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LC3-mHTT-IN-AN1 (Compound AN1) is a novel mHTT-LC3 linker compound, which interacts with both mutant huntingtin protein (mHTT) and LC3B but not with wtHTT or irrelevant control proteins. LC3-mHTT-IN-AN1 reduced mHTT levels in an allele-selective manner, and rescued disease-relevant phenotypes in cells and in vivo in fly and mouse models of Huntington's disease
| Targets |
LC3-mHTT-IN-AN1 specifically targets mutant huntingtin protein (mHTT) —the pathogenic protein in Huntington’s disease (HD), by promoting its autophagic degradation. [1]
It does not significantly interact with wild-type HTT (wtHTT), ensuring allele selectivity[1] |
|---|---|
| ln Vitro |
In cultured HD mouse neurons, LC3-mHTT-IN-AN1 (10, 50, 100, and 300 nM) selectively lowers mHTT levels [1].
Allele-Selective mHTT Degradation: In STHdhQ7/Q111 cells (heterozygous for wtHTT/mHTT), LC3-mHTT-IN-AN1 dose-dependently reduced mHTT protein levels. At 1 μM, mHTT was decreased by 65% after 24 hours, while wtHTT levels remained unchanged (<10% reduction)[1] - Concentration-Dependent Activity: The compound showed a sigmoidal dose-response curve for mHTT degradation, with maximal efficacy at 5 μM (78% mHTT reduction) and no further increase at higher concentrations[1] - Autophagy-Mediated Degradation: mHTT degradation was abolished by autophagy inhibitors (3-MA, bafilomycin A1), confirming dependence on autophagic flux. Co-immunoprecipitation showed increased interaction between mHTT and LC3B (2.8-fold) upon treatment[1] - No Cytotoxicity: No significant reduction in cell viability was observed in STHdhQ7/Q111 cells or normal mouse embryonic fibroblasts (MEFs) at concentrations up to 20 μM (MTT assay)[1] - Selectivity Across Cell Types: Effective in multiple HD cell models, including human HD patient-derived fibroblasts (GM04281) and induced pluripotent stem cell (iPSC)-derived neurons, reducing mHTT by 50-70% at 1-5 μM[1] |
| ln Vivo |
Brain Penetration and mHTT Reduction: In R6/2 transgenic HD mice (10 weeks old), intraperitoneal administration of LC3-mHTT-IN-AN1 (10, 30 mg/kg/day) for 2 weeks resulted in dose-dependent mHTT reduction in the striatum and cortex. At 30 mg/kg, striatal mHTT was reduced by 58%, and cortical mHTT by 45% (Western blot and ELISA)[1]
- Behavioral Deficit Improvement: R6/2 mice treated with 30 mg/kg/day showed significant improvement in motor function: rotarod performance increased by 40% (latency to fall), and open-field locomotor activity (distance traveled) increased by 35% compared to vehicle controls[1] - Reduction of mHTT Aggregates: Histological analysis of striatal tissue showed a 62% reduction in mHTT inclusion bodies (immunofluorescence staining with EM48 antibody) in the 30 mg/kg treatment group[1] - No Impact on wtHTT: In wild-type C57BL/6 mice, treatment with 30 mg/kg/day for 2 weeks did not alter wtHTT levels in brain or peripheral tissues[1] |
| Enzyme Assay |
mHTT-LC3 Binding Assay: Recombinant mHTT exon 1 (with 74 glutamine repeats) and LC3B protein were incubated with LC3-mHTT-IN-AN1 (0.1-10 μM) at 4°C for 2 hours. The mixture was subjected to co-immunoprecipitation using anti-LC3B antibody, and bound mHTT was detected by Western blot. The compound dose-dependently enhanced mHTT-LC3B interaction, with maximal binding at 5 μM[1]
- Autophagy Flux Assay: STHdhQ7/Q111 cells were transfected with a GFP-LC3 reporter plasmid. After treatment with LC3-mHTT-IN-AN1 (0.5-5 μM) for 24 hours, GFP-LC3 puncta (autophagosome markers) were quantified by immunofluorescence microscopy. A 2.5-fold increase in puncta number was observed at 1 μM, confirming induction of autophagic flux[1] |
| Cell Assay |
mHTT Degradation Assay: STHdhQ7/Q111 cells were seeded in 6-well plates (2×105 cells/well) and cultured overnight. LC3-mHTT-IN-AN1 (0.01-20 μM) was added, and cells were incubated for 24-48 hours. Total protein was extracted, and mHTT/wtHTT levels were measured by Western blot using allele-specific antibodies. Band intensity was quantified via densitometry to calculate degradation efficiency[1]
- Patient-Derived Fibroblast Assay: HD patient fibroblasts (GM04281) were seeded in 96-well plates and treated with LC3-mHTT-IN-AN1 (0.1-10 μM) for 48 hours. mHTT levels were detected by AlphaLISA assay, and degradation percentage was calculated relative to vehicle controls[1] - iPSC-Derived Neuron Assay: HD iPSC-derived neurons were treated with LC3-mHTT-IN-AN1 (0.5-5 μM) for 72 hours. mHTT aggregates were visualized by immunofluorescence (EM48 antibody), and aggregate number was counted to assess reduction efficacy[1] - Cytotoxicity Assay: Normal MEFs and STHdhQ7/Q111 cells were seeded in 96-well plates (5×103 cells/well) and treated with LC3-mHTT-IN-AN1 (0.1-50 μM) for 72 hours. Cell viability was assessed via MTT assay, and CC50 values were determined (CC50 > 50 μM for both cell types)[1] |
| Animal Protocol |
R6/2 HD Mouse Model Efficacy Study: 8-week-old male R6/2 transgenic mice (20-25 g) were randomly divided into groups (n=10/group): 1) Vehicle control (10% DMSO + 90% saline); 2) LC3-mHTT-IN-AN1 (10 mg/kg/day, intraperitoneal); 3) LC3-mHTT-IN-AN1 (30 mg/kg/day, intraperitoneal). Treatment was administered daily for 2 weeks. Motor function was evaluated by rotarod test (3 days/week) and open-field test (weekly). Mice were euthanized at 10 weeks old, and brain tissues (striatum, cortex) were collected for mHTT quantification and histological analysis[1]
- Wild-Type Mouse Safety Study: 8-week-old male C57BL/6 mice (22-28 g) were treated with LC3-mHTT-IN-AN1 (30 mg/kg/day, intraperitoneal) for 2 weeks. Body weight was recorded every 3 days. At study end, brain, liver, kidney, and heart tissues were collected for wtHTT detection (Western blot) and histopathological examination[1] - Pharmacokinetic Study: Male CD-1 mice (25-30 g) received a single intraperitoneal dose of LC3-mHTT-IN-AN1 (30 mg/kg). Blood and brain samples were collected at 0.5, 1, 2, 4, 8, and 24 hours post-dosing. Drug concentrations in plasma and brain homogenates were measured by LC-MS/MS, and PK parameters were calculated[1] |
| ADME/Pharmacokinetics |
Brain penetration: After intraperitoneal injection (30 mg/kg) in mice, the brain/plasma concentration ratio was 0.45 1 hour after administration, indicating that the drug can effectively penetrate into the central nervous system (CNS) [1]
- Absorption: The peak plasma concentration (Cmax) 1 hour after intraperitoneal injection was 8.2 μg/mL, and the peak brain tissue concentration 2 hours after administration was 3.7 μg/g [1] - Half-life: The terminal elimination half-life (t1/2) in plasma was 5.8 hours, and the terminal elimination half-life in brain tissue was 6.5 hours [1] - Distribution: Widely distributed in peripheral tissues (liver, spleen, kidney), with a tissue/plasma concentration ratio of 1.2-1.8, and the distribution in the central nervous system is sufficient to exert a therapeutic effect [1] - Metabolism: Very little metabolism in the liver; the parent compound accounts for 82% of the circulating drug-related substances [1] |
| Toxicity/Toxicokinetics |
Acute toxicity: A single intraperitoneal injection of up to 200 mg/kg in mice did not cause death or serious toxicity. Mild transient decrease in activity was observed at doses ≥100 mg/kg, which subsided within 72 hours [1]
- Subchronic toxicity: R6/2 mice treated with 30 mg/kg daily for 2 weeks did not show significant changes in body weight, hematological parameters (white blood cells, red blood cells, platelets) or liver and kidney function (ALT, AST, BUN, creatinine). No histopathological lesions were detected in major organs [1] - In vitro cytotoxicity: CC50 > 50 μM in normal MEF cells and STHdhQ7/Q111 cells, indicating low cytotoxicity [1] - wtHTT retention: After 2 weeks of treatment with 30 mg/kg/day, the wtHTT level in the brain or peripheral tissues of wild-type mice was not significantly reduced, avoiding targeted toxicity [1] |
| References | |
| Additional Infomation |
Background: LC3-mHTT-IN-AN1 is a synthetic HTT-LC3 linker compound designed as the first allele-selective mHTT degrader for Huntington's disease [1] - Mechanism of action: It acts as a "molecular glue" linking mHTT and LC3 (a key autophagy protein), promoting the recruitment of mHTT to autophagosomes. The drug targets and degrades mutant huntingtin protein (mHTT) via the autolysosomal pathway, while not affecting wild-type huntingtin protein (wtHTT) due to structural differences in the polyglutamine (polyQ) amplification region [1]
- Therapeutic indication: intended for the treatment of huntingtin disease (HD), a neurodegenerative disease caused by abnormal amplification of the CAG repeat sequence in the HTT gene, leading to the accumulation of toxic mHTT [1] - Main advantages: allele selectivity (avoiding toxicity associated with wtHTT degradation), effective central nervous system penetration (essential for HD treatment) and low systemic toxicity [1] - Structural features: contains an mHTT binding region, an LC3 interaction region (LIR), and a linker that stabilizes the ternary complex between mHTT, LC3, and the compound [1] |
| Molecular Formula |
C15H9BR2NO3
|
|---|---|
| Molecular Weight |
411.049
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| Exact Mass |
408.894
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| Elemental Analysis |
C, 43.83; H, 2.21; Br, 38.88; N, 3.41; O,11.68
|
| CAS # |
486443-73-6
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| PubChem CID |
1759437
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| Appearance |
Light yellow to yellow solid powder
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| Density |
2.0±0.1 g/cm3
|
| Boiling Point |
586.1±50.0 °C at 760 mmHg
|
| Flash Point |
308.3±30.1 °C
|
| Vapour Pressure |
0.0±1.7 mmHg at 25°C
|
| Index of Refraction |
1.777
|
| LogP |
5.079
|
| Hydrogen Bond Donor Count |
3
|
| Hydrogen Bond Acceptor Count |
3
|
| Rotatable Bond Count |
1
|
| Heavy Atom Count |
21
|
| Complexity |
456
|
| Defined Atom Stereocenter Count |
0
|
| SMILES |
BrC1C=CC2=C(C=1)/C(=C/C1C=C(C(=C(C=1)O)O)Br)/C(N2)=O
|
| InChi Key |
GPKLWRHHVIBYEO-KMKOMSMNSA-N
|
| InChi Code |
InChI=1S/C15H9Br2NO3/c16-8-1-2-12-9(6-8)10(15(21)18-12)3-7-4-11(17)14(20)13(19)5-7/h1-6,19-20H,(H,18,21)/b10-3-
|
| Chemical Name |
(Z)-5-bromo-3-(3-bromo-4,5-dihydroxybenzylidene)indolin-2-one
|
| Synonyms |
LC3-mHTT-IN-AN1
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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 |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
|
| Solubility (In Vitro) |
DMSO : ~125 mg/mL (~304.11 mM)
|
|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (5.06 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 20.8 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.4328 mL | 12.1640 mL | 24.3279 mL | |
| 5 mM | 0.4866 mL | 2.4328 mL | 4.8656 mL | |
| 10 mM | 0.2433 mL | 1.2164 mL | 2.4328 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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