mGlu3 (negative allosteric modulator, noncompetitive antagonist, IC50 = 190 ± 26 nM, efficacy = 100 ± 0.68%); no activity (IC50 >12,500 nM for antagonist mode, >25,000 nM for agonist mode, >12,500 nM for potentiator mode) at mGlu1, mGlu2, mGlu4, mGlu5, mGlu7, mGlu8, and GABAB receptors. [1]

qPCR in real time[2]

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In mixed rat cortical cultures challenged with Aβ(25-35), LY2389575 (0.1 to 1 μM) abolished the neuroprotective activity of the dual mGlu2/3 agonist LY379268 (1 μM).
At the highest concentration (1 μM), LY2389575 alone amplified Aβ(25-35)-induced neurotoxicity, increasing neuronal death beyond that caused by Aβ alone.
LY2389575 (1 μM) did not affect neuronal viability in the absence of Aβ.
In murine mixed cortical cultures with wild-type astrocytes, LY2389575 amplified Aβ toxicity and abolished the neuroprotective activity of LY379268; these effects were absent when astrocytes lacked mGlu3 receptors.
In the presence of LY2389575, the dual agonist LY379268 became neurotoxic, unmasking a toxic activity mediated by mGlu2 receptor activation.
In pure neuronal cultures, LY2389575 prevented the protective effect of glial conditioned medium collected from LY379268-treated astrocytes against Aβ toxicity. [1]

LY2389575 was characterized using fluorometric imaging plate reader (FLIPR) assays in AV-12 cell lines stably expressing human mGlu1, mGlu2, mGlu3, mGlu4, mGlu5, mGlu7, mGlu8, or GABAB receptors, along with the Ga15 subunit (except mGlu1 and mGlu5 which did not require Ga15). Cells were cultured in DMEM with high glucose and pyridoxine hydrochloride, supplemented with 5% heat-inactivated dialyzed fetal bovine serum, 1 mM sodium pyruvate, 10 mM HEPES, 1 mM L-glutamine, and selection agents. Cells were harvested 18-24 h before assay and seeded into 96-well black-walled poly-D-lysine-coated plates at 115,000 cells per well for mGlu3. Cells were incubated with 8 μM Fluo-3AM (50 μl/well) for 90-120 min at 25°C. Dye solution was replaced with assay buffer (Hanks' balanced salt solution + 20 mM HEPES). A single-addition FLIPR assay generated a 10-point concentration-response curve for glutamate to calculate EC90 and EC10 values. LY2389575 was diluted in DMSO using 3-fold dilution series and tested in a two-addition FLIPR assay with a 10-point concentration response profile (starting at 25 μM for agonist assay, 12.5 μM for potentiator/antagonist assays; final DMSO 0.625%). Compound was added first, data collected every second for 30 s then every 3 s for 90 s to detect agonist activity, followed immediately by addition of EC90 or EC10 glutamate. Data were collected every second for 29 images then every 3 s for 15 images. Maximal response defined by 100 μM glutamate. Compound effect measured as maximal-minimal peak heights in relative fluorescent units corrected for basal fluorescence. Agonist effects quantified as % of maximal glutamate response; antagonist effects as % inhibition of EC90 glutamate response; potentiation effects as % increase of EC10 glutamate response relative to ECmax. Data fitted with four-parameter logistic curve. To determine competitive or allosteric nature, 12-point glutamate dose-response curves were generated in antagonist mode in absence and presence of increasing LY2389575 concentrations. LY2389575 behaved as a noncompetitive antagonist of mGlu3 receptors. [1]

Real Time qPCR[2]
Cell Types: Microglial cells
Tested Concentrations: 5 μM
Incubation Duration: 1 h
Experimental Results: Increased levels of Mrc1. LY2389575 Hydrochide (5 μM; 1 h) induces increased levels of Mrc1 in small astrocytes[2] . .

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Mixed cortical cultures from rat embryos: cells dissociated from cortices grown in DMEM/F12 (1:1) with 10% horse serum, 10% fetal calf serum, 2 mM glutamine, 6 mg/ml glucose for 7-10 days, then glial division halted by 10 μM cytosine-D-arabinoside for 3 days, shifted to serum-free medium. Mature cultures contained 35-40% neurons and 60-65% astrocytes. For Aβ toxicity experiments, cultures were challenged with 25 μM Aβ(25-35) or 100 nM Aβ(1-42) oligomers (>50 kDa) for 24 h in presence of 1 μM MK-801 and 30 μM DNQX to block ionotropic glutamate receptors. LY2389575 was added at concentrations 0.1-1 μM. Neuronal injury assessed by phase-contrast microscopy and trypan blue staining (dead neurons counted from three random microscopic fields per well).
Pure neuronal cultures from rat embryonic day 15: cortices dissected in Ca2+/Mg2+-free buffer, mechanically dissociated, plated on poly-D-lysine-coated 35-mm dishes at 2×10^6 cells/dish in DMEM/Ham's F12 (1:1) with BSA, insulin, transferrin, putrescine, progesterone, selenium, glutamine, glucose, penicillin/streptomycin, and 10 μM cytosine-D-arabinofuranoside for 3 days to prevent non-neuronal proliferation. Yields >99% pure neurons. Neuronal injury assessed by MTT assay (0.9 mg/ml MTT for 2 h at 37°C, then 20% SDS for 1 h, formazan measured at 560 nm).
Astrocyte pure cultures from 1-3 day old rats or wild-type/mGlu3(-/-) mice: cortical glial cells dissociated with 0.25% trypsin, plated in DMEM with 10% fetal calf serum, penicillin/streptomycin, 2 mM glutamine, shaken overnight to remove microglia/oligodendrocytes, replated and used after 6-8 days. For glial conditioned medium (GCM) experiments, astrocytes were treated with 1 μM LY379268 ± LY341495 or LY2389575 for 24 h, medium collected and transferred to pure neuronal cultures prior to Aβ challenge. TGF-β1 levels measured by ELISA, and TGF-β1 mRNA by real-time RT-PCR (primers: forward 5'-atagcctgagtggctgtct-3', reverse 5'-tgggactgatcaccattgatt-3', normalized to β-actin). Neutralizing anti-TGF-β1 antibody (2 μg/ml) or type-1 TGF-β receptor inhibitor SB431542 added to GCM. [1]

In mixed cortical cultures, LY2389575 at 1 μM did not affect neuronal viability in the absence of Aβ (number of dead neurons: 41±3 in control vs 42±1 in treated cultures, n=3 per condition). No other toxicity data reported. [1]

[1]. Targeting group II metabotropic glutamate (mGlu) receptors for the treatment of psychosis associated with Alzheimer's disease: selective activation of mGlu2 receptors amplifies beta-amyloid toxicity in cultured neurons, whereas dual activation of mGlu2 and mGlu3 receptors is neuroprotective. Mol Pharmacol. 2011 Mar;79(3):618-26.

[2]. mGlu3 receptor regulates microglial cell reactivity in neonatal rats. J Neuroinflammation. 2021 Jan 6;18(1):13.

[3]. Metabotropic Glutamate Receptors in Alzheimer's Disease Synaptic Dysfunction: Therapeutic Opportunities and Hope for the Future. J Alzheimers Dis. 2020;78(4):1345-1361.

LY2389575 is a selective negative allosteric modulator of mGlu3 receptors (CAS#: 885104-09-6). It was used to demonstrate that activation of glial mGlu3 receptors protects against Aβ neurotoxicity via a paracrine TGF-β1 mechanism, and that selective mGlu2 receptor activation (e.g., by the PAM LY566332) amplifies Aβ toxicity. The compound revealed that dual mGlu2/3 agonists like LY379268 become neurotoxic when mGlu3 receptors are blocked, unmasking mGlu2-mediated toxicity. These findings suggest that selective mGlu2 receptor PAMs may be harmful in Alzheimer's disease, while dual mGlu2/3 agonists could be beneficial for treating psychosis associated with Alzheimer's disease (PAD). [1]

C15H16BRCL3N4

438.57

435.962

885104-09-6

885102-71-6;885104-09-6 (HCl);

11503054

White to light yellow solid powder

2

4

4

23

354

1

C1CN(C[C@H]1NCC2=C(C=C(C=C2)Cl)Cl)C3=NC=C(C=N3)Br.Cl

IRIBLLOEUZMCQY-ZOWNYOTGSA-N

InChI=1S/C15H15BrCl2N4.ClH/c16-11-7-20-15(21-8-11)22-4-3-13(9-22)19-6-10-1-2-12(17)5-14(10)18;/h1-2,5,7-8,13,19H,3-4,6,9H2;1H/t13-;/m0./s1

(3S)-1-(5-bromopyrimidin-2-yl)-N-[(2,4-dichlorophenyl)methyl]pyrrolidin-3-amine;hydrochloride

LY 2389575 LY2389575 LY-2389575

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO : ~100 mg/mL (~228.01 mM)
MEthanol : ~8.33 mg/mL (~18.99 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.70 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.70 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.70 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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