mGluR1/5

NPS2390 decreases proliferation and reverses phenotypic modulation via inhibiting autophagy in PASMCs under hypoxia [2]
To identify whether the function of CaSR is involved with the regulation of autophagy in HPASMCs proliferation and phenotypic modulation under hypoxia, HPASMCs were treated with NPS2390 (10 μM, CaSR inhibitor) for 24 h under hypoxic or normoxic conditions. Our results showed that the addition of NPS2390 reduced the proliferation and reversed phenotypic modulation in HPASMCs under hypoxia (Figure 6A and B). Meanwhile, the expression of LC3-II was also decreased after treating with NPS2390 (Figure 6C). The results indicate that the inhibition of CaSR suppressed the autophagy of HPASMCs under hypoxia (Figure 6D).

---

Changes in [Ca2+]i in different groups [2]
We found that hypoxia markedly enhanced [Ca2+]i, and the addition of R568 (30 μM) raised a cooperativity as the [Ca2+]i further increases in the hypoxia + R568 group (P < 0.05) when compared with the hypoxia group. But NPS2390 can suppress these changes (Figure 7).

---

NPS2390 inhibits PI3K PI3K/Akt/mTOR pathways in HPASMCs under hypoxia [2]
Next, we intend to identify whether the decreasing level of autophagy activated by NPS2390 was attributed to the change in PI3K/Akt/mTOR pathways. After the addition of NPS2390 for 24 h under hypoxia, the expressions of p-PI3K, p-Akt, and p-mTOR were increased (Figure 8A and B). To further verify whether the function of NPS2390 is dependent on the PI3K/Akt pathways, the PI3K inhibitor LY294002 (10 μM) was added together with the CaSR agonist R568 into HPASMCs under hypoxia. LY294002 suppressed the activation of Akt and its downstream mTOR, when compared with the R568 treatment hypoxia group. Furthermore, the function of NPS2390 on autophagy-associated protein was verified by western blot. The expression of LC3-II was upregulated by the addition of R568 at hypoxia for 24 h when compared with the hypoxic group without any treatment (Figure 8C and D). The LY294002 treatment obviously controls the expression of LC3-II when compared to the R568 treatment hypoxia group (Figure 8E). These results demonstrated that the PI3K/Akt signaling pathway targets autophagy activation that is dependent on mTOR inhibition, which may be related to promote the suppression of HPASMC proliferation by NPS2390.

Traumatic brain injury (TBI) initiates a complex cascade of neurochemical and signaling changes that leads to neuronal apoptosis, which contributes to poor outcomes for patients with TBI. Previous study indicates that calcium-sensing receptor (CaSR) activation contributes to neuron death in focal cerebral ischemia-reperfusion mice, however, its role in neuronal apoptosis after TBI is not well-established. Using a controlled cortical impact model in rats, the present study was designed to determine the effect of CaSR inhibitor NPS2390 upon neuronal apoptosis after TBI. Rats were randomly distributed into three groups undergoing the sham surgery or TBI procedure, and NPS2390 (1.5 mg/kg) was infused subcutaneously at 30 min and 120 min after TBI. All rats were sacrificed at 24 h after TBI. Our data indicated that NPS2390 significantly reduced the brain edema and improved the neurological function after TBI. In addition, NPS2390 decreased caspase-3 levels and the number of apoptotic neurons. Furthermore, NPS2390 up-regulated anti-apoptotic protein Bcl-2 expression and down-regulated pro-apoptotic protein Bax, and reduced subsequent release of cytochrome c into the cytosol. In summary, this study indicated that inhibition of CaSR by NPS2390 attenuates neuronal apoptosis after TBI, in part, through modulating intrinsic apoptotic pathway. [3]

---

NPS2390 alleviated brain edema and improved the neurological function after TBI [3]
To confirm the protective effects of NPS2390 at the macroscopic level, we measured brain water content and neurological functional deficits. As shown in Fig. 1B, the TBI groups had a significantly higher brain water content (P < 0.05) when compared with the sham group. However, groups treated with melatonin had significantly less brain water content than the TBI group (P < 0.05). In addition, CCI resulted in neurological functional deficits. The mNSS scores were significantly higher in the TBI group than in the sham group (P < 0.05, Fig. 1C). NPS2390 administration significantly decreased neurological deficits 24 h after TBI (P < 0.05, Fig. 1C) as shown in Fig. 1C.

---

NPS2390 decreased the number of apoptotic neurons [3]
A barely detectable number of TUNEL-positive neurons were found in the cortex of left hemisphere in the sham group (Fig. 2). In the group of rats subjected to TBI, there were an increased number of TUNEL and NeuN double-staining cells when compared with the sham group (P < 0.01, Fig. 2). Importantly, NPS2390 treatment significantly attenuated neuronal apoptosis 24 h post-TBI (P < 0.05, Fig. 2).

---

NPS2390 up-regulated the expression of Bcl-2 and reduced expression of cleaved caspase-1 and bax [3]
To confirm the role of NPS2390 in neuronal apoptosis, the protein level of cleaved caspase-3 was measured by Western blot. The protein level of cleaved caspase-3 was up-regulated in the TBI group when compared with the sham group, while NPS2390 treatment markedly diminished cleaved caspase-3 expression after TBI (P < 0.05, Fig. 3A and B). To investigate the role of NPS2390 in intrinsic apoptotic pathway, the protein levels of Bcl-2 and Bax were measured by Western blot. The protein levels of Bcl-2 were significantly decreased in TBI + vehicle group (P < 0.05, Fig. 3A and C), whereas NPS2390 administration increased the protein expression of Bcl-2 (P < 0.05, Fig. 3A and C). In addition, the protein expression levels of Bax were significantly increased 24 h following TBI in the TBI + vehicle group relative to sham rats (P < 0.05, Fig. 3A and D). Bax protein levels were markedly reduced by minocycline injection compared with vehicle rats (P < 0.05, Fig. 3A and D).

---

NPS2390 decreased the release of cytochrome c into the cytosol [3]
The up-regulation of Bax and down-regulation of Bcl-2 induce the increased permeability of the outer mitochondrial membrane and subsequent cytochrome c release. levels of cytochrome c in the mitochondria and cytosol were significantly decreased and increased, respectively, after TBI induction compared with the sham group (P < 0.05, Fig. 4A and B). NPS2390 administration inhibited the release of cytochrome c into the cytosol compared with the TBI + vehicle group.

Hypoxia treatment of HPASMCs [2]
The original medium of HPASMCs was discarded and 1% fetal bovine serum was added to DMEM. The culture was incubated for 12 h for synchronization and then changed to SMCM culture. The cells were placed in a hypoxic incubator and treated at 1% O2 concentration.

---

BrdU incorporation and detection [2]
BrdU was incorporated into PASMCs, and proliferation of different treatment groups was detected using immunofluorescence technique. Cells from different treatment groups were cultured in 96-well plates for 44 h, and then BrdU was added to these cells and continued culturing for 4 h at 38.5℃. Subsequently, the cells were fixed with 4% paraformaldehyde for 20 min, washed for 3 times with phosphate buffered saline with Tween-20 (PBST) at a concentration of 2 mol/L HCl, 37 ºC and denatured for 30 min. After adding 0.1 mol/L of sodium tetraborate, the cells were kept for 5 min and washed with PBST for 3 times; then the cells were blocked with ready-to-use goat serum for 20 min at room temperature. After adding BrdU primary antibody G3G4 (1:200), the cells were incubated overnight at 4 ºC and washed for 3 times with PBST. TRIFC-labeled goat anti-mouse secondary antibody (1:500) was added to the cells and incubated for 1 h at 37ºC and washed for 3 times with PBST after incubation. Finally, DAPI was added and the cells were kept at room temperature for 30 min, after which the cells were washed for 3 times with PBST. The BrdU incorporation index was calculated by BrdU-labeled nuclei and DAPI-stained nuclei ratio under I×70 fluorescence microscopy.

---

Cell cycle assays [2]
For cell cycle analysis, the PASMCs to be tested were washed with a phosphate buffered saline solution, placed in a centrifuge tube, and centrifuged at 1,000 rpm for 5 min. The supernatant was discarded, and the cells were resuspended in 80% ethanol at 4 ºC. The cells were centrifuged again for 24 min at 1,000 rpm for 24 min. The fixative was discarded, and the cells were resuspended in propidium iodide. Cell cycle analysis was performed with a Cy-toFLEX S flow cytometer after 30 min at room temperature.

---

Western blot [2]
The cells were collected and 300 μL of protein lysate was added. The cells were placed on ice for 15 min and then centrifuged at 15 ºC/12,000 rpm for 15 min. The supernatant was collected. After adjusting the concentration, the protein was boiled in boiling water for 10 min. SDS-PAGE gels were prepared and filled with 40 μg of sample per well for electrophoresis. After 300-mA wet rotation for 120 min, the PVDF membrane was blocked at room temperature for 1 h and then added to the antibody dilution of CaSR (1:800), calponin (1:500), SMA-α (1:1000), OPN (1:500), PCNA (1:1000), Ki67(1:1000), LC3 (1:1000), caspase-3(1:1000), AKT (1:1000), p-AKT (1:1000), PI3K (1:1000), p-PI3K (1:1000), mTOR (1:1000), p-mTOR (1:1000), and GAPDH (1:1000) at 4℃ overnight. The gel was wash with TBST for 3 times, each time for 10 min, and then horseradish-labeled goat anti-rabbit secondary antibody (1: 50,000) was added, The gel was incubate for 1 h at room temperature and washed with TBST for 3 times; each protein expression was detected by ECL chemiluminescence at 10 min. The resulting protein strip image, using Quality one software to analyze the image to GAPDH light density. The degree value is used as an internal parameter to correct the optical density value of the target protein.

---

Measurement of intracellular calcium in HPASMCs [2]
Fluo-4 AM calcium ion fluorescent probe was used to detect the intracellular calcium ion density in each group. The cell density was 2 × 105/mL. The laser confocal small dish was inserted. After treating the cells according to the above group, 10 μmol/L of Fluo-4 AM was added according to the instructions. The cells were then incubated in the dark for 30 min, after which they were rinsed in PBS with calcium-free buffer repeatedly, centrifuged for 2 times, and observed under laser confocal microscope.

Animals grouping and drug administration [3]
In total, 51 rats were used in this study. The rats were randomly assigned to three groups: the sham group, the TBI + vehicle group, the TBI + NPS2390 group. NPS2390(1.5 mg/kg) was infused subcutaneously at 30 min and 120 min after TBI. The SAH + vehicle group was subjected to SAH and treated with vehicle. The dose of NPS 2390 and the time point were chosen based on a previous study.

---

Evaluation of neurological deficits [3]
The modified neurological severity scores (mNSS) was used to examine the effects of NPS2390 on the neurological deficits of animals after TBI. The neurological functions of each group were blindly evaluated at 24 h after TBI using motor, sensory, reflex, and balance tests. The total score ranged from 0 to 18, and the higher scores indicated decreases in function.

[1]. Positive and negative modulation of group I metabotropic glutamate receptors. J Med Chem. 2008 Feb 14;51(3):634-47.

[2]. NPS2390, a Selective Calcium-sensing Receptor Antagonist Controls the Phenotypic Modulation of Hypoxic Human Pulmonary Arterial Smooth Muscle Cells by Regulating Autophagy. J Transl Int Med. 2019 Jul 11;7(2):59-68.

[3]. Calcium-sensing receptor antagonist NPS2390 attenuates neuronal apoptosis though intrinsic pathway following traumatic brain injury in rats. Biochem Biophys Res Commun. 2017 Apr 29;486(2):589-594.

A discriminating pharmacophore model for noncompetitive metabotropic glutamate receptor antagonists of subtype 1 (mGluR1) was developed that facilitated the discovery of moderately active mGluR1 antagonists. One scaffold was selected for the design of several focused libraries where different substitution patterns were introduced. This approach facilitated the discovery of potent mGluR1 antagonists, as well as positive and negative mGluR5 modulators, because both receptor subtypes share similar binding pockets. For mGluR1 antagonists, a homology model of the mGlu1 receptor was established, and a putative binding mode within the receptor's transmembrane domain was visualized. [1]

---

Background and objectives: Calcium-sensing receptor (CaSR) is known to regulate hypoxia-induced pulmonary hypertension (HPH) and vascular remodeling via the phenotypic modulation of pulmonary arterial smooth muscle cells (PASMCs) in small pulmonary arteries. Moreover, autophagy is an essential modulator of VSMC phenotype. But it is not clear whether CaSR can regulate autophagy involving the phenotypic modulation under hypoxia.
Methods: The viability of human PASMCs was detected by cell cycle and BrdU. The expressions of proliferation protein, phenotypic marker protein, and autophagy protein in human PASMCs were determined by western blot.
Results: Our results showed that hypoxia-induced autophagy was considerable at 24 h. The addition of NPS2390 decreased the expression of autophagy protein and synthetic phenotype marker protein osteopontin and increased the expression of contractile phenotype marker protein SMA-ɑ and calponin via suppressing downstream PI3K/Akt/mTOR signal pathways.
Conclusions: Our study demonstrates that treatment of NPS2390 was conducive to inhibit the proliferation and reverse phenotypic modulation of PASMCs by regulating autophagy levels. [2]

---

The present study provides the evidence that CaSR antagonist NPS2390 exert protective effects against the TBI-induced brain injury. First, NPS2390 alleviated brain edema and improved neurological function. Second, NPS2390 down-regulated the protein levels of caspase-3 and reduced the number of TUNEL-positive neurons. In addition, NPS2390 up-regulated the anti-apoptotic protein Bcl-2 and down-regulated the pro-apoptotic protein Bax, and reduced the subsequent release of cytochrome c, indicating the inhibitory effect of NPS2390 on intrinsic apoptotic pathway.
In this study, neurological deficits were evaluated by mNSS and brain edema were determined by the wet weight/dry weight method. Neuronal apoptosis was measured by western blot of cleaved caspase-3 and double immunofluorescence staining of NeuN and TUNEL. Our results showed that TBI could significantly induce neurological dysfunction and severe brain edema, and obvious neuronal apoptosis. These changes were consistent with previous studies. These changes were improved by NPS2390. These findings suggest that CaSR activation is closely related to neuronal apoptosis and may contributed to the development of brain injury after TBI.[3]

---

There are several limitations to our study. First, this study only reveals the neuroprotective effects of NPS2390 at early time point and further work needs to be performed to evaluate the long-term effects of NPS2390 after TBI. In addition, the therapeutic time window, the optimal dosage, and other drug administration routes of NPS2390 treatment in TBI need to be addressed. Finally, the mechanisms by which CaSR regulates intrinsic apoptotic pathway in TBI remain to be explored. In conclusion, this study demonstrated the CaSR antagonist NPS2390 attenuates cortical neuronal apoptosis, alleviates brain edema and improves the neurological function following TBI in rats. The intrinsic apoptotic pathway may involve in the neuroprotective effects of NPS2390.

C19H21N3O

307.389544248581

307.168

C, 74.24; H, 6.89; N, 13.67; O, 5.20

226878-01-9

7067728

White to light yellow solid powder

1.3±0.1 g/cm3

542.4±30.0 °C at 760 mmHg

281.9±24.6 °C

0.0±1.4 mmHg at 25°C

1.658

3.65

1

3

2

23

450

0

O=C(C1C=NC2C=CC=CC=2N=1)NC12CC3CC(CC(C3)C1)C2

ZKFVOZCCAXQXBU-UHFFFAOYSA-N

InChI=1S/C19H21N3O/c23-18(17-11-20-15-3-1-2-4-16(15)21-17)22-19-8-12-5-13(9-19)7-14(6-12)10-19/h1-4,11-14H,5-10H2,(H,22,23)

N-(1-adamantyl)quinoxaline-2-carboxamide

NPS-2390; NPS 2390; NPS2390

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~6.25 mg/mL (~20.33 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

 (Please use freshly prepared in vivo formulations for optimal results.)