FTO (fat mass and obesity-associated protein, an RNA m6A demethylase) – Kd = 185 ± 77 nM; IC50 = 1.46 μM [1]

Compound 2 inhibited FTO demethylase activity with an IC50 of 1.46 μM (tested at concentrations ranging from 1 nM to 100 μM). [1]
Compound 2 bound directly to FTO protein with a dissociation constant Kd of 185 ± 77 nM as measured by microscale thermophoresis (MST) using unlabeled titrated compound 2 (0–10 μM) interacting with Alexa647-labeled His-tagged FTO (20 nM). [1]
In primary mouse midbrain dopamine neuron cultures, compound 2 promoted neuronal survival and protected against growth factor deprivation-induced apoptosis. At concentrations of 10 nM, 100 nM, and 1000 nM, compound 2 dose-dependently increased the number of TH-positive neurons. Statistically significant neuroprotective effects were observed at 100 nM and 1000 nM (p < 0.05, p < 0.01, p < 0.001, one-way ANOVA with Dunnett’s posthoc test). The effect was comparable to GDNF (3 nM, 100 ng/mL) positive control. No signs of toxicity were observed. [1]
Compound 2 also protected dopamine neurons from 6-OHDA-induced neuronal cell death (data not shown). [1]
In an artificial in vitro blood-brain barrier (BBB) model, compound 2 demonstrated good penetration ability with 31.7 ± 3.3% penetration (defined as the ratio of compound concentration in the 'brain' side well to the concentration in the insert). [1]

FTO demethylase inhibition assay: The enzymatic assay was modified from Huang et al. Experiments were conducted in reaction buffer (50 mM Tris-HCl, pH 7.5, 300 μM 2OG, 280 μM (NH4)2Fe(SO4)2, and 2 mM L-ascorbic acid). The reaction mixture contained 200 ng methylated N6-adenine RNA probe (5′-CUUGUCAm6ACAGCAGA-3′) and 10 nM FTO protein with different concentrations of ligands (1 nM to 100 μM). Reactions were incubated on a 96-well plate for 2 h at room temperature. The amount of m6A was measured using an m6A RNA methylation quantification colorimetric kit. The inhibitory effect was calculated as the enhancement of m6A amount compared to negative control (DMSO) relative to the difference between positive control (max inhibition) and negative control. [1]
Microscale thermophoresis (MST) binding assay: Recombinant human FTO protein was labeled through His-tag using a labeling kit. The labeled FTO protein (target) was used at 20 nM. Unlabeled titrated compound 2 (0–10 μM) was used. Measurements were done in buffer containing 10 mM Na-phosphate buffer pH 7.4, 1 mM MgCl2, 3 mM KCl, 150 mM NaCl, 0.05% Tween-20 using red LED source (power 100%) and medium MST power at 25°C. Data analysis was performed using MO.Affinity Analysis software. Each data point represents mean fraction bound values from n = 3 independent experiments per binding pair ± S.D. [1]
Molecular docking: The crystal structure of FTO in complex with 5-carboxy-8-hydroxyquinoline IOX1 (pdb:4IE4) was used. AutoDock 4.2 was used for docking studies. The active site was surrounded with a grid-box sized 80×80×80 points with spacing of 0.375 Å. Docking efficiency was calculated as DE = ΔG_dock / N, where N is the number of non-hydrogen atoms. [1]
Molecular dynamics simulation: Desmond simulation package was used. NPT ensemble with temperature 300 K and pressure 1 bar was applied. Five simulation runs of 10 ns each with relaxation time 1 ps were carried out. OPLS_2005 force field parameters were used. The simulation showed a very strong hydrogen bond between the pyridine nitrogen atom of compound 2 and the ammonium group of Arg96 residue of FTO. Important interactions included hydrogen bonds with residues Arg96, Glu234, Arg322, and Asp233, as well as additional hydrophobic interactions. [1]

Primary cultures of midbrain dopamine neurons: Midbrain floors were dissected from ventral mesencephalic tissue of 13-day-old NMRI strain mouse embryos. Tissues were incubated with 0.5% trypsin in HBSS for 20 min at 37°C, then mechanically dissociated. Cells were plated onto 96-well plates coated with poly-L-ornithine. Dopamine neurons were cultured for five days in Dulbecco's MEM/Nut mix F12 with N2 serum supplement, 33 mM D-glucose, 0.5 mM L-glutamine, and 100 μg/mL Primocin. On day 6, to deprive growth factors, cultures were washed three times with normal medium and compounds were applied. Cells were grown for five days with different concentrations of FTO inhibitors (10, 100, 1000 nM). Human recombinant GDNF (100 ng/mL) was used as positive control. After five days, cultures were fixed and stained with anti-tyrosine hydroxylase antibody. Images were acquired by high-content imaging equipment. Immunopositive neurons were counted using image analysis software. Results are expressed as percentage of cell survival compared to GDNF-maintained neurons. [1]

Blood-brain barrier penetration: In an artificial in vitro BBB model (murine endothelial cells bEnd3 co-cultured with murine HIFko astrocytes on hanging cell culture inserts), compound 2 (10 μM final concentration) was added to the insert ('blood' side). After 1 h incubation, samples were taken from the insert and the well ('brain' side). Penetration % was defined as the ratio of compound concentration in the well to the concentration in the insert. Compound 2 showed 31.7 ± 3.3% BBB penetration. Compound concentrations were analyzed using LC-MS with an electrospray ion source in positive ionization mode. [1]

The authors noted that in neuronal survival experiments, there were "no signs of toxicity of the tested compounds." [1]

[1]. Small-Molecule Inhibitors of the RNA M6A Demethylases FTO Potently Support the Survival of Dopamine Neurons. Int J Mol Sci. 2021 Apr 26;22(9):4537.

Compound 2 is 4-amino-8-chloroquinoline-3-carboxylic acid (purity >90%). It was identified through high-throughput virtual screening of the ZINC compound library using known FTO inhibitors as templates. The docking free energy ΔG was -7.70 kcal/mol with a docking efficiency of 0.51. This is the first demonstration that inhibitors of FTO can support the survival and protect dopamine neurons from growth factor deprivation-induced death in vitro. The compound has the potential to penetrate the blood-brain barrier, making it a candidate for systemic delivery in neurodegenerative disease treatment. [1]

C10H7CLN2O2

222.63

222.02

955328-22-0

17028176

Typically exists as solid at room temperature

1.551g/cm3

440.5ºC at 760 mmHg

220.2ºC

1.746

2.749

2

4

1

15

262

0

C1=CC2=C(C(=CN=C2C(=C1)Cl)C(=O)O)N

DMQKTDVVSMSBCL-UHFFFAOYSA-N

InChI=1S/C10H7ClN2O2/c11-7-3-1-2-5-8(12)6(10(14)15)4-13-9(5)7/h1-4H,(H2,12,13)(H,14,15)

4-amino-8-chloroquinoline-3-carboxylic acid

4-Amino-8-chloroquinoline-3-carboxylic acid; FTO-IN-12; 955328-22-0; DTXSID60588692; RefChem:290051; DTXCID50539456;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.

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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).

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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)

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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).

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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

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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.

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