Agonist-selektive Phosphorylierung und Dephosphorylierung des humanen Somatostatinrezeptors 5

Die Gruppe der Somatostatinrezeptoren, die aus fünf Subtypen besteht und zur Superfamilie der G-Protein-gekoppelten Rezeptoren gehört, bildet für die Diagnose und Therapie von neuroendokrinen Tumoren eine der wichtigsten pharmazeutischen Ziele. Besonders dem Somatostatinrezeptor 2 (sst2) kommt eine große Bedeutung als Zielstruktur bei der Therapie von Akromegalie-Patienten zu. Da der natürliche Ligand Somatostatin durch seine kurze Halbwertszeit als therapeutisches Mittel limitiert ist, wurden in den vergangenen Jahrzehnten Somatostatinanaloga entwickelt, die zum Teil eine höhere Affinität und eine besseres Wirkprofil am Rezeptor aufweisen als der natürliche Ligand. In den vergangenen Jahren zeigte sich jedoch, dass es häufig nach Langzeittherapien zu einem Wirkungsverlust von z.B. Octreotid auf die Karzinoide oder Hypophysenadenome kommen kann, was eng mit einem Verlust der sst2-Dichte auf den Zellen einhergeht. Da der Somatostatinrezeptor 5 (sst5) häufig mit dem sst2 auf diesen Tumoren koexprimiert wird, wurden neue Somatostatinanaloga wie Pasireotid (SOM230) entwickelt, die über ein breiteres Wirkspektrum und Bindungsprofil verfügen als Octreotid. Im Gegensatz zum sehr gut untersuchten molekularen Wirkmechanismen des sst2 ist nur sehr wenig über die Regulierung des sst5 bekannt. Deshalb wurden in dieser Arbeit phosphorylierungsspezifische Antikörper generiert und charakterisiert, die zur Aufklärung der Regulierung des sst5 nach Stimulierung mit verschiedenen Agonisten, wie SS-14, Octreotid oder Pasireotid, beitragen sollten. Es wurden durch Inhibitor- und siRNA-Knockdown-Untersuchungen sowie durch Western Blot und immunzytochemische Studien gezeigt, dass SS-14, den stärksten, Pasireotid und L817,818 (sst5-Agonist) einen partiellen und Octeotid und KE108 gar keinen Effekt auf die Phosphorylierung der zwei untersuchten Threonine an den Positionen 333 und 347 des C-Terminus sowie auf das Internalisierungsverhalten des sst5 haben. Erstmals konnte für den sst5 nachgewiesen werden, dass er homolog durch die GRK2 an der agonisten-induzierbaren Stelle T333 phosphoryliert wird, während die Position T347 konstitutiv phosphoryliert war. Auch konnte gezeigt werden, dass es beim sst5 im Vergleich zum sst2 sowohl zu einer schnelleren Phosphorylierung als auch zu einer rascheren Dephosphorylierung nach Agonistengabe kam. Es konnte in dieser Arbeit auch das verantwortliche dephosphorylierende Enzym und die zugehörige katalytische Untereinheit, die PP1γ, identifiziert werden

A Transplantable Phosphorylation Probe for Direct Assessment of G Protein-Coupled Receptor Activation

The newly developed multireceptor somatostatin analogs pasireotide (SOM230), octreotide and somatoprim (DG3173) have primarily been characterized according to their binding profiles. However, their ability to activate individual somatostatin receptor subtypes (sst) has not been directly assessed so far. Here, we transplanted the carboxyl-terminal phosphorylation motif of the sst2 receptor to other somatostatin receptors and assessed receptor activation using a set of three phosphosite-specific antibodies. Our comparative analysis revealed unexpected efficacy profiles for pasireotide, octreotide and somatoprim. Pasireotide was able to activate sst3 and sst5 receptors but was only a partial agonist at the sst2 receptor. Octreotide exhibited potent agonistic properties at the sst2 receptor but produced very little sst5 receptor activation. Like octreotide, somatoprim was a full agonist at the sst2 receptor. Unlike octreotide, somatoprim was also a potent agonist at the sst5 receptor. Together, we propose the application of a phosphorylation probe for direct assessment of G protein-coupled receptor activation and demonstrate its utility in the pharmacological characterization of novel somatostatin analogs

Agonist-selective internalization of human somatostatin receptors.

<p>HEK293 cells stably expressing sst₂, sst₃ or sst₅ receptors were treated with either 1 µM SS-14, octreotide, pasireotide or somatoprim for 0 or 30 min. Cells were fixed, immunoflurescently stained with anti-sst₂ {UMB-1}, anti-sst₃ {UMB-5} or anti-sst₅ {UMB-4} antibodies, and examined by confocal microscopy. Shown are representative images from one of three independent experiments performed in duplicate. <i>Scale bar</i>, 20 µm.</p

Agonist-selective phosphorylation of the sst₃-sst₂CT chimera.

<p>(<i>Top</i>) Schematic representation of the rat sst₃-sst₂CT chimera indicating the phosphate acceptor sites S341/343, T353/354 and T356/359 within the carboxyl-terminal tail. (<i>Bottom</i>) HEK293 cells stably expressing the rat sst₃-sst_2ACT receptor were either not exposed or exposed to concentrations of 10⁻¹² to 10⁻⁵ M SS-14, octreotide, pasireotide or somatoprim for 5 min. The levels of phosphorylated rsst₃-sst_2ACT receptors were then determined using anti-pS341/pS343 {3157}, anti-pT353/pT354 {0521} or anti-pT356/pT359 {0522}. Western blots shown are representative of three to five independent experiments for each condition. The positions of the molecular mass markers are indicated on the left (in kDa).</p

Agonist-selective phosphorylation of the human sst₅ and sst₅-sst₂CT chimera.

<p>(<i>A</i>,<i>Top panel</i>) Schematic representation of the sst₅-sst₂CT receptor indicating the phosphate acceptor sites S341/343, T353/354 and T356/359 within the carboxyl-terminal tail. (<i>A</i>, <i>Bottom</i>) HEK293 cells stably expressing the sst₅-sst_2ACT receptor were either not exposed or exposed for 5 min to SS-14, octreotide, pasireotide or somatoprim in concentrations ranging from 10⁻¹² to 10⁻⁵ M. The levels of phosphorylated sst₅-sst_2ACT receptors were then determined using the phosphosite-specific antibodies anti-pS341/pS343 {3157}, anti-pT353/pT354 {0521} and anti-pT356/pT359 {0522}. (<i>B</i>,<i>Top panel</i>) Schematic representation of the human sst₅ receptor indicating the phosphate acceptor site T333 within its carboxyl-terminal tail. (<i>B</i>, <i>Bottom</i>) HEK293 cells stably expressing the human sst₅ receptors were either not exposed or exposed for 5 min to SS-14, octreotide, pasireotide or somatoprim in concentrations ranging from 10⁻¹² to 10⁻⁵ M. The levels of phosphorylated sst₅ receptors were then determined using the phosphosite-specific antibodies anti-pT333 {3567}. Western blots shown are representative of three to five independent experiments for each condition. The positions of the molecular mass markers are indicated on the left (in kDa).</p

Agonist-stimulated ³⁵S-GTPγS binding.

<p>Stimulation of [³⁵S]GTPγS binding by SS-14, Octreotide, Pasireotide and Somatoprim in the concentration range of 10⁻¹² to 10⁻⁶ M. Membranes wer prepared from HEK293 cells stably expressing either the human sst₂, sst₅ and sst₅-sst_2ACT or the rat sst₃-sst_2ACT receptor. Values represent means of triplicate determinations. SE values were smaller than 15%. Three replicate experiments gave similar results.</p

Ligand binding properties of sst₅-sst₂CT receptors.

<p>Ligand binding assays were carried out as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039458#s2" target="_blank">Materials and Methods</a>”. The half-maximal inhibitory concentrations (IC₅₀) were analyzed by nonlinear regression curve fitting using the computer program GraphPad Prism. Data are presented as the mean of three independent experiments performed in triplicate.</p

Agonist-selective phosphorylation of the human sst₂ receptor.

<p>(<i>Top</i>) Schematic representation of the human sst₂ receptor indicating the phosphate acceptor sites S341/343, T353/354 and T356/359 within its carboxyl-terminal tail. (<i>Bottom</i>) HEK293 cells stably expressing the sst₂ receptor were either not exposed or exposed for 5 min to SS-14, octreotide, pasireotide or somatoprim in concentrations ranging from 10⁻¹² to 10⁻⁵ M. The levels of phosphorylated sst₂ receptors were then determined using the phosphosite-specific antibodies anti-pS341/pS343 {3157}, anti-pT353/pT354 {0521} and anti-pT356/pT359 {0522}. Western blots shown are representative of three to five independent experiments for each condition. The positions of the molecular mass markers are indicated on the left (in kDa).</p

Effect of anakinra versus usual care in adults in hospital with COVID-19 and mild-to-moderate pneumonia (CORIMUNO-ANA-1): a randomised controlled trial

International audienc

Effect of Tocilizumab vs Usual Care in Adults Hospitalized With COVID-19 and Moderate or Severe Pneumonia

International audienceImportance Severe pneumonia with hyperinflammation and elevated interleukin-6 is a common presentation of coronavirus disease 2019 (COVID-19).Objective To determine whether tocilizumab (TCZ) improves outcomes of patients hospitalized with moderate-to-severe COVID-19 pneumonia.Design, Setting, and Particpants This cohort-embedded, investigator-initiated, multicenter, open-label, bayesian randomized clinical trial investigating patients with COVID-19 and moderate or severe pneumonia requiring at least 3 L/min of oxygen but without ventilation or admission to the intensive care unit was conducted between March 31, 2020, to April 18, 2020, with follow-up through 28 days. Patients were recruited from 9 university hospitals in France. Analyses were performed on an intention-to-treat basis with no correction for multiplicity for secondary outcomes.Interventions Patients were randomly assigned to receive TCZ, 8 mg/kg, intravenously plus usual care on day 1 and on day 3 if clinically indicated (TCZ group) or to receive usual care alone (UC group). Usual care included antibiotic agents, antiviral agents, corticosteroids, vasopressor support, and anticoagulants.Main Outcomes and Measures Primary outcomes were scores higher than 5 on the World Health Organization 10-point Clinical Progression Scale (WHO-CPS) on day 4 and survival without need of ventilation (including noninvasive ventilation) at day 14. Secondary outcomes were clinical status assessed with the WHO-CPS scores at day 7 and day 14, overall survival, time to discharge, time to oxygen supply independency, biological factors such as C-reactive protein level, and adverse events.Results Of 131 patients, 64 patients were randomly assigned to the TCZ group and 67 to UC group; 1 patient in the TCZ group withdrew consent and was not included in the analysis. Of the 130 patients, 42 were women (32%), and median (interquartile range) age was 64 (57.1-74.3) years. In the TCZ group, 12 patients had a WHO-CPS score greater than 5 at day 4 vs 19 in the UC group (median posterior absolute risk difference [ARD] −9.0%; 90% credible interval [CrI], −21.0 to 3.1), with a posterior probability of negative ARD of 89.0% not achieving the 95% predefined efficacy threshold. At day 14, 12% (95% CI −28% to 4%) fewer patients needed noninvasive ventilation (NIV) or mechanical ventilation (MV) or died in the TCZ group than in the UC group (24% vs 36%, median posterior hazard ratio [HR] 0.58; 90% CrI, 0.33-1.00), with a posterior probability of HR less than 1 of 95.0%, achieving the predefined efficacy threshold. The HR for MV or death was 0.58 (90% CrI, 0.30 to 1.09). At day 28, 7 patients had died in the TCZ group and 8 in the UC group (adjusted HR, 0.92; 95% CI 0.33-2.53). Serious adverse events occurred in 20 (32%) patients in the TCZ group and 29 (43%) in the UC group (P = .21).Conclusions and Relevance In this randomized clinical trial of patients with COVID-19 and pneumonia requiring oxygen support but not admitted to the intensive care unit, TCZ did not reduce WHO-CPS scores lower than 5 at day 4 but might have reduced the risk of NIV, MV, or death by day 14. No difference on day 28 mortality was found. Further studies are necessary for confirming these preliminary results.Trial Registration ClinicalTrials.gov Identifier: NCT0433180