Synthesis of the calcilytic ligand NPS 2143

Johansson, Henrik; Cailly, Thomas; Thomsen, A.; Bräuner‐Osborne, Hans; Pedersen, Daniel Sejer

doi:10.3762/bjoc.9.154

Cited by 9 publications

(11 citation statements)

References 20 publications

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“…Importantly the inhibition of vascular reactivity by Calhex-231 and NPS 2143 occurs at concentrations previously shown to reduce CaSR-mediated responses in other cell and tissue types, with IC 50 values of ~0.4 µm reported for both of these compounds, and maximum inhibition of CaSR responses achieved between ~3–10 µm (Faure et al, 2009, Jensen and Bräuner-Osborne, 2007, Johansson et al, 2013, Nemeth et al, 2001, Petrel et al, 2004, Petrel et al, 2003, Saidak et al, 2009, Yamamura et al, 2015). …”

Section: Resultsmentioning

confidence: 87%

“…1 A (i), (ii) and B show that increasing [Ca 2+ ] o from 1 mM to 6 mM induced concentration-dependent vasorelaxations of methoxamine pre-contracted arteries compared to vehicle controls, with 6 mM [Ca 2+ ] o inducing almost complete relaxation. To confirm that [Ca 2+ ] o -induced vasorelaxations were mediated by stimulation of CaSRs, vessel segments were pre-treated with the calcilytics Calhex-231 and NPS 2143 at concentrations commonly used to study CaSR responses (Faure et al, 2009, Johansson et al, 2013, Nemeth et al, 2001, Petrel et al, 2004, Petrel et al, 2003, Saidak et al, 2009, Yamamura et al, 2015, Yamamura et al, 2013, Yamamura et al, 2012). Fig.…”

Section: Resultsmentioning

confidence: 99%

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The calcilytics Calhex-231 and NPS 2143 and the calcimimetic Calindol reduce vascular reactivity via inhibition of voltage-gated Ca2+ channels

Greenberg

Jahan

Shi

et al. 2016

European Journal of Pharmacology

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The present study investigates the effect of commonly used negative and positive allosteric modulators of the calcium-sensing receptor (CaSR) on vascular reactivity. In wire myography studies, increasing [Ca2+]o from 1 mM to 6 mM induced concentration-dependent relaxations of methoxamine-induced pre-contracted rabbit mesenteric arteries, with 6 mM [Ca2+]o producing almost complete relaxation. [Ca2+]o-induced relaxations were attenuated in the presence of the calcilytics Calhex-231 and NPS 2143, and abolished by the removal of the endothelium. In addition to their calcilytic effects, Calhex-231 and NPS 2143 also produced concentration-dependent inhibitions of methoxamine- or KCl-induced precontracted tone, which were unaffected by removal of the endothelium and unopposed in the presence of the calcimimetic Calindol. In vessels with depleted Ca2+ stores, contractions mediated by Ca2+ influx via voltage-gated Ca2+ channels (VGCCs) were inhibited by Calhex231. In freshly isolated single rabbit mesenteric artery smooth muscle cells, Calhex-231 and NPS 2143 inhibited whole-cell VGCC currents. Application of Calindol also inhibited methoxamine- and KCl-induced pre-contracted tone, and inhibited whole-cell VGCC currents. In conclusion, in addition to their CaSR-mediated actions in the vasculature, Calhex-231, NPS 2143 and Calindol reduce vascular contractility via direct inhibition of VGCCs.

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Section: Resultsmentioning

confidence: 87%

Section: Resultsmentioning

confidence: 99%

The calcilytics Calhex-231 and NPS 2143 and the calcimimetic Calindol reduce vascular reactivity via inhibition of voltage-gated Ca2+ channels

Greenberg

Jahan

Shi

et al. 2016

European Journal of Pharmacology

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“…Dulbecco's modified Eagle's medium, dialyzed FBS, a mixture of 10,000 U/ml penicillin and 10,000 mg/ml streptomycin, Dulbecco's PBS without Ca 21 and Mg 21 , Opti-MEM, Hanks' balanced salt solution (HBSS) without Ca 21 , Mg 21 and phenol red, Lipofectamine 2000, and pertussis toxin (PTX) were obtained from Thermo Fisher Scientific (Waltham, MA). The allosteric modulator N-(3-[2-chlorophenyl]propyl)-(R)-a-methyl-3-methoxybenzylamine (NPS R-568) hydrochloride was purchased from Tocris Bioscience (Bristol, UK) and 2-chloro-6-[(2R)-2-hydroxy-3-[(2-methyl-1-naphthalen-2-ylpropan-2-yl)amino]propoxy]benzonitrile (NPS 2143) hydrochloride was synthesized in-house as previously published (Johansson et al, 2013). The selective G q/11 inhibitor YM-254890 (CAS Number 568580-02-9) was purchased from Wako Chemicals GmbH (Neuss, Germany).…”

Section: Methodsmentioning

confidence: 99%

Calcium-Sensing Receptor Internalization Is β-Arrestin–Dependent and Modulated by Allosteric Ligands

Mos

Jacobsen

Foster

et al. 2019

Molecular Pharmacology

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G protein-coupled receptor (GPCR) internalization is crucial for the termination of GPCR activity, and in some cases is associated with G protein-independent signaling and endosomal receptor signaling. To date, internalization has been studied in great detail for class A GPCRs; whereas it is not well established to what extent the observations can be generalized to class C GPCRs, including the extracellular calcium-sensing receptor (CaSR). The CaSR is a prototypical class C GPCR that maintains stable blood calcium (Ca 21) levels by sensing minute changes in extracellular free Ca 21. It is thus necessary that the activity of the CaSR is tightly regulated, even while continuously being exposed to its endogenous agonist. Previous studies have used overexpression of intracellular proteins involved in GPCR trafficking, pathway inhibitors, and cell-surface expression or functional desensitization as indirect measures to investigate CaSR internalization. However, there is no general consensus on the processes involved, and the mechanism of CaSR internalization remains poorly understood. The current study provides new insights into the internalization mechanism of the CaSR. We have used a state-of-the-art time-resolved fluorescence resonance energy transfer-based internalization assay to directly measure CaSR internalization in real-time. We demonstrate that the CaSR displays both constitutive and concentration-dependent Ca 21-mediated internalization. For the first time, we conclusively show that CaSR internalization is sensitive to immediate positive and negative modulation by the CaSR-specific allosteric modulators N-(3-[2-chlorophenyl]propyl)-(R)-a-methyl-3-methoxybenzylamine (NPS R-568) and 2-chloro-6-[(2R)-2hydroxy-3-[(2-methyl-1-naphthalen-2-ylpropan-2-yl)amino] propoxy]benzonitrile (NPS 2143), respectively. In addition, we provide compelling evidence that CaSR internalization is b-arrestin-dependent while interestingly being largely independent of G q/11 and G i/o protein signaling. SIGNIFICANCE STATEMENT A novel highly efficient cell-based real-time internalization assay to show that calcium-sensing receptor (CaSR) internalization is b-arrestin-dependent and sensitive to modulation by allosteric ligands.

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“…NPS R-568 hydrochloride was purchased from Tocris Bioscience (Bristol, UK), while NPS 2143 hydrochloride was synthesized as previously published50 and kindly provided by Dr. Daniel Sejer Pedersen, University of Copenhagen, DK. All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated.…”

Section: Methodsmentioning

confidence: 99%

Investigating the molecular mechanism of positive and negative allosteric modulators in the calcium-sensing receptor dimer

Jacobsen

Gether

Bräuner‐Osborne

2017

Sci Rep

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Allosteric modulators that are targeting the calcium-sensing receptor (CaSR) hold great therapeutic potential, and elucidating the molecular basis for modulation would thus benefit the development of novel therapeutics. In the present study, we aimed at investigating the mechanism of allosteric modulation in CaSR by testing dimers carrying mutations in the allosteric site of one or both of the subunits. To ensure measurements on a well-defined dimer composition, we applied a trans-activation system in which only the specific heterodimer of two loss-of-function mutants responded to agonist. Although one of these mutants was potentiated by a positive allosteric modulator, we showed that receptor activity was further potentiated in a trans-activation heterodimer containing a single allosteric site, however only when the allosteric site was located in the subunit responsible for G protein coupling. On the contrary, preventing activation in both subunits was necessary for obtaining full inhibition by a negative allosteric modulator. These findings correlate with the proposed activation mechanism of the metabotropic glutamate receptors (mGluRs), in which only a single transmembrane domain is activated at a time. CaSR and mGluRs belong to the class C G protein-coupled receptors, and our findings thus suggest that the activation mechanism is common to this subfamily.

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Synthesis of the calcilytic ligand NPS 2143

Cited by 9 publications

References 20 publications

The calcilytics Calhex-231 and NPS 2143 and the calcimimetic Calindol reduce vascular reactivity via inhibition of voltage-gated Ca2+ channels

The calcilytics Calhex-231 and NPS 2143 and the calcimimetic Calindol reduce vascular reactivity via inhibition of voltage-gated Ca2+ channels

Calcium-Sensing Receptor Internalization Is β-Arrestin–Dependent and Modulated by Allosteric Ligands

Investigating the molecular mechanism of positive and negative allosteric modulators in the calcium-sensing receptor dimer

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