Assessing activation of the human neuropeptide FF2 receptor with a non-radioactive GTP binding assay

We have evaluated a novel, time-resolved fluorometric GTP binding assay for its suitability for functional screening of neuropeptide FF (NPFF) receptor ligands. Our results suggest that this assay, which relies on the use of a europium-labeled GTP analogue, Eu-GTP, provides a powerful alternative to the [³⁵S]guanosine-5′-O-(3-thio)triphosphate binding assay for assessing the functional properties of NPFF analogs. Further, we demonstrate that the tetrapeptide PMRF-NH₂ exhibited high agonist potency at the NPFF2 receptor, and that the efficacies of this peptide and another shortened NPFF analog were greater than that of NPFF.     

Discovery of pyrrolo[1,2-b]pyridazine-3-carboxamides as Janus kinase (JAK) inhibitors

A new class of Janus kinase (JAK) inhibitors was discovered using a rationally designed pyrrolo[1,2-b]pyridazine-3-carboxamide scaffold. Preliminary studies identified (R)-(2,2-dimethylcyclopentyl)amine as a preferred C4 substituent on the pyrrolopyridazine core (3b). Incorporation of amino group to 3-position of the cyclopentane ring resulted in a series of JAK3 inhibitors (4g–4j) that potently inhibited IFNγ production in an IL2-induced whole blood assay and displayed high functional selectivity for JAK3–JAK1 pathway relative to JAK2. Further modifications led to the discovery of an orally bioavailable (2-fluoro-2-methylcyclopentyl)amino analogue 5g which is a nanomolar inhibitor of both JAK3 and TYK2, functionally selective for the JAK3–JAK1 pathway versus JAK2, and active in a human whole blood assay.     

The discovery of 2,5-isomers of triazole-pyrrolopyrimidine as selective Janus kinase 2 (JAK2) inhibitors versus JAK1 and JAK3

Members of the Janus kinase (JAK) family are potential therapeutic targets. Abnormal signaling by mutant JAK2 is related to hematological malignancy, such as myeloproliferative neoplasms (MPNs), and tyrosine kinase inhibitor (TKI)-resistance in non-small cell lung cancer (NSCLC). We discovered a potent and highly selective inhibitor of JAK2 over JAK1 and -3 based on the structure of 4-(2,5-triazole)-pyrrolopyrimidine. Among all triazole compounds tested, 2,5-triazole regioisomers more effectively inhibited JAK2 kinase activity than isomers with substitutions of various alkyl groups at the R₂ position, except for methyl-substituted 1,5-triazole, which was more potent than the corresponding 1,4- and 2,5-triazoles. None of the synthesized 1,4-isomers inhibited all three JAK family members. Compounds with phenyl or tolyl group substituents at the R₁ position were completely inactive compared with the corresponding analogues with a methyl substituted at the R₁ position. As a result of this structure–activity relationship, 54, which is substituted with a cyclopropylmethyl moiety, exhibited significant inhibitory activity and selectivity (IC₅₀ = 41.9 nM, fold selectivity JAK1/2 10.6 and JAK3/2 58.1). Compound 54 also exhibited an equivalent inhibition of wild type JAK2 and the V617F mutant. Moreover, 54 inhibited the proliferation of HEL 92.1.7 cells, which carry JAK2 V617F, and gefitinib-resistant HCC827 cells. Compound 54 also suppressed STAT3 phosphorylation at Y705.     

Functional properties of Pfr(Tic)amide and BIBP3226 at human neuropeptide FF2 receptors

The functional characteristics of two putative neuropeptide FF (NPFF) antagonists, BIBP3226 and PFR(Tic)amide, on the human neuropeptide FF receptor subtype 2 (hNPFF2) were investigated. Surprisingly, PFR(Tic)amide was shown to exhibit agonist properties in the [³⁵S]guanosine-5′-O-(3-thio)triphosphate ([³⁵S]GTPγS) binding assay. The efficacy of PFR(Tic)amide was significantly greater than that of (1DMe)Y8Fa, a stable analog of NPFF, and PFR(Tic)amide can therefore be classified as a ‘super-agonist’. BIBP3226 did act as a reversible competitive antagonist on the hNPFF2 receptor. However, high concentrations of BIBP3226 also non-specifically increased [³⁵S]GTPγS binding. The usefulness of BIBP3226 as an antagonist tool on the NPFF receptor is thus limited.     

Phenolic derivatives from Wigandia urens with weak activity against the chemokine receptor CCR5

Three compounds, 2,3-dihydroxy-4-methoxy-6,6,9-trimethyl-6H-dibenzo[b,d]pyran (1), 8-methoxy-2-methyl-2-(4-methyl-3-pentenyl)-2H-1-benzopyran-6-ol (2) and 4-methoxy-3-(3-methyl-2-butenyl)-benzoic acid (3), have been isolated from Wigandia urens. The structures of compounds 1, 2 and 3 were determined from spectroscopic data and showed activity in a CCR5 assay with IC(50) values of 33, 46 and 26 muM respectively.     

3D-QSAR studies of substituted 1-(3, 3-diphenylpropyl)-piperidinyl amides and ureas as CCR5 receptor antagonists

3D-QSAR studies on the derivatives of 1-(3,3-diphenylpropyl)-piperidinyl amide and urea as CCR5 receptor antagonists were performed by comparative molecular field analysis (CoMFA) and comparative molecular similarity indices (CoMSIA) methods to rationalize the structural requirements responsible for the inhibitory activity of these compounds. The global minimum energy conformer of the template molecule, the most active and pharmacokinetically stable molecule of the series, was obtained by systematic search and used to build structures of the molecules in the dataset. The best predictions for the CCR5-receptor were obtained with the CoMFA standard model (q ² = 0.787, r ² = 0.962) and CoMSIA model combined steric, electrostatic and hydrophobic fields (q ² = 0.809, r ² = 0.951). The predictive ability of CoMFA and CoMSIA were determined using a test set of 12 compounds giving predictive correlation coefficients of 0.855 and 0.83, respectively, indicating good predictive power. Further, the robustness of the model was verified by bootstrapping analysis. The contour maps produced by the CoMFA and CoMSIA models were used to identify the structural features relevant to the biological activity in this series. Based on the CoMFA and CoMSIA analysis, we have identified some key features in the series that are responsible for CCR5 antagonistic activity which may be used to design more potent 1-(3,3-diphenylpropyl)-piperidinyl derivatives and predict their activity prior to synthesis. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Mechanisms of ligand binding and efficacy at the human D2(short) dopamine receptor

Mechanisms of ligand binding and receptor activation for the human D2(short) dopamine receptor have been probed using two homologous series of monohydroxylated and dihydroxylated agonists (phenylethylamines and 2-dipropylaminotetralins). In ligand binding studies, the majority of compounds exhibited competition curves versus [3H]spiperone that were best fitted using a two site binding model. The compounds had different abilities (potencies and maximal effects) to stimulate [35S]GTPgammaS binding and to inhibit forskolin-stimulated cAMP accumulation. From the data it can be concluded that: (i) the ability of an agonist to stabilize receptor/G protein coupling can be used to predict agonist efficacy for some groups of compounds (2-dipropylaminotetralins) but not for others (phenylethylamines); (ii) the receptor may be activated by unhydroxylated compounds; (iii) single hydroxyl groups or pairs of hydroxyl groups on the agonist may contribute to binding affinity, potency and efficacy; and (iv) for the 2-dipropylaminotetralin series two modes of agonist/receptor interaction have been identified associated with different relative efficacy.     

Agonist Action at D2(short) Dopamine Receptors Determined in Ligand Binding and Functional Assays

Abstract: Mechanisms of agonist action at the G protein-coupled D_2(short) dopamine receptor expressed in Chinese hamster ovary cells have been investigated. Agonist binding was assayed in the presence and absence of GTP (100 µM). Data in the absence of GTP were fitted best by a two-site model (apomorphine, dopamine, 10,11-dihydroxy-N-n-propylnorapomorphine hydrochloride, and quinpirole) or a one-site model [bromocriptine, dihydroergocristine, and (?)-3-(3-hydroxyphenyl)-N-propylpiperidine hydrochloride], whereas in the presence of GTP a one-site model was the best fit for all compounds. Agonist binding parameters were used to provide a measure of the ability of the agonist to stabilise the ternary complex of agonist/receptor/G protein. Agonist stimulation of [³⁵S]guanosine 5′-O-(3-thiotriphosphate) ([³⁵S]-GTPγS) binding for a range of agonist concentrations was measured and the EC₅₀ and maximal effects determined. The initial rates of [³⁵S]GTPγS binding induced by maximally stimulating agonist concentrations were also recorded. Simultaneous inhibition of agonist-stimulated [³⁵S]GTPγS binding and receptor occupancy by spiperone was determined. Agonist inhibition of forskolin-stimulated cyclic AMP accumulation was determined for a range of agonist concentrations and the EC₅₀ and maximal inhibition recorded. The data on the maximal agonist responses showed that it was possible to detect a spectrum of agonist efficacy (partial and full agonism) in both functional assays. The data on the apparent potencies of agonists to elicit the functional responses showed that different extents of amplification of response were seen for different agonists in both assays. The maximal activity data have been compared with the stabilisation of the agonist/receptor/G protein ternary complex as measured in binding assays.     

Loss of cannabinoid-stimulated guanosine 5'-O-(3-[(35)S]Thiotriphosphate) binding without receptor down-regulation in brain regions of anandamide-tolerant rats

The endogenous cannabinoid anandamide has been reported to produce well-defined behavioral tolerance, but studies on the possible mechanisms underlying this process are few and often contradictory. The present study was designed to survey the cellular events involved in anandamide tolerance, in terms of the effects on receptor number, coupling with G proteins, and activation of the cyclic AMP (cAMP) cascade. Chronic treatment of rats with anandamide (20 mg/kg i.p. for 15 days) resulted in behavioral tolerance without any change in cannabinoid receptor binding in the brain regions studied (striatum, cortex, hippocampus, and cerebellum), suggesting that receptor down-regulation was not involved in the development of anandamide behavioral tolerance. In contrast, prolonged exposure to anandamide significantly reduced agonist-stimulated guanosine 5'-O:-(3-[(35)S]thiotriphosphate) binding in the same areas, with losses of >50%, suggesting that receptor desensitization may be part of the molecular mechanism underlying this tolerance. Finally, concerning the cAMP cascade-the most well-known intracellular signaling pathways activated by CB(1) receptors-in the brain regions from rats tolerant to anandamide, we found no alteration in cAMP levels or in protein kinase A activity. We propose that anandamide, unlike Delta(9)-tetrahydrocannabinol and other cannabinoids, does not alter the receptor system at multiple levels but that desensitization of the CB(1) receptor might account for behavioral tolerance to the drug.     

JAK1 N-terminus binds to conserved Box 1 and Box 2 motifs of cytokine receptor common beta subunit but signal activation requires JAK1 C-terminus

The human interleukin-3 receptor (hIL-3R) consists of a unique alpha subunit (hIL-3Ralpha) and a common beta subunit (betac). Binding of IL-3 to IL-3R activates Janus kinases JAK1 and JAK2. Our previously study showed that JAK2 and JAK1 were constitutively associated with the hIL-3Ralpha and betac subunits, respectively. In this study, we further demonstrate that JAK2 binds to the intracellular domain of hIL-3Ralpha and JAK1 binds to the Box 1 and Box 2 motifs of betac using GST-hIL-3R fusion proteins in pull-down assays. JAK1 mutational analysis revealed that its JH7-3 domains bound directly to the Box 1 and Box 2 motifs of betac. We further examined the role of JAK1 JH7-3 domains in JAK1 and JAK2-mediated signaling using the CDJAKs fusion proteins, which consisted of a CD16 extracellular domain, a CD7 transmembrane domain, and either JAK1 (CDJAK1), JAK2 (CDJAK2), or JAK1-JH7-3 domains (CDJAK1-JH7-3) as intracellular domains. Anti-CD16 antibody crosslinking of wild type fusion proteins CDJAK1 with CDJAK2 could mimic IL-3 signaling, however, the crosslinking of fusion proteins CDJAK1-JH7-3 with CDJAK2 failed to activate downstream proteins. These results suggest that the JAK1-JH7-3 domains are required for betac interaction and abolish wild type JAK1 and JAK2-mediated signaling.     

In Vitro Evaluation of 11C-Labeled (S)-Nicotine, (S)-3-Methyl-5-(1-Methyl-2-Pyrrolidinyl)isoxazole, and (R,S)-1-Methyl-2-(3-Pyridyl)azetidine as Nicotinic Receptor Ligands for Positron Emission Tomography Studies

Abstract: The binding characteristics of the novel ¹¹C-labeled nicotinic ligands (R,S)-1-methyl-2-(3-pyridyl) azetidine (MPA) and (S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole (ABT-418) were investigated in comparison with those of (S)-[¹¹C]nicotine in vitro in the rat brain to be able to predict the binding properties of the new ligands for positron emission tomography studies in vivo. The data from time-resolved experiments for all ligands indicated fast binding kinetics, with the exception of a slower dissociation of [¹¹C]MPA in comparison with (S)-[¹¹C]nicotine and [¹¹C]ABT-418. Saturation experiments revealed for all ligands two nicotinic receptor binding sites with affinity constants (K_D values) of 2.4 and 560 nM and binding site densities (B_max values) of 65.5 and 223 fmol/mg of protein for (S)-[¹¹C]nicotine, K_D values of 0.011 and 2.2 nM and B_max values of 4.4 and 70.7 fmol/mg of protein for [¹¹C]MPA, and K_D values of 1.3 and 33.4 nM and B_max values of 8.8 and 69.2 fmol/mg of protein for [¹¹C]ABT-418. In competing with the ¹¹C-ligands, epibatidine was most potent, followed by cytisine. A different rank order of potencies was found for (?)-nicotine, (+)-nicotine, MPA, and ABT-418 displacing each of the ¹¹C-ligands. Autoradiograms displayed a similar pattern of receptor binding for all ligands, whereby [¹¹C]MPA showed the most distinct binding pattern and the lowest nonspecific binding. We conclude that the three ¹¹C-labeled nicotinic ligands were suitable for characterizing nicotinic receptors in vitro. The very high affinity of [¹¹C]MPA to nicotinic acetylcholine receptors, its low nonspecific binding, and especially the slower dissociation kinetics of the [¹¹C]MPA from the putative high-affinity nicotinic acetylcholine receptor binding site compared with (S)-[¹¹C]nicotine and [¹¹C]ABT-418 raise the level of interest in [¹¹C]MPA for application in positron emission tomography.     

Enantiospecific enzymatic preparation of (2S,3S)-3-alkylaspartic acids of current interest in the synthesis of stereoregular poly[β-(2S,3S)-3-alkylmalic acids] as new optically active functional polyesters

(2S,3S)-3-methyl- and 3-isopropylaspartic acids were synthesized by bioconversion of the corresponding alkylfumarates (mesaconate and 3-isopropylfumarate) using β-methylaspartase from cell-free extracts of Clostridium tetanomorphum. Optically pure (2S,3S)-3-alkylaspartic acids were transformed in several steps to benzyl (3S,4R)-3-alkylmalolactonates without any racemization of the two chiral centers. These optically active α,β-substituted-β-lactones were polymerized by anionic ring opening polymerization yielding optically active semi-crystalline polyesters. ¹³C NMR analysis of poly[benzyl β-3-isopropylmalate] in CDCl₃ has shown that only the iso-type stereosequence is present in the polymer, indicating that the macromolecular chain is constituted by the only units of benzyl β-(2S,3S)-3-isopropylmalate monomer. The polymerization reaction was done without any racemization of the two stereogenic centers as in the case of benzyl (3S,4R)-3-methylmalolactonate. © 1996 Wiley-Liss, Inc.     

Stimulation of Guanosine 5'-O-(3-[35S]Thiotriphosphate) Binding by Cholinergic Muscarinic Receptors in Membranes of Rat Olfactory Bulb

Abstract: In membranes of rat olfactory bulb, a brain region in which muscarinic agonists increase cyclic AMP formation, the muscarinic stimulation of guanosine 5'- O -(3-[³⁵S]thiotriphosphate) ([³⁵S]GTPγS) binding was used as a tool to investigate the receptor interaction with the guanine nucleotide-binding regulatory proteins (G proteins). The stimulation of the radioligand binding by carbachol (CCh) was optimal (threefold increase) in the presence of micromolar concentrations of GDP and 100 m M NaCl. Exposure to N -ethylmaleimide and pertussis toxin markedly inhibited the CCh effect, whereas it increased the relative stimulation of [³⁵S]GTPγS binding elicited by pituitary adenylate cyclase-activating polypeptide (PACAP). On the other hand, membrane treatment with cholera toxin curtailed the PACAP stimulation of [³⁵S]GTPγS binding but did not affect the response to CCh. Like CCh, a number of cholinergic agonists stimulated [³⁵S]GTPγS binding in a concentration-dependent and saturable manner. The antagonist profile of the muscarinic stimulation of [³⁵S]GTPγS binding was highly correlated with that displayed by the muscarinic stimulation of adenylyl cyclase. These data indicate that the olfactory bulb muscarinic receptors couple to G_i/G_o, but not to G_s, and support the possibility that activation of G_i/G_o mediates the stimulatory effect on adenylyl cyclase activity.     

CB(2) cannabinoid receptor antagonist SR144528 decreases mu-opioid receptor expression and activation in mouse brainstem: role of CB(2) receptor in pain

Formerly considered as an exclusively peripheral receptor, it is now accepted that CB(2) cannabinoid receptor is also present in limited amounts and distinct locations in the brain of several animal species, including mice. However, the possible roles of CB(2) receptors in the brain need to be clarified. The aim of our work was to study the mu-opioid receptor (MOR) mRNA expression level and functional activity after acute in vivo and in vitro treatments with the endocannabinoid noladin ether (NE) and with the CB(2) receptor antagonist SR144528 in brainstem of mice deficient in either CB(1) or CB(2) receptors. This study is based on our previous observations that noladin ether (NE) produces decrease in the activity of MOR in forebrain and this attenuation can be antagonized by the CB(2) cannabinoid antagonist SR144528, suggesting a CB(2) receptor mediated effect. We used quantitative real-time PCR to examine the changes of MOR mRNA levels, [(35)S]GTPgammaS binding assay to analyze the capability of mu-opioid agonist DAMGO to activate G-proteins and competition binding assays to directly measure the ligand binding to MOR in mice brainstem. After acute NE administration no significant changes were observed on MOR signaling. Nevertheless pretreatment of mice with SR144528 prior to the administration of NE significantly decreased MOR signaling suggesting the involvement of SR144528 in mediating the effect of MOR. mRNA expression of MORs significantly decreased both in CB(1) wild-type and CB(1) knockout mice after a single injection of SR144528 at 0.1mg/kg when compared to the vehicle treated controls. Consequently, MOR-mediated signaling was attenuated after acute in vivo treatment with SR144528 in both CB(1) wild-type and CB(1) knockout mice. In vitro addition of 1microM SR144528 caused a decrease in the maximal stimulation of DAMGO in [(35)S]GTPgammaS binding assays in CB(2) wild-type brainstem membranes whereas no significant changes were observed in CB(2) receptor knockouts. Radioligand binding competition studies showed that the noticed effect of SR144528 on MOR signaling is not mediated through MORs. Our data demonstrate that the SR144528 caused pronounced decrease in the activity of MOR is mediated via CB(2) cannabinoid receptors.     

Dual coupling of opioid receptor-like (ORL1) receptors to adenylyl cyclase in the different layers of the rat main olfactory bulb

The coupling of opioid receptor-like (ORL1) receptors to adenylyl cyclase has been investigated in specific layers of the rat main olfactory bulb. Membranes prepared from the olfactory nerve-glomerular layer (ON-G layer), external plexiform layer (EP layer) and granule cell layer (GR layer) displayed specific binding sites for [(3)H]-nociceptin/orphanin FQ ([(3)H]Noc/OFQ). In each layer, the presence of high-and low-affinity binding sites, with K(D) values in the picomolar and nanomolar range, respectively, was detected. The binding of [(3)H]Noc/OFQ was displaced by unlabelled Noc/OFQ, but not by opioid antagonists. In each layer, Noc/OFQ significantly stimulated [(35)S]GTPgammaS binding with nanomolar potencies. In ON-G layer, Noc/OFQ inhibited basal adenylyl cyclase activity and the enzyme stimulations by corticotropin releasing hormone (CRH), Ca(2+)/calmodulin (Ca(2+)/CaM) and forskolin (FSK). In EP layer, Noc/OFQ inhibited Ca(2+)/CaM-and FSK-stimulated enzyme activities. Conversely, in GR layer the peptide stimulated basal cyclase activity and potentiated the enzyme activation by CRH. The Noc/OFQ stimulation was counteracted by the GDP-bound form of the alpha subunit of transducin and was mimicked by transducin betagamma subunits. In the same tissue layer, Ca(2+)/CaM-and FSK-stimulated enzyme activities were inhibited. Naloxone failed to antagonize all the actions of Noc/OFQ. Western blot and RT-PCR analysis revealed the expression of Ca(2+)-insensitive and -sensitive adenylyl cyclases in the three layers. These results demonstrate that in rat main olfactory bulb ORL1 receptors can differentially affect distinct forms of adenylyl cyclase in a layer specific manner.     

Anatomical location of LPA1 activation and LPA phospholipid precursors in rodent and human brain

Lysophosphatidic acid (LPA) is a signaling molecule that binds to six known G protein‐coupled receptors: LPA₁–LPA₆. LPA evokes several responses in the CNS, including cortical development and folding, growth of the axonal cone and its retraction process. Those cell processes involve survival, migration, adhesion proliferation, differentiation, and myelination. The anatomical localization of LPA₁ is incompletely understood, particularly with regard to LPA binding. Therefore, we have used functional [³⁵S]GTPγS autoradiography to verify the anatomical distribution of LPA₁ binding sites in adult rodent and human brain. The greatest activity was observed in myelinated areas of the white matter such as corpus callosum, internal capsule and cerebellum. MaLPA₁‐null mice (a variant of LPA₁‐null) lack [³⁵S]GTPγS basal binding in white matter areas, where the LPA₁ receptor is expressed at high levels, suggesting a relevant role of the activity of this receptor in the most myelinated brain areas. In addition, phospholipid precursors of LPA were localized by MALDI‐IMS in both rodent and human brain slices identifying numerous species of phosphatides and phosphatidylcholines. Both phosphatides and phosphatidylcholines species represent potential LPA precursors. The anatomical distribution of these precursors in rodent and human brain may indicate a metabolic relationship between LPA and LPA₁ receptors.