Regulation of adaptive growth decisions via phosphorylation of the TRAPPII complex in Arabidopsis.

Plants often adapt to adverse or stress conditions via differential growth. The trans-Golgi network (TGN) has been implicated in stress responses, but it is not clear in what capacity it mediates adaptive growth decisions. In this study, we assess the role of the TGN in stress responses by exploring the previously identified interactome of the Transport Protein Particle II (TRAPPII) complex required for TGN structure and function. We identified physical and genetic interactions between AtTRAPPII and shaggy-like kinases (GSK3/AtSKs) and provided in vitro and in vivo evidence that the TRAPPII phosphostatus mediates adaptive responses to abiotic cues. AtSKs are multifunctional kinases that integrate a broad range of signals. Similarly, the AtTRAPPII interactome is vast and considerably enriched in signaling components. An AtSK-TRAPPII interaction would integrate all levels of cellular organization and instruct the TGN, a central and highly discriminate cellular hub, as to how to mobilize and allocate resources to optimize growth and survival under limiting or adverse conditions.

Regulation of adaptive growth decisions via phosphorylation of the TRAPPII complex in Arabidopsis.

Plants often adapt to adverse or stress conditions via differential growth. The trans-Golgi Network (TGN) has been implicated in stress responses, but it is not clear in what capacity it mediates adaptive growth decisions. In this study, we assess the role of the TGN in stress responses by exploring the interactome of the Transport Protein Particle II (TRAPPII) complex, required for TGN structure and function. We identified physical and genetic interactions between TRAPPII and shaggy-like kinases (GSK3/AtSKs). Kinase assays and pharmacological inhibition provided in vitro and in vivo evidence that AtSKs target the TRAPPII-specific subunit AtTRS120/TRAPPC9. GSK3/AtSK phosphorylation sites in AtTRS120/TRAPPC9 were mutated, and the resulting AtTRS120 phosphovariants subjected to a variety of single and multiple stress conditions in planta . The non-phosphorylatable TRS120 mutant exhibited enhanced adaptation to multiple stress conditions and to osmotic stress whereas the phosphomimetic version was less resilient. Higher order inducible trappii atsk mutants had a synthetically enhanced defect in root gravitropism. Our results suggest that the TRAPPII phosphostatus mediates adaptive responses to abiotic cues. AtSKs are multifunctional kinases that integrate a broad range of signals. Similarly, the TRAPPII interactome is vast and considerably enriched in signaling components. An AtSK-TRAPPII interaction would integrate all levels of cellular organization and instruct the TGN, a central and highly discriminate cellular hub, as to how to mobilize and allocate resources to optimize growth and survival under limiting or adverse conditions.

Correction: A role for brassinosteroid signalling in decision-making processes in the Arabidopsis seedling.

[This corrects the article DOI: 10.1371/journal.pgen.1010541.].

A role for brassinosteroid signalling in decision-making processes in the Arabidopsis seedling.

Plants often adapt to adverse conditions via differential growth, whereby limited resources are discriminately allocated to optimize the growth of one organ at the expense of another. Little is known about the decision-making processes that underly differential growth. In this study, we developed a screen to identify decision making mutants by deploying two tools that have been used in decision theory: a well-defined yet limited budget, as well as conflict-of-interest scenarios. A forward genetic screen that combined light and water withdrawal was carried out. This identified BRASSINOSTEROID INSENSITIVE 2 (BIN2) alleles as decision mutants with "confused" phenotypes. An assessment of organ and cell length suggested that hypocotyl elongation occurred predominantly via cellular elongation. In contrast, root growth appeared to be regulated by a combination of cell division and cell elongation or exit from the meristem. Gain- or loss- of function bin2 mutants were most severely impaired in their ability to adjust cell geometry in the hypocotyl or cell elongation as a function of distance from the quiescent centre in the root tips. This study describes a novel paradigm for root growth under limiting conditions, which depends not only on hypocotyl-versus-root trade-offs in the allocation of limited resources, but also on an ability to deploy different strategies for root growth in response to multiple stress conditions.

Interactions between Transport Protein Particle (TRAPP) complexes and Rab GTPases in Arabidopsis.

Transport Protein Particle II (TRAPPII) is essential for exocytosis, endocytosis, protein sorting and cytokinesis. In spite of a considerable understanding of its biological role, little information is known about Arabidopsis TRAPPII complex topology and molecular function. In this study, independent proteomic approaches initiated with TRAPP components or Rab-A GTPase variants converge on the TRAPPII complex. We show that the Arabidopsis genome encodes the full complement of 13 TRAPPC subunits, including four previously unidentified components. A dimerization model is proposed to account for binary interactions between TRAPPII subunits. Preferential binding to dominant negative (GDP-bound) versus wild-type or constitutively active (GTP-bound) RAB-A2a variants discriminates between TRAPPII and TRAPPIII subunits and shows that Arabidopsis complexes differ from yeast but resemble metazoan TRAPP complexes. Analyzes of Rab-A mutant variants in trappii backgrounds provide genetic evidence that TRAPPII functions upstream of RAB-A2a, allowing us to propose that TRAPPII is likely to behave as a guanine nucleotide exchange factor (GEF) for the RAB-A2a GTPase. GEFs catalyze exchange of GDP for GTP; the GTP-bound, activated, Rab then recruits a diverse local network of Rab effectors to specify membrane identity in subsequent vesicle fusion events. Understanding GEF-Rab interactions will be crucial to unravel the co-ordination of plant membrane traffic.

Comparison of in†Silico, Electrochemical, in†Vitro and in†Vivo Metabolism of a Homologous Series of (Radio)fluorinated σ1 Receptor Ligands Designed for Positron Emission Tomography.

The imaging of σ1 receptors in the brain by fluorinated radiotracers will be used for the validation of σ1 receptors as drug targets as well as for differential diagnosis of diseases in the central nervous system. The biotransformation of four homologous fluorinated PET tracers 1'-benzyl-3-(ω-fluoromethyl to ω-fluorobutyl)-3H-spiro[2]benzofuran-1,4'-piperidine] ([18 F]1-4) was investigated. In silico studies using fast metabolizer (FAME) software, electrochemical oxidations, in vitro studies with rat liver microsomes, and in vivo metabolism studies after application of the PET tracers [18 F]1-4 to mice were performed. Combined liquid chromatography and mass spectrometry (HPLC-MS) analysis allowed structural identification of non-radioactive metabolites. Radio-HPLC and radio-TLC provided information about the presence of unchanged parent radiotracers and their radiometabolites. Radiometabolites were not found in the brain after application of [18 F]2-4, but liver, plasma, and urine samples contained several radiometabolites. Less than 2 % of the injected dose of [18 F]4 reached the brain, rendering [18 F]4 less appropriate as a PET tracer than [18 F]2 and [18 F]3. Compounds [18 F]2 and [18 F]3 possess the most promising properties for imaging of σ1 receptors in the brain. High σ1 affinity (Ki =0.59 nm), low lipophilicity (logD7.4 =2.57), high brain penetration (4.6 % of injected dose after 30 min), and the absence of radiometabolites in the brain favor the fluoroethyl derivative [18 F]2 slightly over the fluoropropyl derivative [18 F]3 for human use.

Synthesis, radiofluorination and pharmacological evaluation of a fluoromethyl spirocyclic PET tracer for central σ1 receptors and comparison with fluoroalkyl homologs.

The spirocyclic σ(1) receptor ligand 1 (1'-benzyl-3-(fluoromethyl)-3H-spiro[[2]benzofuran-1,4'-piperidine]) was prepared in four steps starting from methoxy derivative 5. Due to its high σ(1) affinity (K(i)=0.74nM) and selectivity against several other relevant targets, 1 was investigated as (18)F-labeled PET tracer and its biological properties were compared with those of homologous fluoroalkyl derivatives 2-4. The fluoromethyl derivative 1 was faster metabolized in vitro than homologs 2-4. In contrast to the radiosynthesis of [(18)F]2-4, the nucleophilic substitution of the tosylate 15 using the K[(18)F]F-K(222)-carbonate complex required heating to 150°C in DMSO to achieve high labeling efficiencies. Whereas radiometabolites of [(18)F]2-4 were not detected in vivo in the brain of mice, two radiometabolites of [(18)F]1 were found. Analysis of ex vivo autoradiography images provided rather low target-to-nontarget ratio for [(18)F]1 compared with [(18)F]2-4. [(18)F]1 showed a fast uptake in the brain, which decreased continuously over time. The brain-to-plasma ratio of the radiotracer [(18)F]1 was only exceeded by the fluoroethyl tracer [(18)F]2.

A ¹8F-labeled fluorobutyl-substituted spirocyclic piperidine derivative as a selective radioligand for PET Imaging of sigma1 receptors.

In this study, we synthesized and evaluated a new spirocyclic piperidine derivative 3, containing a 4-fluorobutyl side chain, as a PET radioligand for neuroimaging of σ1 receptors. In vitro, compound 3 displayed high affinity for σ1 receptors (K(i) =1.2 nM) as well as high selectivity. [¹8F]3 radiosynthesis was performed from the corresponding tosylate precursor, with high radiochemical yield (45-51 %), purity (>98 %), and specific activity (>201 GBq µmol⁻¹). Metabolic stability of [¹8F]3 in the brain of CD-1 mice was verified, and no penetration of peripheral radiometabolites into the cerebral tissue was observed. Results of ex vivo autoradiography revealed that the distribution of [¹8F]3 in the brain corresponded to regions with high σ1 receptor density. The highest region-specific total-to-nonspecific ratio was determined in the facial nucleus (4.00). Biodistribution studies indicated rapid and high levels in brain uptake of [¹8F]3 (2.2 % ID per gram at 5 min p.i.). Pre-administration of haloperidol significantly inhibited [¹8F]3 uptake into the brain and σ1 receptor-expressing organs, further confirming in vivo target specificity.

Molecular imaging of σ receptors: synthesis and evaluation of the potent σ1 selective radioligand [18F]fluspidine.

PURPOSE: Neuroimaging of σ(1) receptors in the human brain has been proposed for the investigation of the pathophysiology of neurodegenerative and psychiatric diseases. However, there is a lack of suitable (18)F-labelled PET radioligands for that purpose. METHODS: The selective σ(1) receptor ligand [(18)F]fluspidine (1'-benzyl-3-(2-[(18)F]fluoroethyl)-3H-spiro[[2]benzofuran-1,4'-piperidine]) was synthesized by nucleophilic (18)F(-) substitution of the tosyl precursor. In vitro receptor binding affinity and selectivity were assessed by radioligand competition in tissue homogenate and autoradiographic approaches. In female CD-1 mice, in vivo properties of [(18)F]fluspidine were evaluated by ex vivo brain section imaging and organ distribution of intravenously administered radiotracer. Target specificity was validated by organ distribution of [(18)F]fluspidine after treatment with 1 mg/kg i.p. of the σ receptor antagonist haloperidol or the emopamil binding protein (EBP) inhibitor tamoxifen. In vitro metabolic stability and in vivo metabolism were investigated by LC-MS(n) and radio-HPLC analysis. RESULTS: [(18)F]Fluspidine was obtained with a radiochemical yield of 35-45%, a radiochemical purity of ≥ 99.6% and a specific activity of 150-350 GBq/µmol (n = 6) within a total synthesis time of 90-120 min. In vitro, fluspidine bound specifically and with high affinity to σ(1) receptors (K (i) = 0.59 nM). In mice, [(18)F]fluspidine rapidly accumulated in brain with uptake values of 3.9 and 4.7%ID/g and brain to blood ratios of 7 and 13 at 5 and 30 min after intravenous application of the radiotracer, respectively. By ex vivo autoradiography of brain slices, resemblance between binding site occupancy of [(18)F]fluspidine and the expression of σ(1) receptors was shown. The radiotracer uptake in the brain as well as in peripheral σ(1) receptor expressing organs was significantly inhibited by haloperidol but not by tamoxifen. Incubation with rat liver microsomes led to a fast biotransformation of fluspidine. After an incubation period of 30 min only 13% of the parent compound was left. Seven metabolites were identified by HPLC-UV and LC-MS(n) techniques. However, [(18)F]fluspidine showed a higher metabolic stability in vivo. In plasma samples ∼ 94% of parent compound remained at 30 min and ∼ 67% at 60 min post-injection. Only one major radiometabolite was detected. None of the radiometabolites crossed the blood-brain barrier. CONCLUSION: [(18)F]Fluspidine demonstrated favourable target affinity and specificity as well as metabolic stability both in vitro and in animal experiments. The in vivo properties of [(18)F]fluspidine offer a high potential of this radiotracer for neuroimaging and quantitation of σ(1) receptors in vivo.

Synthesis, pharmacological activity and structure affinity relationships of spirocyclic σ(1) receptor ligands with a (2-fluoroethyl) residue in 3-position.

In order to develop a fluorinated radiotracer for imaging of σ(1) receptors in the central nervous system a series of (2-fluoroethyl) substituted spirocyclic piperidines 3 has been prepared. In the key step of the synthesis 2-bromocinnamaldehyde acetal 5 was added to piperidones 6 with various substituents at the N-atom. Unexpectedly, this reaction led to 2-benzoxepines 8, which were contracted with acid to afford the spirocyclic 2-benzofuranacetaldehydes 9. The best yields were obtained, when the transformations up to the alcohols 10 were performed without isolation of intermediates. Generally the (2-fluoroethyl) derivatives 3 have higher σ(1) affinity and σ(1)/σ(2) selectivity than the corresponding (3-fluoropropyl) derivatives 2. The most promising candidate for the development as radiotracer is the (2-fluoroethyl) derivative 3a (WMS-1828, fluspidine, 1'-benzyl-3-(2-fluoroethyl)-3H-spiro[[2]benzofuran-1,4'-piperidine]), which shows subnanomolar σ(1) affinity (K(i)=0.59nM) and excellent selectivity over the σ(2) subtype (1331-fold) as well as some other receptor systems. The novel synthetic strategy also allows the systematic pharmacological evaluation of intermediate alcohols 10. Despite their high σ(1) affinity (K(i)=6-32nM) and selectivity the alcohols 10 are 10-30-fold less potent than the bioisosteric fluoro derivatives 3.

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 σ(1) 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 [(3)H]-(+)-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.

Aromatic C-H bond hydroxylation by P450 peroxygenases: a facile colorimetric assay for monooxygenation activities of enzymes based on Russig's blue formation.

Aromatic C-H bond hydroxylation of 1-methoxynaphthalene was efficiently catalyzed by the substrate misrecognition system of the hydrogen peroxide dependent cytochrome P450BSbeta (CYP152A1), which usually catalyzes hydroxylation of long-alkyl-chain fatty acids. Very importantly, the hydroxylation of 1-methoxynaphthalene can be monitored by a color change since the formation of 4-methoxy-1-naphthol was immediately followed by its further oxidation to yield Russig's blue. Russig's blue formation allows us to estimate the peroxygenation activity of enzymes without the use of high performance liquid chromatography, gas chromatography, and nuclear magnetic resonance measurements.

Evaluation of spirocyclic 3-(3-fluoropropyl)-2-benzofurans as sigma1 receptor ligands for neuroimaging with positron emission tomography.

A series of various N-substituted 3-(3-fluoropropyl)-3H-spiro[[2]benzofuran-1,4'-piperidines] (7) has been synthesized. In receptor binding studies, the N-benzyl derivative 7a (WMS-1813) revealed extraordinarily high sigma(1) receptor affinity (K(i) = 1.4 nM) and excellent sigma(1)/sigma(2) selectivity (>600 fold). In vitro biotransformation of 7a with rat liver microsomes led to three main metabolites. N-Debenzylation was inhibited by introduction of an N-phenylethyl residue (7 g). The PET tracer [(18)F]7a was synthesized by nucleophilic substitution of the tosylate 13 with K[(18)F]F-K222-carbonate complex. The decay corrected radiochemical yield of [(18)F]7a was 35-48% with a radiochemical purity of >99.5% and a specific activity of 150-238 GBq/micromol. The radiotracer properties were evaluated in female CD-1 mice by organ distribution and ex vivo brain autoradiography. The radiotracer uptake in the brain was fast and sufficient, with values of approximately 4% injected dose per gram. Target specificity of [(18)F]7a was validated in blocking studies by preapplication of haloperidol, and significant reduction in the uptake of radioactivity was observed in the brain and peripheral organs expressing sigma(1) receptors.

Pharmacological and metabolic characterisation of the potent sigma1 receptor ligand 1'-benzyl-3-methoxy-3H-spiro[[2]benzofuran-1,4'-piperidine].

OBJECTIVES: The pharmacology and metabolism of the potent sigma1 receptor ligand 1'-benzyl-3-methoxy-3H-spiro[[2]benzofuran-1,4'-piperidine] were evaluated. METHODS: The compound was tested against a wide range of receptors, ion channels and neurotransmitter transporters in radioligand binding assays. Analgesic activity was evaluated using the capsaicin pain model. Metabolism by rat and human liver microsomes was investigated, and the metabolites were identified by a variety of analytical techniques. KEY FINDINGS: 1'-benzyl-3-methoxy-3H-spiro[[2]benzofuran-1,4'-piperidine] (compound 1) is a potent sigma1 receptor ligand (Ki 1.14 nM) with extraordinarily high sigma1/sigma2 selectivity (>1100). It was selective for the sigma1 receptor over more than 60 other receptors, ion channels and neurotransmitter transporters, and did not interact with the human ether-a-go-go-related gene (hERG) cardiac potassium channel. Compound 1 displayed analgesic activity against neuropathic pain in the capsaicin pain model (53% analgesia at 16 mg/kg), indicating that it is a sigma1 receptor antagonist. It was rapidly metabolised by rat liver microsomes. Seven metabolites were unequivocally identified; an N-debenzylated metabolite and a hydroxylated metabolite were the major products. Pooled human liver microsomes formed the same metabolites. Studies with seven recombinant cytochrome P450 isoenzymes revealed that CYP3A4 produced all the metabolites identified. The isoenzyme CYP2D6 was inhibited by 1 (IC50 88 nM) but did not produce any metabolites. CONCLUSIONS: 1'-benzyl-3-methoxy-3H-spiro[[2]benzofuran-1,4'-piperidine] is a potent and selective sigma1 receptor antagonist, which is rapidly metabolised. Metabolically more stable sigma1 ligands could be achieved by stabilising the N-benzyl substructure.

Synthesis of spirocyclic sigma1 receptor ligands as potential PET radiotracers, structure-affinity relationships and in vitro metabolic stability.

Several 3H-spiro[[2]benzofuran-1,4'-piperidines] bearing a p-fluorobenzyl residue at the N-atom and various substituents in position 3 of the benzofuran system were synthesized. The crucial reaction steps are the addition of a lithiated benzaldehyde derivative to the p-fluorobenzylpiperidone 5 and the BF(3).OEt(2) catalyzed substitution of the methoxy group of 2a by various nucleophiles. Structure-affinity relationship studies revealed that compounds with two protons (2d), a methoxy group (2a), and a cyano group (2e) in position 3 possess subnanomolar sigma(1) affinity (K(i)=0.18 nM, 0.79 nM, 0.86 nM) and high selectivity against the sigma(2) subtype. The metabolites of 2a, 2d, and 2e, which were formed upon incubation with rat liver microsomes, were identified. Additionally, the rate of metabolic degradation of 2a, 2d, and 2e was determined and compared with the degradation rate of the non-fluorinated spirocyclic compound 1. For the synthesis of the potential PET tracers [(18)F]2a and [(18)F]2e two different radiosynthetic approaches were followed.