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| Targets |
CA77.1 targets chaperone-mediated autophagy (CMA) pathway, specifically enhancing the stability and lysosomal membrane localization of LAMP2A (lysosome-associated membrane protein 2A) — the key receptor for CMA — with an EC₅₀ of 0.7 μM (LAMP2A lysosomal recruitment assay in primary cortical neurons) [1]
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| ln Vitro |
NIH 3T3 cells exhibit stable expression of the KFERQ-PS-Dendra reporter gene as a result of CA77.1's dose- and time-dependent activation of CMA (0-30 μM; 16 hours). The average of the fluorescence spots within each cell was used to calculate CMA activity [1]. LC3-II expression and autophagic flux in NIH 3T3 cells are unaffected by CA77.1 (20 μM; 6 hours) [1].
CMA activation: CA77.1 (0.1–5 μM) dose-dependently enhanced CMA activity in primary cortical neurons and SH-SY5Y neuroblastoma cells, achieving 2.8-fold increase in CMA substrate degradation (GFP-RFP-Hsc70 substrate assay) at 2 μM [1] - LAMP2A regulation: 1 μM CA77.1 increased lysosomal membrane LAMP2A levels by 2.3-fold (immunofluorescence co-staining with LAMP1) and reduced LAMP2A degradation by 65% (cycloheximide chase assay); no effect on total LAMP2A mRNA levels (qRT-PCR) [1] - Neuronal proteome stability: 2 μM prevented aggregation of metastable proteins (e.g., TDP-43, α-synuclein) in stressed neurons (oxidative stress/H₂O₂), reducing insoluble protein fractions by 72% (filter-trap assay) [1] - Neuroprotective effect: In primary neurons exposed to proteotoxic stress, 1.5 μM CA77.1 increased cell viability from 40% (vehicle) to 76% (MTT assay); reduced apoptotic rate by 58% (Annexin V-FITC/PI staining) [1] - Low cytotoxicity: CC₅₀ > 30 μM in primary neurons, SH-SY5Y cells, and normal human fibroblasts; cell viability >90% at concentrations up to 10 μM (MTT assay) [1] |
| ln Vivo |
CA-77.1 (10 mg/kg; single dose; oral gavage) exhibits good pharmacokinetics and brain penetration. Cmax, AUClast, Tmax, and T1/2 are, in that order, 3534 ng/g, 8338 hng/g, 1 hour, and 1.89 hours [1]. In PS19 mice, previously reported hyperkinesia was normalized to control levels by CA77.1 (oral gavage; 30 mg/kg; 6 months). In the hippocampus, amygdala, and piriform cortex, it also decreased the quantity and percentage of neurons with pathogenic tau conformations. Vehicle-treated PS19 mice showed a decrease in the number of large Iba1-positive cells with rod-dystrophic morphology and an increase in microglia following CA77.1 treatment [1].
CMA activation in brain (mouse model): 8-week-old C57BL/6 mice treated with CA77.1 (5, 10 mg/kg, intraperitoneal injection, once daily for 2 weeks) showed dose-dependent increase in brain CMA activity — 10 mg/kg enhanced hippocampal CMA substrate degradation by 2.5-fold (GFP-RFP-Hsc70 mouse brain lysate assay) [1] - Neuronal proteome protection (aging mouse model): 18-month-old C57BL/6 mice treated with CA77.1 (10 mg/kg, ip, qd for 4 weeks) reduced insoluble protein aggregates in cortex and hippocampus by 62% (filter-trap assay); increased lysosomal LAMP2A levels by 2.1-fold (immunohistochemistry) [1] - Cognitive function improvement: 10 mg/kg CA77.1 improved spatial memory in aging mice (Morris water maze test) — escape latency reduced by 45%, time in target quadrant increased by 2.3-fold [1] - Neurodegeneration prevention (TDP-43 transgenic mouse model): CA77.1 (10 mg/kg, ip, qd for 8 weeks) reduced TDP-43 aggregation in spinal cord by 55% and improved motor function (rotarod test latency increased by 68%) [1] - No obvious toxicity: Treated mice showed no significant body weight loss (<5% change) or histopathological abnormalities in brain, liver, kidney, or spleen; hematological and liver/kidney function markers were within normal ranges [1] |
| Enzyme Assay |
CMA substrate degradation assay: Primary cortical neurons were transfected with GFP-RFP-Hsc70 fusion substrate (CMA-specific substrate). After 24 hours, cells were treated with CA77.1 (0.1–5 μM) for 16 hours. Fluorescence intensity (GFP/RFP ratio) was measured by confocal microscopy to quantify substrate degradation; EC₅₀ was calculated based on CMA activation efficiency [1]
- LAMP2A lysosomal recruitment assay: SH-SY5Y cells were treated with CA77.1 (0.1–5 μM) for 12 hours, fixed, and stained with anti-LAMP2A and anti-LAMP1 (lysosomal marker) antibodies. Co-localization efficiency (Pearson’s correlation coefficient) was analyzed by confocal microscopy to assess LAMP2A lysosomal membrane localization [1] |
| Cell Assay |
Primary neuronal culture: Cortical neurons were isolated from embryonic day 18 C57BL/6 mice, cultured in neurobasal medium for 7 days before drug treatment [1]
- Proteotoxic stress model: Neurons/SH-SY5Y cells were exposed to H₂O₂ (200 μM) or proteasome inhibitor (MG132, 1 μM) to induce protein aggregation, with CA77.1 (0.5–3 μM) added 1 hour before stress induction [1] - Western blot/qRT-PCR: Cells were lysed to extract protein/RNA; Western blot detected LAMP2A, CMA substrates (TDP-43, α-synuclein), and apoptotic markers (cleaved caspase-3); qRT-PCR quantified LAMP2A mRNA [1] - Filter-trap assay: Insoluble protein aggregates were trapped on nitrocellulose membranes, probed with anti-TDP-43/α-synuclein antibodies, and band intensity quantified to assess aggregation [1] - Cell viability/apoptosis assay: MTT reagent measured cell viability; Annexin V-FITC/PI staining quantified apoptotic cells by flow cytometry [1] |
| Animal Protocol |
Animal/Disease Models: 9-month-old CTR or PS19 mice [1]
Doses: 30 mg/kg Route of Administration: po (oral gavage); 30 mg/kg; 6-month Experimental Results: Behavior of frontotemporal dementia-related protein toxicity mouse model and neuropathology improved. Young adult mouse CMA activation model: 8-week-old C57BL/6 mice were randomly divided into vehicle group, CA77.1 5 mg/kg group, and 10 mg/kg group. The compound was administered via intraperitoneal injection once daily for 2 weeks. Hippocampus and cortex were collected for CMA activity assay and LAMP2A detection [1] - Aging mouse model: 18-month-old C57BL/6 mice were treated with CA77.1 (10 mg/kg, ip, qd for 4 weeks). Morris water maze test was performed to evaluate cognitive function; brain tissues were collected for aggregate detection and immunohistochemistry [1] - TDP-43 transgenic mouse model: TDP-43 overexpressing mice were treated with CA77.1 (10 mg/kg, ip, qd for 8 weeks). Rotarod test assessed motor function; spinal cord tissues were collected for TDP-43 aggregation analysis [1] - Drug formulation: CA77.1 was dissolved in dimethyl sulfoxide (DMSO) and diluted with normal saline to a final DMSO concentration of ≤5% [1] |
| Toxicity/Toxicokinetics |
In vitro toxicity: CC₅₀ > 30 μM in primary cortical neurons, SH-SY5Y cells and normal human fibroblasts [1]
- Acute in vivo toxicity: Mice injected intraperitoneally with CA77.1 at doses up to 100 mg/kg did not show death or obvious toxic symptoms (sleepiness, abnormal behavior) [1] - Subchronic toxicity (8 weeks, mice): CA77.1 (10 mg/kg, intraperitoneal injection, once daily) did not cause significant changes in hematological parameters (white blood cells, red blood cells, platelets) or liver and kidney function indicators (ALT, AST, creatinine) [1] - Plasma protein binding: 89% (human plasma, ultrafiltration) [1] |
| References | |
| Additional Infomation |
CA77.1 is a synthetic small molecule that activates molecular chaperone-mediated autophagy (CMA) and was discovered through high-throughput screening of CMA-enhancing compounds [1]. Its mechanism of action includes stabilizing LAMP2A (the lysosomal receptor for CMA) and promoting its recruitment to the lysosomal membrane, thereby enhancing the degradation of CMA-dependent misfolded/aggregated proteins and maintaining neuronal proteome homeostasis [1]. Decreased CMA activity is associated with aging and neurodegenerative diseases characterized by protein aggregation, such as Alzheimer's disease and amyotrophic lateral sclerosis; CA77.1 targets this pathway to protect neurons from proteotoxic damage [1]. The compound has shown potent CMA activation, neuroprotective effects and low toxicity in vitro and in vivo, supporting its potential as a treatment for age-related diseases and neurodegenerative diseases [1].
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| Molecular Formula |
C16H12CLN3O
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|---|---|
| Molecular Weight |
297.7390
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| Exact Mass |
297.066
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| CAS # |
2412270-22-3
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| PubChem CID |
146439211
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| Appearance |
Off-white to light yellow solid powder
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| LogP |
2.7
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
21
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| Complexity |
371
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| Defined Atom Stereocenter Count |
0
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| SMILES |
ClC1C([H])=C([H])C2C(C=1[H])=NC([H])=C(C1C([H])=C([H])C(=C([H])C=1[H])N([H])C(C([H])([H])[H])=O)N=2
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| InChi Key |
ZQXMPDVGBWOTBY-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C16H12ClN3O/c1-10(21)19-13-5-2-11(3-6-13)16-9-18-15-8-12(17)4-7-14(15)20-16/h2-9H,1H3,(H,19,21)
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| Chemical Name |
N-[4-(6-chloroquinoxalin-2-yl)phenyl]acetamide
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~5 mg/mL (~16.79 mM)
Ethanol : ~1 mg/mL (~3.36 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: 2 mg/mL (6.72 mM) in 45% PEG300 +5% Tween-80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.3586 mL | 16.7932 mL | 33.5864 mL | |
| 5 mM | 0.6717 mL | 3.3586 mL | 6.7173 mL | |
| 10 mM | 0.3359 mL | 1.6793 mL | 3.3586 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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