Mechanismen der agonist-selektiven Phosphorylierung und Dephosphorylierung von Somatostatin-Rezeptoren

Die Familie der G-Protein-gekoppelten Rezeptoren (GPCRs) stellt die größte Gruppe der Signalproteine dar und zählt zu den wichtigsten Zielstrukturen in der heutigen Arzneimittelwelt. Die Entwicklung neuer Medikament gegen GPCRs ist getrieben von der Suche nach Verbindungen welche nanomolare und subnanomolare Bindungsaffinitäten aufweisen. Immer mehr deutet darauf hin, dass für viele GPCRs mehrere aktive Konformationen existieren und somit selektiv spezifische Signalwege aktiviert werden können. Die Desensitisierung des GPCR Signals ist essentiell für die Aufrechterhaltung der zellulären Homöostase. Viele GPCRs werden agonist-abhängig über die Phosphorylierung von intrazellulären Serin- und Threoninresten im C-Terminus des Rezeptors koordiniert. Diese Phosphorylierung erleichtert die Bindung von β-Arrestin und führt zur Desensitisierung der G-Protein-abhängigen Signaltransduktion. β-Arrestin dient darüber hinaus als Scaffold- und Adapterprotein und ist somit in der Lage die Rezeptor-Internalisierung zu erleichtern und eine zweite Welle der Signaltransduktion auszulösen. Die Entwicklung neuer Pan-Somatostatin-Analoga war bisher darauf gerichtet, Substanzen zu entwickeln, die möglichst hohe Affinitäten zu mehr als einem der fünf Somatostatin-Rezeptoren haben (sst1-sst5). Die für den klinischen Einsatz neu entwickelten Somatostain-Analoga Pasireotid (SOM230), Octreotid und Somatoprim (DG3173) sind Multirezeptor-Liganden und wurden bis jetzt nur durch ihr Bindungsprofil charakterisiert. Ihre Fähigkeit einzelne Somatostatin-Rezeptorsubtypen zu aktivieren wurde bis jetzt noch nicht direkt untersucht. Um die Rezeptoraktivierung der einzelnen Somatostatin-Rezeptoren messen zu können wurde der C-Terminus des sst2-Rezeptors mit dem von anderen Rezeptor-Subtypen ausgetauscht. Dieses Vorgehen erlaubte die Verwendung der phospho-sst2 Antikörper zur Messung der sst5- und sst3-Aktivierung

Epigenetic-Like Stimulation of Receptor Expression in SSTR2 Transfected HEK293 Cells as a New Therapeutic Strategy

Simple Summary Neuroendocrine tumors (NETs) expressing the somatostatin receptor subtype 2 (SSTR2) are promising targets for peptide receptor radionuclide therapy (PRRT) using the somatostatin analogue Lu-177-DOTATATE. Patients expressing low levels of SSTR2 do not benefit from PRRT. Therefore, an approach to increase the efficacy of PRRT utilizing the effects of 5-aza-2′-deoxycytidine (5-aza-dC) and valproic acid (VPA) on the SSTR2 expression levels is investigated. The cell lines HEKsst 2 and PC3 are incubated with 5-aza-dC and VPA in different combinations. The drug pretreatment of HEKsst 2 cells leads to increased Lu-177-DOTATATE uptake values (factor 28) and lower cell survival (factor 4) in comparison to unstimulated cells; in PC3 cells, the effects are negligible. Further, for the stimulated cell types, the maintenance of the intrinsic radiosensitivity in each cell type is confirmed by X-ray irradiation. The increased SSTR2 expression induced by VPA and 5-aza-dC stimulation in HEKsst 2 cells might improve treatment strategies for patients with NETs. Abstract The aim of the study was to increase the uptake of the SSTR2-targeted radioligand Lu-177-DOTATATE using the DNA methyltransferase inhibitor (DNMTi) 5-aza-2′-deoxycytidine (5-aza-dC) and the histone deacetylase inhibitor (HDACi) valproic acid (VPA). The HEKsst 2 and PC3 cells were incubated with variable concentrations of 5-aza-dC and VPA to investigate the uptake of Lu-177-DOTATATE. Cell survival, subsequent to external X-rays (0.6 or 1.2 Gy) and a 24 h incubation with 57.5 or 136 kBq/mL Lu-177-DOTATATE, was investigated via colony formation assay to examine the effect of the epidrugs. In the case of stimulated HEKsst 2 cells, the uptake of Lu-177-DOTATATE increased by a factor of 28 in comparison to the unstimulated cells. Further, stimulated HEKsst 2 cells demonstrated lower survival fractions (factor 4). The survival fractions of the PC3 cells remained almost unchanged. VPA and 5-aza-dC did not induce changes to the intrinsic radiosensitivity of the cells after X-ray irradiation. Clear stimulatory effects on HEKsst 2 cells were demonstrated by increased cell uptake of the radioligand and enhanced SST2 receptor quantity. In conclusion, the investigated approach is suitable to stimulate the somatostatin receptor expression and thus the uptake of Lu-177-DOTATATE, enabling a more efficient treatment for patients with poor response to peptide radionuclide therapy (PRRT)

Critical assessment of G protein-biased agonism at the µ opioid receptor

G protein-biased agonists of the µ-opioid receptor have been proposed to be an improved class of opioid analgesics. Recent studies have been unable to reproduce the original experiments in the β-arrestin2 knockout mouse that led to this proposal, and alternative genetic models do not support the G protein-biased MOPr agonist hypothesis. Further, assessment of putatively biased ligands has been confounded by several factors, including assay amplification. As such, the extent to which current lead compounds represent mechanistically novel, extremely G protein-biased agonists is in question, as is the underlying assumption that β-arrestin2 mediates deleterious opioid effects. Addressing these current challenges represents a pressing issue in order to successfully advance drug development at this receptor and improve upon current opioid analgesics

Low intrinsic efficacy for G protein activation can explain the improved side-effect profile of new opioid agonists

Biased agonism at G protein–coupled receptors describes the phenomenon whereby some drugs can activate some downstream signaling activities to the relative exclusion of others. Descriptions of biased agonism focusing on the differential engagement of G proteins versus β-arrestins are commonly limited by the small response windows obtained in pathways that are not amplified or are less effectively coupled to receptor engagement, such as β-arrestin recruitment. At the μ-opioid receptor (MOR), G protein–biased ligands have been proposed to induce less constipation and respiratory depressant side effects than opioids commonly used to treat pain. However, it is unclear whether these improved safety profiles are due to a reduction in β-arrestin–mediated signaling or, alternatively, to their low intrinsic efficacy in all signaling pathways. Here, we systematically evaluated the most recent and promising MOR-biased ligands and assessed their pharmacological profile against existing opioid analgesics in assays not confounded by limited signal windows. We found that oliceridine, PZM21, and SR-17018 had low intrinsic efficacy. We also demonstrated a strong correlation between measures of efficacy for receptor activation, G protein coupling, and β-arrestin recruitment for all tested ligands. By measuring the antinociceptive and respiratory depressant effects of these ligands, we showed that the low intrinsic efficacy of opioid ligands can explain an improved side effect profile. Our results suggest a possible alternative mechanism underlying the improved therapeutic windows described for new opioid ligands, which should be taken into account for future descriptions of ligand action at this important therapeutic target

Susceptibility of optimal train schedules to stochastic disturbances of process times

This work focuses on the stochastic evaluation of train schedules computed by a microscopic scheduler of railway operations based on deterministic information. The research question is to assess the degree of sensitivity of various rescheduling algorithms to variations in process times (running and dwell times). In fact, the objective of railway traffic management is to reduce delay propagation and to increase disturbance robustness of train schedules at a network scale. We present a quantitative study of traffic disturbances and their effects on the schedules computed by simple and advanced rescheduling algorithms. Computational results are based on a complex and densely occupied Dutch railway area; train delays are computed based on accepted statistical distributions, and dwell and running times of trains are subject to additional stochastic variations. From the results obtained on a real case study, an advanced branch and bound algorithm, on average, outperforms a First In First Out scheduling rule both in deterministic and stochastic traffic scenarios. However, the characteristic of the stochastic processes and the way a stochastic instance is handled turn out to have a serious impact on the scheduler performance

Pregnane-X-receptor mediates the anti-inflammatory activities of rifaximin on detoxification pathways in intestinal epithelial cells

International audienceThe pregnane-X-receptor (PXR) is master gene overseeing detoxification of wide number of xenobiotics and is critical for maintenance of intestinal integrity. The intestinal expression of genes involved in cellular detoxification is down-regulated in patients with inflammatory bowel diseases (IBD). Rifaximin, is a non absorbable antibiotic endowed with a PXR agonistic activity. In the present study we have investigated whether rifaximin activates PXR in primary human colon epithelial cells and human colon biopsies and assessed whether this antibiotic antagonizes the effect of Tumor necrosis factor (TNF)-α on expression of PXR and PXR-related genes. Present results demonstrate that primary colon epithelial cells express PXR and that their exposure to rifaximin induces the expression of genes involved in cellular detoxification. Exposure to TNFα reduces the expression of PXR mRNA as well as expression of its target genes. This inhibitory effect was prevented by that co-treatment with rifaximin. Knocking down the expression of PXR in colon epithelial cells by an anti-PXR siRNA, abrogated the counter-regulatory effects exerted by rifaximin on cell exposed to TNFα. Finally, exposure of colon biopsies obtained from ulcerative colitis patients to rifaximin increased the expression of genes involved in xenobiotics metabolism. In aggregate, these data illustrate that rifaximin increases the expression of PXR and PXR-regulated genes involved in the metabolism and excretion of xenobiotics and antagonized the effects of TNFα in intertsinal epithelial cells and colon biopsies. These non-antibiotic effects of rifaximin could contribute to the maintenance of the intestinal barrier integrity against xenobiotics and products generated by luminal bacteria

Microglia modulate blood flow, neurovascular coupling, and hypoperfusion via purinergic actions

Microglia, the main immunocompetent cells of the brain, regulate neuronal function, but their contribution to cerebral blood flow (CBF) regulation has remained elusive. Here, we identify microglia as important modulators of CBF both under physiological conditions and during hypoperfusion. Microglia establish direct, dynamic purinergic contacts with cells in the neurovascular unit that shape CBF in both mice and humans. Surprisingly, the absence of microglia or blockade of microglial P2Y12 receptor (P2Y12R) substantially impairs neurovascular coupling in mice, which is reiterated by chemogenetically induced microglial dysfunction associated with impaired ATP sensitivity. Hypercapnia induces rapid microglial calcium changes, P2Y12R-mediated formation of perivascular phylopodia, and microglial adenosine production, while depletion of microglia reduces brain pH and impairs hypercapnia-induced vasodilation. Microglial actions modulate vascular cyclic GMP levels but are partially independent of nitric oxide. Finally, microglial dysfunction markedly impairs P2Y12R-mediated cerebrovascular adaptation to common carotid artery occlusion resulting in hypoperfusion. Thus, our data reveal a previously unrecognized role for microglia in CBF regulation, with broad implications for common neurological diseases

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

A comparison of Power Doppler with conventional sonographic imaging for the evaluation of renal artery stenosis

BACKGROUND: Power Doppler (PD) has improved diagnostic capabilities of vascular sonography, mainly because it is independent from the angle of insonation. We evaluated this technique in a prospective comparison with conventional imaging, consisting in Duplex and Color Doppler, for the evaluation of Renal Artery (RA) stenosis. METHODS: Sensitivity, specificity and predictive values of PD and conventional imaging were assessed in a blinded fashion on eighteen patients, 9 with angiographic evidence of unilateral RA stenosis (hypertensive patients) and 9 with angiographically normal arteries (control group). PD images were interpreted with an angiography-like criteria. RESULTS: In the control group both techniques allowed correct visualization of 16 out of the 18 normal arteries (93% specificity). Only in five hypertensive patients RA stenosis was correctly identified with conventional technique (56% sensitivity and 86% negative predictive value); PD was successful in all hypertensive patients (100% sensitivity and negative predictive value), since the operators could obtain in each case of RA stenosis a sharp color signal of the whole vessel with a clear "minus" at the point of narrowing of the lumen. All results were statistically significant (p < 0.01). CONCLUSIONS: This study demonstrates that PD is superior to conventional imaging, in terms of sensitivity and specificity, for the diagnosis of RA stenosis, because it allows a clear visualization of the whole stenotic vascular lumen. Especially if it is used in concert with the other sonographic techniques, PD can enable a more accurate imaging of renovascular disease with results that seem comparable to selective angiography