The target of AR-7 is retinoic acid receptor alpha (RARα), acting as an antagonist of this receptor. [1]
- AR-7 targets retinoic acid receptor alpha (RARα) as an antagonist, and it functions as a specific activator of chaperone-mediated autophagy (CMA) by inhibiting RARα signaling. [3]

In both WT and LRRK2R1441G KI mutant MEFs, treatment with RARA antagonist AR7 (20 μM; for 16 h) boosted lysosomal activity[1]. In NIH 3T3 cells, AR7 (10, 20, 30 uM; 12 hours) has no influence on macroautophagy[2]. By selectively degrading cytosolic proteins in lysosomes, chaperone-mediated autophagy (CMA) supports cellular quality control and the response of the cell to stress. Reduced CMA activity is a common feature of aging and age-related diseases. CMA is inhibited by signaling via the retinoic acid receptor alpha (RARα). Mouse fibroblasts with AR7 exhibit a considerable increase in CMA activity. Combining GR1 and AR7 results in a significant increase in CMA-activating potency, confirming their cooperative impact. The stimulatory effect of AR7 on CMA is partially reduced upon treatment with the transcriptional repressor Actinomycin D, which is consistent with transcriptional modifications that contribute to CMA upregulation[3].

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Pre-incubation of AR-7 at a concentration of 20 μM significantly increased the total lysosomal activity in both wild-type (WT) and LRRK2^{R1441G} knockin (KI) mouse embryonic fibroblasts (MEFs) expressing either SNCA or the KFERQ-peptide. LRRK2^{R1441G} KI MEFs had lower overall lysosomal degradation activity compared with WT controls, and AR-7 treatment effectively elevated lysosomal activity in these mutant cells (N = 4, p < 0.05, p < 0.01). Additionally, AR-7 induced neuronal LAMP2A transcription in MEFs, and treatment with AR-7 for 24 h dose-dependently upregulated Lamp2a mRNA expression in both WT and KI cells (N = 3) [1]
- In mouse fibroblasts, AR-7 at a concentration of 20 μM promoted the accumulation of CMA substrates in lysosomes (visualized as puncta formation) when the cells were stably expressing PAmCherry-KFERQ-NE. The treatment with AR-7 for 16 h in complete culture medium induced lysosomal accumulation of CMA substrates, similar to the effect of serum deprivation (a known inducer of CMA). Moreover, AR-7 increased the degradation rate of long-lived proteins in mouse fibroblasts, and this effect was dependent on RARα and LAMP2A. In LAMP2A-knockdown fibroblasts, the stimulatory effect of AR-7 on protein degradation was abolished. AR-7 also protected mouse fibroblasts from oxidative stress induced by paraquat (PQ): when exposed to 2 mM PQ, control fibroblasts treated with AR-7 showed higher cellular viability compared with untreated cells; in LAMP2A-knockdown fibroblasts exposed to 0.5 mM PQ, AR-7 also improved cell viability (N = 3). Furthermore, AR-7 at 20 μM reduced the toxicity of α-synuclein overexpression in mouse fibroblasts treated with 1 mM PQ, and it decreased the level of α-synuclein oligomers in these cells [3]

Adult male Wistar rats fasted for 48h before sacrifice were used for isolation of lysosomes from liver. All animal work was approved and performed according to the guidelines set by the Albert Einstein College of Medicine Institutional Animal Care and Use Committee.

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In primary cortical neurons from LRRK2^{R1441G} KI mice, treatment with AR-7 at concentrations of 10 μM and 20 μM from DIV9 to DIV21 dose-dependently reduced the accumulation of both intracellular and extracellular SNCA oligomers, as measured by ELISA. LRRK2^{R1441G} KI primary neurons had significantly higher levels of SNCA oligomers than WT neurons at all time points (DIV9, 14, 21), and AR-7 treatment significantly prevented the progressive accumulation of these oligomers (p < 0.05, p < 0.01). Additionally, AR-7 significantly reduced total intracellular SNCA levels in both WT and LRRK2^{R1441G} KI neurons compared with vehicle-treated control groups (N = 4), while no significant changes in the protein levels of LAMP2A and HSPA8 were observed in either WT or KI cultures at DIV21 after AR-7 treatment [1]

Molecular docking experiments were conducted to investigate the binding of AR-7 to the RARα-binding pocket. AR-7 was docked to a hydrophobic region of the RARα-binding pocket formed by helices h3, h10, and h12 in the lowest-energy conformation for docking pose I. The docking analysis revealed the interaction mode of AR-7 with RARα, though no specific binding affinity values (e.g., Ki, Kd) were determined. Additionally, a luciferase reporter assay was performed to evaluate the effect of AR-7 on RARα transcriptional activity: mouse fibroblasts were co-transfected with the hRARα receptor, a reporter luciferase plasmid, and a renilla reporter (for transfection control). AR-7 was tested at different concentrations alone or in combination with 100 nM all-trans-retinoic acid (ATRA). AR-7 inhibited ATRA-induced RARα transcriptional activity, and the Ki value for this inhibition was determined (n = 4–6) [3]

Lysosomal activity assay in MEFs: WT and LRRK2^{R1441G} KI MEFs expressing SNCA or KFERQ-peptide were pre-incubated with AR-7 at 20 μM for a specific period. Total lysosomal activity was measured using an enzymatic assay kit based on the degradation of a self-quenched lysosomal substrate, followed by flow cytometry analysis. The fluorescence intensity of the substrate was detected to quantify lysosomal activity, and the data were compared between AR-7-treated and untreated groups [1]
- Lamp2a mRNA expression assay: WT and LRRK2^{R1441G} KI MEFs were treated with AR-7 at different concentrations (0, 10, 20 μM) for 24 h. Total RNA was extracted from the cells, and reverse transcription-polymerase chain reaction (RT-PCR) was performed to amplify Lamp2a mRNA. The expression level of Lamp2a mRNA was quantified and normalized to a reference gene, and the dose-dependent effect of AR-7 on Lamp2a transcription was analyzed [1]
- SNCA oligomer measurement in primary cortical neurons: Primary cortical neurons from WT and LRRK2^{R1441G} KI mice were cultured from DIV9 to DIV21 with AR-7 at 0, 10, or 20 μM. At DIV9, 14, and 21, total cell lysates and conditioned media were collected. The levels of SNCA oligomers were measured using an enzyme-linked immunosorbent assay (ELISA) with a standard curve generated from purified recombinant SNCA oligomer standards. The intracellular and extracellular SNCA oligomer levels were quantified and compared between AR-7-treated and untreated groups [1]
- CMA substrate accumulation assay: Mouse fibroblasts stably expressing PAmCherry-KFERQ-NE were treated with AR-7 at 20 μM for 16 h in complete culture medium. A control group was subjected to serum deprivation to induce CMA. After treatment, the cells were photoactivated with UV-A (405 nm) for 5 min to induce mCherry fluorescence emission. The formation of fluorescent puncta (indicating lysosomal accumulation of CMA substrates) was observed under a fluorescence microscope, and the number of puncta per cell was quantified in more than 50 cells from at least 4 different fields [3]
- Long-lived protein degradation assay: Mouse fibroblasts (control, RARα-knockdown, or LAMP2A-knockdown) were labeled with radioactive amino acids to mark long-lived proteins. The cells were then treated with AR-7 at 20 μM for 12 h, and the release of radioactive amino acids was measured to determine the rate of protein degradation. The percentage of proteolysis was calculated, and the effect of AR-7 on protein degradation in different cell groups was compared [3]
- Cell viability assay under oxidative stress: Control and LAMP2A-knockdown mouse fibroblasts were exposed to paraquat (PQ) at concentrations of 2 mM and 0.5 mM, respectively. AR-7 was added to the culture medium 12 h before or after PQ treatment. Cell viability was assessed using a standard cell viability assay (e.g., MTT or CCK-8 assay), and the absorbance values were measured to calculate the viability percentage of the cells [3]
- α-synuclein toxicity assay: Mouse fibroblasts were transfected with different concentrations of an α-synuclein-expressing plasmid. The cells were then treated with 1 mM PQ alone or in combination with 20 μM AR-7. Cell viability was measured, and the level of α-synuclein (including monomers and oligomers) was detected by western blotting. The intensity of the protein bands was quantified by densitometry to analyze the effect of AR-7 on α-synuclein-induced toxicity [3]

Adult male Wistar rats were used for isolation of lysosomes from liver

In the long-term culture of primary cortical neurons of LRRK2^{R1441G} KI mice, treatment with AR-7 at concentrations of 10 μM and 20 μM from day 9 to day 21 did not cause serious changes in cell morphology or cytotoxicity. This was assessed by cell morphology observation and lactate dehydrogenase (LDH) release assay (Figure S7 in the original literature) [1].

[1]. Age-dependent accumulation of oligomeric SNCA/α-synuclein from impaired degradation in mutant LRRK2 knockin mouse model of Parkinson disease: role for therapeutic activation of chaperone-mediated autophagy (CMA). Autophagy. 2020 Feb;16(2):347-370.

[2]. Chaperone-mediated autophagy prevents collapse of the neuronal metastable proteome. Cell. 2021 May 13;184(10):2696-2714.e25.

[3]. Chemical modulation of chaperone-mediated autophagy by retinoic acid derivatives. Nat Chem Biol. 2013 Jun;9(6):374-82.

AR-7 (7-chloro-3-(4-methylphenyl)-2H-1,4-benzoxazine) is a molecular chaperone-mediated autophagy (CMA)-specific activator and an antagonist of retinoic acid receptor α (RARα). It exerts its therapeutic effect by activating CMA, thereby enhancing the degradation of misfolded proteins, such as SNCA/α-synuclein, which are key pathogenic proteins in Parkinson's disease (PD). AR-7 activation of CMA represents a viable disease-modifying therapeutic strategy for PD because it reduces the accumulation of age-related pathogenic SNCA oligomers in the brain [1]. AR-7 is a synthetic derivative of all-trans retinoic acid (ATRA) designed, through structure-based chemical modification, to specifically neutralize the inhibitory effect of RARα signaling on CMA. Unlike ATRA (which inhibits CMA), AR-7 enhances cellular protein quality control by blocking RARα activation of CMA. AR-7’s chemoenhancing effect on CMA can protect cells from oxidative stress and protein toxicity, making it a promising drug for treating age-related diseases characterized by impaired CMA function, such as neurodegenerative diseases (like Parkinson’s disease and Alzheimer’s disease) and diabetes[3].

C15H12CLNO

257.72

257.061

80306-38-3

44250175

White to off-white solid powder

3.597

0

2

1

18

323

0

MVOZLTFXYGHZPM-UHFFFAOYSA-N

InChI=1S/C15H12ClNO/c1-10-2-4-11(5-3-10)14-9-18-15-8-12(16)6-7-13(15)17-14/h2-8H,9H2,1H3

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)

Solubility in Formulation 1: 2.5 mg/mL (9.70 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 (9.70 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 (9.70 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.)