These data suggest that metabotropic EAA receptors exist in hippocampus that are coupled to PLD activation and are pharmacologically distinct from phosphoinositide hydrolysis-coupled mGluRs.     

Inhibition of neuronal membrane GABAB receptor binding by GABA structural analogues

1. A number of compounds structurally related to GABA were tested as inhibitors of baclofen-sensitive GABAB receptor binding to membranes from mouse brain. 2. In addition to two known inhibitors--baclofen and 5-aminovaleric acid--two analogues were shown to possess inhibitory activity. These compounds were 4-aminobutyryl-DL-alanine hydrobromide (IC50 = 3 microM) and trans-2-(aminomethyl)cyclopropane carboxylic acid (IC50 = 90 microM). 3. Both drugs also exhibited affinity for GABAA binding sites. 4. Further experiments are needed to establish if these analogues exert agonist or antagonist action at the GABAB receptor.     

cAMP analogs and selective inhibitors used to study low Km Mucor rouxii cAMP phosphodiesterase.

1. The sensitivity of partially purified low Km phosphodiesterase (PDE) from Mucor rouxii to pharmacological agents and cAMP analogs was studied. The IC50 obtained were compared with those reported for PDEs from higher eukaryotes. 2. The best inhibitors of the hydrolysis of 1 microM cAMP were SQ 65.442 (IC50 c 10 microM), dipyridamol and CI 930. cGMP was not an inhibitor (IC50 greater than 1000 microM). 3. The cAMP analogs were tested as inhibitors of the hydrolysis of 0.1 microM cAMP. 8-Aminohexylamino cAMP was the best inhibitor with an IC50 of c 1 microM. 4. A sedimentation profile of Mucor PDE was assayed in the presence of several pharmacological inhibitors and cAMP analogs. No isoforms with different sensitivity towards the inhibitors were detected. Forms with slightly different behaviour towards some cAMP analogs were observed.     

Novel alkylpolyaminoguanidines and alkylpolyaminobiguanides with potent antitrypanosomal activity

A series of polyaminoguanidines and polyaminobiguanides were synthesized and evaluated as potential antitrypanosomal agents. These analogues inhibit trypanothione reductase (TR) with IC50 values as low as 0.95 microM, but do not inhibit the closely related human enzyme glutathione reductase (GR). The most effective analogues, 7a, 7b and 8d, inhibited parasitic growth in vitro with IC50 values of 0.18, 0.09 and 0.18 microM, respectively. These agents represent a promising new class of potential antitrypanosomal agents.     

Synthesis of trans-caffeate analogues and their bioactivities against HIV-1 integrase and cancer cell lines

Forty caffeate analogues were synthesized via a convenient method starting from vanillin with moderate to good yields. The testing of biological activity of these compounds against HIV-1 integrase indicates that four compounds: bornyl caffeate, bornyl 2-nitrocaffeate, 5-nitrocaffeic acid and 5-nitrocaffeic acid phenethyl ester (5-nitroCAPE) possess a good HIV integrase inhibitory activity, IC(50) 19.9, 26.8, 25.0 and 13.5 microM , respectively. Twelve caffeate analogues were tested by MTT assay on growth of human hepatocellular carcinoma BEL-7404, human breast MCF-7 adenocarcinoma, human lung A549 adenocarcinoma and human gastric cancer BCG823 cell lines, respectively. And the best result is IC(50) 5.5 microM for CAPE against BEL-7404.     

Muscarinic Cholinergic Stimulation of Exogenous Phosphatidylinositol Hydrolysis Is Regulated by Guanine Nucleotides in Rabbit Brain Cortical Membranes

Rabbit brain cortical membranes, which have been extracted with 2 M KCl, hydrolyze exogenously added [3H]phosphatidylinositol [( 3H]PI) in a guanine nucleotide- and carbachol-dependent manner. Both oxotremorine-M and carbachol are full agonists with EC50 values of 8 and 73 microM, respectively. Pirenzepine and atropine inhibit carbachol-stimulated [3H]PI hydrolysis. The hydrolysis-resistant guanine nucleotide analog guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) is the most potent in supporting carbachol-stimulated hydrolysis of PI. There is no effect of carbachol in the absence of guanine nucleotides or in the presence of 100 microM adenosine 5'-O-(3-thiotriphosphate), adenosine-5'-(beta, gamma-imido)triphosphate, or sodium pyrophosphate. Guanylyl-5'-(beta,gamma-imido)triphosphate [Gpp(NH)p] in the presence of carbachol also stimulates PI hydrolysis although much less than that seen with GTP gamma S. GDP and Gpp(NH)p are potent antagonists of the GTP gamma S-dependent carbachol response. Optimal stimulation by carbachol and GTP gamma S was observed at 0.3-1 microM free Ca2+ and 6 mM MgCl2. Limited trypsinization resulted in loss of receptor-regulated PI breakdown and a slight decrease in basal activity. These results demonstrate that phospholipase C hydrolysis of exogenous PI by rabbit cortical membranes may be stimulated by carbachol in a guanine nucleotide-dependent manner.     

Novel pyrrolo-quinoline derivatives as potent inhibitors for PI3-kinase related kinases.

Several pyrrolo-quinoline gamma-lactones were found as novel inhibitors for two members of the PI3-kinase related kinase (PIKK) family, Ataxia-Telangiectasia-mutated (ATM) protein and the mammalian Target of Rapamycin (mTOR). Preliminary structure-activity relationship studies indicated that an electrophilic exocyclic double bond conjugated to the carbonyl group of the gamma-lactone ring was crucial for the PIKK inhibitory potency. One of the best ATM inhibitors in this series, DK8G557, showed IC(50) values of 0.6 and 7.0 microM for ATM and mTOR, respectively. This compound exhibited potent and selective growth inhibition activities in the NCI 60 human tumor cell line screen with a GI(50) MG-MID value of 2.69 microM. The best mTOR inhibitor in this series, HP9912, exhibited IC(50) values of 0.5 and 6.5 microM for mTOR and ATM, respectively. These compounds suggest novel leads for the discovery of potent small molecule inhibitors of PIKKs as potential anticancer drugs, with therapeutic activities as either single, or as sensitizing agents to conventional radio-, or chemo-therapeutic strategies.     

Second messengers in platelet aggregation evoked by serotonin and A23187, a calcium ionophore.

We investigated the combined effect of 5-hydroxytryptamine (5-HT, serotonin) and calcium ionophore (A23187) on human platelet aggregation. Aggregation, monitored at 37 degrees C using a Dual-channel Lumi-aggregometer, was recorded for 5 min after challenge by a change in light transmission as a function of time. 5-HT (2-200 microM) alone did not cause platelet aggregation, but markedly potentiated A23187 (low dose) induced aggregation. Inhibitory concentration (IC50) values for a number of compounds were calculated as means +/- SEM from dose-response determinations. Synergism between 5-HT (2-5 microM) and A23187 (0.5-2 microM) was inhibited by 5-HT receptor blockers, methysergide (IC50 = 18 microM) and cyproheptadine (IC50 = 20 microM), and calcium channel blockers (verapamil and diltiazem, IC50 = 20 microM and 40 microM respectively). Interpretation of the effects of these blockers is complicated by their lack of specificity. Similarly, U73122, an inhibitor of phospholipase C (PLC), blocked the synergistic effect at an IC50 value of 9.2 microM. Wortmannin, a phosphatidylinositide 3-kinase (PI 3-K) inhibitor, also blocked the response (IC50 = 2.6 microM). However, neither genistein, a tyrosine-specific protein kinase inhibitor, nor chelerythrine, a protein kinase C inhibitor, affected aggregation at concentrations up to 10 microM. We conclude that the synergistic interaction between 5-HT and ionophore may be mediated by activation of PLC/Ca2+ and PI 3-kinase signalling pathways, but definitive proof will require other enzyme inhibitors with greater specificity.     

Synthesis of (bis)sulfonic acid, (bis)benzamides as follicle-stimulating hormone (FSH) antagonists

Screening efforts identified (bis)sulfonic acid, (bis)benzamides (1-3) as compounds that interact with the follicle stimulating-hormone receptor (FSHR) and inhibit FSH-stimulated cAMP accumulation with IC(50) values in the low micromolar range. Structure-activity relationship studies using novel analogues of 1-3 revealed that two phenylsulfonic acid moieties were necessary for activity and that the carbon-carbon double bond of the stilbene sub-series was the optimum spacer connecting these groups. Selected analogues (2, 14, and 50) were also able to block FSHR-dependent estradiol production in rat primary ovarian granulosa cells and progesterone secretion in a clonal mouse adrenal Y1 cell line. IC(50) values for these compounds in these assays were in the low micromolar range. Optimization of the benzoic acid side chains of 1-3 led to gains in selectivity versus activity at the thyroid stimulating hormone (TSH) receptor (TSHR). For instance, while stilbene (bis)sulfonic acid congener 2 was only 10-fold selective for FSHR over TSHR, analogue 50 with an IC(50) value of 0.9 microM in the FSHR-cAMP assay was essentially inactive at 30 microM in the TSHR-cAMP assay.