Enantioselective σ1 receptor binding and biotransformation of the spirocyclic PET tracer 1′‐benzyl‐3‐(3‐fluoropropyl)‐3H‐spiro[[2]benzofuran‐1,4′‐piperidine]

It was shown that racemic (±)‐ 2 [1′‐benzyl‐3‐(3‐fluoropropyl)‐3H‐spiro[[2]benzofuran‐1,4′‐piperidine], WMS‐1813 ] represents a promising positron emission tomography (PET) tracer for the investigation of centrally located σ₁ receptors. To study the pharmacological activity of the enantiomers of 2 , a preparative HPLC separation of (R)‐2 and (S)‐2 was performed. The absolute configuration of the enantiomers was determined by CD‐spectroscopy together with theoretical calculations of the CD‐spectrum of a model compound. In receptor binding studies with the radioligand [³H]‐(+)‐pentazocine, (S)‐2 was thrice more potent than its (R)‐configured enantiomer (R)‐2 . The metabolic degradation of the more potent (S)‐enantiomer was considerably slower than the metabolism of (R)‐2 . The structures of the main metabolites of both enantiomers were elucidated by determination of the exact mass using an Orbitrap‐LC‐MS system. These experiments showed a stereoselective biotransformation of the enantiomers of 2 . Chirality, 2011. © 2010 Wiley‐Liss, Inc.     

Radiolabeling and in vitro /in vivo evaluation of N‐(1‐adamantyl)‐8‐methoxy‐4‐oxo‐1‐phenyl‐1,4‐dihydroquinoline‐3‐carboxamide as a PET probe for imaging cannabinoid type 2 receptor

The cannabinoid type 2 (CB₂) receptor plays an important role in neuroinflammatory and neurodegenerative diseases such as multiple sclerosis, amyotrophic lateral sclerosis, and Alzheimer's disease and is therefore a very promising target for therapeutic approaches as well as for imaging. Based on the literature, we identified one 4‐oxoquinoline derivative (designated KD2) as the lead structure. It was synthesized, radiolabeled and evaluated as a potential imaging tracer for CB₂. [¹¹C]KD2 was obtained in 99% radiochemical purity. Moderate blood–brain barrier (BBB) passage was predicted for KD2 from an in vitro transport assay with P‐glycoprotein‐transfected Madin Darby canine kidney cells. No efflux of KD2 by P‐glycoprotein was detected. In vitro autoradiography of rat and mouse spleen slices demonstrated that [¹¹C]KD2 exhibits high specific binding towards CB₂. High spleen uptake of [¹¹C]KD2 was observed in dynamic positron emission tomography (PET) studies with Wistar rats and its specificity was confirmed by displacement study with a selective CB₂ agonist, GW405833. A pilot autoradiography study with post‐mortem spinal cord slices from amyotrophic lateral sclerosis (ALS) patients with [¹¹C]KD2 suggested the presence of CB₂ receptors under disease conditions. Specificity of [¹¹C]KD2 binding could also be demonstrated on these human tissues. In conclusion, [¹¹C]KD2 shows good in vitro and in vivo properties as a potential PET tracer for CB₂.

     

The role of 5‐HT2A receptor and 5‐HT2A/5‐HT1A receptor interaction in the suppression of catalepsy

In the present study, the 5‐HT_2A and 5‐HT_1A receptors functional activity and 5‐HT_2A receptor gene expression were examined in the brain of ASC/Icg and congenic AKR.CBAD13Mit76C mouse strains (genetically predisposed to catalepsy) in comparison with the parental catalepsy‐resistant AKR/J and catalepsy‐prone CBA/Lac mouse strains. The significantly reduced 5‐HT_2A receptor functional activity along with decreased 5‐HT_2A receptor gene expression in the frontal cortex was found in all mice predisposed to catalepsy compared with catalepsy‐resistant AKR/J. 5‐HT_2A agonist DOI (0.5 and 1 mg/kg, i.p.) significantly reduced catalepsy in ASC/Icg and CBA/Lac, but not in AKR.CBAD13Mit76C mice. Essential increase in 5‐HT_1A receptor functional activity was shown in catalepsy‐prone mouse strains in comparison with catalepsy‐resistant AKR/J mice. However, in AKR.CBAD13Mit76C mice it was lower than in ASC/Icg and CBA/Lac mice. The inter‐relation between 5‐HT_2A and 5‐HT_1A receptors in the regulation of catalepsy was suggested. This suggestion was confirmed by prevention of DOI anticataleptic effect in ASC/Icg and CBA/Lac mice by pretreatment with 5‐HT_1A receptor antagonist p‐MPPI (3 mg/kg, i.p.). At the same time, the activation of 5‐HT_2A receptor led to the essential suppression of 5‐HT_1A receptor functional activity, indicating the opposite effect of 5‐HT_2A receptor on pre‐ and postsynaptic 5‐HT_1A receptors. Thus, 5‐HT_2A/5‐HT_1A receptor interaction in the mechanism of catalepsy suppression in mice was shown.     

Relationships between serotonergic and cannabinoid system in depressive‐like behavior: a PET study with [11C]‐DASB

Chronic stress represents a major environmental risk factor for mood disorders in vulnerable individuals. The neurobiological mechanisms underlying these disorders involve serotonergic and endocannabinoid systems. In this study, we have investigated the relationships between these two neurochemical systems in emotional control using genetic and imaging tools. CB₁ cannabinoid receptor knockout mice (KO) and wild‐type littermates (WT) were exposed to chronic restraint stress. Depressive‐like symptoms (anhedonia and helplessness) were produced by chronic stress exposure in WT mice. CB₁ KO mice already showed these depressive‐like manifestations in non‐stress conditions and the same phenotype was observed after chronic restraint stress. Chronic stress similarly impaired long‐term memory in both genotypes. In addition, brain levels of serotonin transporter (5‐HTT) were assessed using positron emission tomography. Decreased brain 5‐HTT levels were revealed in CB₁ KO mice under basal conditions, as well as in WT mice after chronic stress. Our results show that chronic restraint stress induced depressive‐like behavioral alterations and brain changes in 5‐HTT levels similarly to those revealed in CB₁ KO mice in non‐stressed conditions. These results underline the relevance of chronic environmental stress on serotonergic and endocannabinoid transmission for the development of depressive symptoms.

     

In Vitro and In Vivo Evaluation of N‐{2‐[4‐(3‐Cyanopyridin‐2‐yl)piperazin‐1‐yl]ethyl}‐3‐[11C]methoxybenz­amide,a Positron Emission Tomography (PET) Radioligand for Dopamine D4 Receptors,in Rodents

The D₄ dopamine receptor belongs to the D₂‐like family of dopamine receptors, and its exact regional distribution in the central nervous system is still a matter of considerable debate. The availability of a selective radioligand for the D₄ receptor with suitable properties for positron emission tomography (PET) would help resolve issues of D₄ receptor localization in the brain, and the presumed diurnal change of expressed protein in the eye and pineal gland. We report here on in vitro and in vivo characteristics of the high‐affinity D₄ receptor‐selective ligand N‐{2‐[4‐(3‐cyanopyridin‐2‐yl)piperazin‐1‐yl]ethyl}‐3‐[¹¹C]methoxybenzamide ([¹¹C] 2 ) in rat. The results provide new insights on the in vitro properties that a brain PET dopamine D₄ radioligand should possess in order to have improved in vivo utility in rodents.     

Alanine‐(87)‐threonine polymorphism impairs signaling and internalization of the human P2Y11 receptor,when co‐expressed with the P2Y1 receptor

The P2Y₁₁ nucleotide receptor detects high extracellular ATP concentrations. Mutations of the human P2RY₁₁ gene can play a role in brain autoimmune responses, and the P2Y₁₁ receptor alanine‐87‐threonine (A87T) polymorphism has been suggested to affect immune‐system functions. We investigated receptor functionality of the P2Y₁₁A87T mutant using HEK293 and 1321N1 astrocytoma cells. In HEK293 cells, the P2Y₁₁ receptor agonist 3′‐O‐(4‐benzoylbenzoyl)adenosine 5′‐triphosphate (BzATP) was completely inactive in evoking intracellular calcium release while the potency of ATP was reduced. ATP was also less potent in triggering cAMP generation. However, 1321N1 astrocytoma cells, which lack any endogenous P2Y₁ receptors, did not display a reduction. Only when 1321N1 cells were co‐transfected with P2Y₁₁A87T and P2Y₁ receptors, the calcium responses to the P2Y₁₁ receptor‐specific agonist BzATP were reduced. It is already known that P2Y₁ and P2Y₁₁ receptors interact. We thus conclude that the physiological impact of A87T mutation of the P2Y₁₁ receptor derives from detrimental effects on P2Y₁–P2Y₁₁ receptor interaction. We additionally investigated alanine‐87‐serine and alanine‐87‐tyrosine P2Y₁₁ receptor mutants. Both mutations rescue the response to BzATP in HEK293 cells, thus ruling out polarity of amino acid‐87 to be the molecular basis for altered receptor characteristics. We further found that the P2Y₁₁A87T receptor shows complete loss of nucleotide‐induced internalization in HEK293 cells. Thus, we demonstrate impaired signaling of the P2Y₁₁ A87T‐mutated receptors when co‐operating with P2Y₁ receptors.

     

Characterization of 1-(2-[18F] fluoro-3-pyridyl)-4-(2-isopropyl-1-oxo- isoindoline-5-yl)-5-methyl-1H-1,2,3-triazole, a PET ligand for imaging the metabotropic glutamate receptor type 1 in rat and monkey brains

Neurovascular degeneration contributes to the pathogenesis of Alzheimer's disease (AD). Because erythropoietin (EPO) promotes endothelial regeneration, we investigated the therapeutic effects of EPO in animal models of AD. In aged Tg2576 mice, EPO receptors (EPORs) were expressed in the cortex and hippocampus. Tg2576 mice were treated with daily injection of EPO (5000 IU/kg/day) for 5 days. At 14 days, EPO improved contextual memory as measured by fear-conditioning test. EPO enhanced endothelial proliferation and the level of synaptophysin expression in the brain. EPO also increased capillary density, and decreased the level of the receptor for advanced glycation endproducts (RAGE) in the brain, while decreasing in the amount of amyloid plaque and amyloid-β (Aβ). In cultured human endothelial cells, EPO enhanced angiogenesis and suppressed the expression of the RAGE. These results show that EPO improves memory and ameliorates endothelial degeneration induced by Aβ in AD models. This pre-clinical evidence suggests that EPO may be useful for the treatment of AD.     

3-(Cyclopropylmethyl)-7-((4-(4-[11C]methoxyphenyl)piperidin-1-yl)methyl)-8-(trifluoromethyl)-[1,2,4]triazolo[4,3-a]pyridine: Synthesis and preliminary evaluation for PET imaging of metabotropic glutamate receptor subtype 2

Selective metabotropic glutamate receptor 2 (mGluR2) inhibitors have been demonstrated to show therapeutic effects by improving alleviating symptoms of schizophrenic patients in clinical studies. Herein we report the synthesis and preliminary evaluation of a ¹¹C-labeled positron emission tomography (PET) tracer originating from a mGluR2 inhibitor, 3-(cyclopropylmethyl)-7-((4-(4-methoxyphenyl)piperidin-1-yl)methyl)-8-(trifluoromethyl)-[1,2,4]triazolo[4,3-a]pyridine (CMTP, 1a). [¹¹C]CMTP ([¹¹C]1a) was synthesized by O-[¹¹C]methylation of desmethyl precursor 1b with [¹¹C]methyl iodide in 19.7 ± 8.9% (n = 10) radiochemical yield (based on [¹¹C]CO₂) with >98% radiochemical purity and >74 GBq/μmol molar activity. Autoradiography study showed that [¹¹C]1a possessed moderate in vitro specific binding to mGluR2 in the rat brain, with a heterogeneous distribution of radioactive accumulation in the mGluR2-rich brain tissue sections, such as the cerebral cortex and striatum. PET study indicated that [¹¹C]1a was able to cross the blood–brain barrier and enter the brain, but had very low specific binding in the rat brain. Further optimization for the chemical structure of 1a is necessary to increase binding affinity to mGluR2 and then improve in vivo specific binding in brain.     

A Deletion in the α2B‐Adrenergic Receptor Gene and Autonomic Nervous Function in Central Obesity

Objective: We investigated the impact of a three‐amino acid deletion (12Glu9) polymorphism in the α_2B‐adrenergic receptor gene on autonomic nervous function. The short form (Glu⁹/Glu⁹) of the polymorphism has previously been associated with a reduced basal metabolic rate in obese subjects. Because autonomic nervous function participates in the regulation of energy metabolism, there could be a link between this polymorphism and autonomic nervous function. Research Methods and Procedures: Data of a 10‐year follow‐up study with 126 nondiabetic control subjects and 84 type 2 diabetic patients were used to determine the effects of the 12Glu9 polymorphism on autonomic nervous function. A deep breathing test and an orthostatic test were used to investigate parasympathetic and sympathetic autonomic nervous function. In addition, cardiovascular autonomic function was studied using power spectral analysis of heart rate variability. Results: No significant differences were found in the frequency of the 12Glu9 deletion polymorphism between nondiabetic and diabetic subjects. The nondiabetic men with the Glu⁹/Glu⁹ genotype, especially those with abdominal obesity, had significantly lower total and low‐frequency power values in the power spectral analysis when compared with other men. Furthermore, in a longitudinal analysis of 10 years, the decrease in parasympathetic function was greater in nondiabetic men with the Glu⁹/Glu⁹ genotype than in the men with the Glu⁹/Glu¹² or Glu¹²/Glu¹² genotypes. Discussion: The results of the present study suggest that the 12Glu9 polymorphism of the α_2B‐adrenergic receptor gene modulates autonomic nervous function in Finnish nondiabetic men. In the nondiabetic men with the Glu⁹/Glu⁹ genotype, the general autonomic tone is depressed, and vagal activity especially becomes impaired with time. Furthermore, this association is accentuated by central obesity.     

Intrinsic α‐helical and β‐sheet conformational preferences: A computational case study of alanine

A fundamental question in protein science is what is the intrinsic propensity for an amino acid to be in an α-helix, β-sheet, or other backbone dihedral angle (-ψ) conformation. This question has been hotly debated for many years because including all protein crystal structures from the protein database, increases the probabilities for α-helical structures, while experiments on small peptides observe that β-sheet-like conformations predominate. We perform molecular dynamics (MD) simulations of a hard-sphere model for Ala dipeptide mimetics that includes steric interactions between nonbonded atoms and bond length and angle constraints with the goal of evaluating the role of steric interactions in determining protein backbone conformational preferences. We find four key results. For the hard-sphere MD simulations, we show that (1) β-sheet structures are roughly three and half times more probable than α-helical structures, (2) transitions between α-helix and β-sheet structures only occur when the backbone bond angle τ (N–C_α–C) is greater than 110°, and (3) the probability distribution of τ for Ala conformations in the “bridge” region of-ψ space is shifted to larger angles compared to other regions. In contrast, (4) the distributions obtained from Amber and CHARMM MD simulations in the bridge regions are broader and have increased τ compared to those for hard sphere simulations and from high-resolution protein crystal structures. Our results emphasize the importance of hard-sphere interactions and local stereochemical constraints that yield strong correlations between -ψ conformations and τ.     

Adenosine A2A receptors are required for glutamate mGluR5‐ and dopamine D1 receptor‐evoked ERK1/2 phosphorylation in rat hippocampus: involvement of NMDA receptor

Interaction between mGluR5 and NMDA receptors (NMDAR ) is vital for synaptic plasticity and cognition. We recently demonstrated that stimulation of mGluR5 enhances NMDAR responses in hippocampus by phosphorylating NR2B(Tyr1472) subunit, and this reaction was enabled by adenosine A_2A receptors (A_2AR) (J Neurochem, 135, 2015, 714). In this study, by using in vitro phosphorylation and western blot analysis in hippocampal slices of male Wistar rats, we show that mGluR5 stimulation or mGluR5/NMDAR s co‐stimulation synergistically activate ERK 1/2 signaling leading to c‐Fos expression. Interestingly, both reactions are under the permissive control of endogenous adenosine acting through A_2ARs. Moreover, mGluR5‐mediated ERK 1/2 phosphorylation depends on NMDAR , which however exhibits a metabotropic way of function, since no ion influx through its ion channel is required. Furthermore, our results demonstrate that mGluR5 and mGluR5/NMDAR ‐evoked ERK 1/2 activation correlates well with the mGluR5/NMDAR ‐evoked NR2B(Tyr1472) phosphorylation, since both phenomena coincide temporally, are Src dependent, and are both enabled by A_2ARs. This indicates a functional involvement of NR2B(Tyr1472) phosphorylation in the ERK 1/2 activation. Our biochemical results are supported by electrophysiological data showing that in CA 1 region of hippocampus, the theta burst stimulation (TBS)‐induced long‐term potentiation coincides temporally with an increase in ERK 1/2 activation and both phenomena are dependent on the tripartite A_2A, mGlu5, and NMDAR s. Furthermore, we show that the dopamine D1 receptors evoked ERK 1/2 activation as well as the NR2B(Tyr1472) phosphorylation are also regulated by endogenous adenosine and A_2ARs. In conclusion, our results highlight the A_2ARs as a crucial regulator not only for NMDAR responses, but also for regulating ERK 1/2 signaling and its downstream pathways, leading to gene expression, synaptic plasticity, and memory consolidation.

     

Structure and dynamic properties of the single disulfide-deficient α-amylase inhibitor [C45A/C73A]tendamistat: An NMR study

Covalent linkages such as disulfide bonds are important for the stabilization of proteins. In the present NMR study we compare the structure and the dynamics of the single disulfide-deficient variant C45A/C73A of the α-amylase inhibitor tendamistat and the wild-type protein, which contains two disulfide bonds (C11-C27 and C45-C73). Complete proton assignment was achieved by standard homonuclear 2D techniques for the variant. Chemical shift differences, intra-strand NOE effects and protected amide proton were used to compare the connectivity of the secondary structure elements of variant and wild-type. Dynamic properties of the wild-type protein were studied by ¹³C_α heteronuclear NOE experiments with carbon in natural abundance. ¹⁵N isotope labeling was necessary to obtain the relaxation parameters of the variant, because of sample degradation. The ¹⁵N resonance assignment was achieved by a ¹⁵N 3D-NOESY-HMQC. Removal of the C45-C73 bond by the C45A/C73A mutation has no influence upon the β-barrel structure of tendamistat beside very local changes at the mutation site. The relaxation data revealed only subtle differences between variant and wild-type on a subnanosecond time scale. Only the N-terminus and G62 in the connecting loop between the anti-parallel β-sheets showed an increased mobility. The results are discussed in respect to thermodynamic stability and the secretion efficiency of tendamistat. Proteins 33:285–294, 1998. © 1998 Wiley-Liss, Inc.     

Biotransformation of 4‐fluoro‐N‐(1‐{2‐[(propan‐2‐yl)phenoxy]ethyl}‐8‐azabicyclo[3.2.1]octan‐3‐yl)‐benzenesulfonamide,a novel potent 5‐HT7 receptor antagonist with antidepressant‐like and anxiolytic properties: In vitro and in silico approach

The aim of the study was to investigate the metabolism of 4‐fluoro‐N‐(1‐{2‐[(propan‐2‐yl)phenoxy]ethyl}‐8‐azabicyclo[3.2.1]octan‐3‐yl)‐benzenesulfonamide (PZ‐1150), a novel 5‐HT₇ receptor antagonist with antidepressant‐like and anxiolytic properties, by the following three ways: in vitro with microsomes; in vitro employing Cunninghamella echinulata, and in silico using MetaSite. Biotransformation of PZ‐1150 with microsomes resulted in five metabolites, while transformation with C. echinulata afforded two metabolites. In both models, the predominant metabolite occurred due to hydroxylation of benzene ring. In silico data coincide with in vitro experiments, as three MetaSite metabolites matched compounds identified in microsomal samples. In human liver microsomes PZ‐1150 exhibited in vitro half‐life of 64 min, with microsomal intrinsic clearance of 54.1 μL/min/mg and intrinsic clearance of 48.7 mL/min/kg. Therefore, PZ‐1150 is predicted to be a high‐clearance agent. The study demonstrated the applicability of using microsomal model coupled with microbial model to elucidate the metabolic pathways of compounds and comparison with in silico metabolite predictions.     

AT2R − 1332 G:A polymorphism and its interaction with AT1R 1166 A:C,ACE I/D and MMP-9 − 1562 C:T polymorphisms: Risk factors for susceptibility to preeclampsia

The possible association of angiotensin type 2 receptor (AT2R) − 1332 G:A polymorphism with susceptibility to preeclampsia was studied in 252 women consisted of 155 women with preeclampsia and 97 healthy pregnant women. Also, the interaction of this polymorphism with angiotensin type 1 receptor (AT1R) 1166 A:C, angiotensin converting enzyme insertion/deletion (ACE I/D) and also with matrix metalloproteinase-9 (MMP-9) − 1562 C:T polymorphism was investigated. The AT2R − 1332 G:A polymorphism was detected using PCR–RFLP method. Significantly higher frequencies of GG+GA genotype and G allele of AT2R were observed in mild (80.2%, p = 0.003 and 47.5%, p = 0.012, respectively) and severe (77.8%, p = 0.034 and 48.1%, p = 0.026, respectively) preeclampsia compared to controls (60.8% and 35.1%, respectively). The presence of G allele was associated with 1.69-fold increased risk of preeclampsia (p = 0.005). In severe preeclamptic women, systolic and diastolic blood pressures in the presence of GG+GA genotype were significantly higher compared to those in the presence of AA genotype. The concomitant presence of both alleles of AT2R G and AT1R C was associated with 1.3 times increased risk of mild preeclampsia (p = 0.03). There was an interaction between AT2R G and ACE D alleles that significantly increased the risk of mild and severe preeclampsia by 1.38- and 1.3-fold, respectively. Also, interaction between MMP-9 T and AT2R G alleles increased the risk of severe preeclampsia 1.39-fold (p = 0.028). Our study demonstrated that the G allele of AT2R − 1332 G:A polymorphism is associated with an increased risk of preeclampsia. Also, epistatic interaction of G allele and each allele of the AT1R C, ACE D and MMP-9 T was associated with the risk of preeclampsia. Our findings suggest that the renin–angiotensin system (RAS) variants and gene–gene interactions affect the risk of preeclampsia.     

A C-NMR study on the influxes into the tricarboxylic acid cycle of a renal epithelial cell line, LLC-PK1/Cl4: the metabolism of [2-C]glycine, l-[3-C]alanine and l-[3-C]aspartic acid in renal epithelial cells

Perchloric acid extracts of LLC-PK₁/Cl₄ cells, a renal epithelial cell line, incubated with either [2-¹³C]glycine l-[3-¹³C]alanine, or d,l-[3-¹³C]aspartic acid were investigated by ¹³C-NMR spectroscopy. All amino acids, except labelled glycine, gave rise to glycolytic products and tricarboxylic acid cycle (TCA) intermediates. For the first time we also observed activity of γ-glutamyltransferase activity and glutathione synthetase activity in LLC-PK₁ cells, as is evident from enrichment of reduced glutathione. Time courseS showed that only 6% of the labelled glycine was utilized in 30 min, whereas 31% of l-alanine and 60% of l-aspartic acid was utilized during the same period. ¹³C-NMR was also shown to be a useful tool for the determination of amino acid uptake in LLC-PK₁ cells. These uptake experiments indicated that glycine alanine and aspartic acid are transported into Cl₄ cells via a sodium-dependent process. From the relative enrichment of the glutamate carbons, we calculated the activity of pyruvate dehydrogenase to be about 61% of when labelled l-alanine was the only carbon source for LLC-PK₁/Cl₄ cells. Experiments with labelled d,l-aspartic, however, showed that about 40% of C-3-enriched oxaloacetate (arising from a de-amination of aspartic acid) reached the pyruvate pool.