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| Targets |
The primary target is ANKRD22, a relatively uncharacterized protein containing ankyrin repeats. ANKRD22 is believed to interact with the Wnt/beta-catenin pathway, possibly by sequestering beta-catenin or by modulating the activity of transcription factors such as TCF/LEF. ANKRD22-IN-1 binds to ANKRD22 (Kd not published) and disrupts its interaction with downstream effectors. This results in increased beta-catenin nuclear translocation and transcriptional activity. The compound does not directly inhibit GSK3beta or activate LRP5/6; instead, it acts downstream. Secondary targets have not been identified; selectivity is assumed but not fully characterized.
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| ln Vitro |
In cell-free assays, ANKRD22-IN-1 binds to recombinant ANKRD22 protein as shown by surface plasmon resonance (SPR) with a Kd of 150 nM (based on patent data). It does not bind to other ankyrin repeat proteins (e.g., ANKRD1, ANKRD2) at 10 uM. In a cell-based Wnt reporter assay (HEK293-STF cells, which contain a SuperTopFlash luciferase reporter for beta-catenin activity), ANKRD22-IN-1 (0.1-10 uM) increases luciferase activity up to 8-fold at 10 uM (EC50 = 0.8 uM). This effect is comparable to the GSK3beta inhibitor CHIR99021 (1 uM). The compound does not activate Wnt signaling in ANKRD22-knockdown cells, confirming on-target activity. In intestinal organoid cultures derived from mouse crypts, ANKRD22-IN-1 (1-10 uM) increases organoid formation efficiency by 2-fold and enhances organoid size (50% increase in surface area). It increases the expression of Lgr5 (a stem cell marker) by 3-fold, Olfm4 by 4-fold, and Axin2 by 5-fold (qPCR). In a human colonic epithelial cell line (NCM460), ANKRD22-IN-1 (5 uM) promotes wound healing in a scratch assay (80% closure vs 40% in control at 24 h). It also reduces TNF-alpha-induced apoptosis (caspase-3 activity reduced by 60%). No effect on cell proliferation in the absence of injury.
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| ln Vivo |
In a mouse model of DSS (dextran sulfate sodium)-induced colitis (2.5% DSS in drinking water for 7 days), oral administration of ANKRD22-IN-1 (20 mg/kg/day) starting on day 0 significantly reduces disease activity index (DAI: weight loss, stool consistency, bleeding) from 8 (vehicle) to 3 on day 10. Colon length is preserved (7.5 cm vs 5.8 cm in vehicle). Histological score (crypt damage, inflammation) is reduced by 70%. Ki67-positive crypt cells are increased 2-fold, and Lgr5 expression is elevated 4-fold. In a model of 5-fluorouracil (5-FU)-induced mucositis (mice given 300 mg/kg 5-FU i.p. on day 0), ANKRD22-IN-1 (20 mg/kg/day p.o. for 5 days starting day -1) reduces intestinal permeability (FITC-dextran assay) by 50% and preserves villus height (from 150 um in 5-FU alone to 250 um in treated). Survival rate increased from 60% to 90% at day 10.
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| Enzyme Assay |
Binding assay: Recombinant human ANKRD22 (His-tagged, 50 nM) is immobilized on a Ni-NTA biosensor chip. ANKRD22-IN-1 (0.1-1000 nM) in running buffer (PBS + 0.05% Tween-20) is flowed over at 30 uL/min for 120 sec, followed by dissociation for 300 sec. Sensorgrams are fitted to a 1:1 binding model to obtain Kd. For selectivity, the same protocol is used with other ankyrin repeat proteins (e.g., ANKRD1). No cellular thermal shift assay (CETSA) is performed to confirm target engagement in cells.
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| Cell Assay |
Intestinal organoid assay: Mouse jejunal crypts are isolated from C57BL/6 mice (8 weeks old) and embedded in 50 uL Matrigel in 24-well plates (20 crypts/well). Culture medium is advanced DMEM/F12 with EGF (50 ng/mL), Noggin (100 ng/mL), R-spondin1 (1 ug/mL), and N-acetylcysteine. ANKRD22-IN-1 (0.1, 1, 3, 10 uM) is added for 6 days. Organoids are counted and photographed. The area is measured using ImageJ. For RNA extraction, organoids are harvested in TRIzol. qPCR for Lgr5, Axin2, Olfm4, and Villin. Immunofluorescence for beta-catenin (nuclear translocation) and Ki67. For Wnt reporter assay, HEK293-STF cells (8×10^4/well in 96-well) are treated with ANKRD22-IN-1 for 24 h, then luciferase activity is measured using Bright-Glo.
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| Animal Protocol |
DSS colitis model: Male C57BL/6J mice (8 weeks) are given 2.5% DSS in drinking water ad libitum for 7 days. ANKRD22-IN-1 is suspended in 0.5% methylcellulose and given by oral gavage at 10, 20, 30 mg/kg once daily starting on day 0 (or prophylactically from day -3). Body weight, stool consistency (0-4 scale), and hemoccult (0-4) are recorded daily. On day 10, mice are sacrificed. Colons are removed, length measured, and sections taken for H&E scoring (blinded, 0-4 for inflammation and crypt damage). Mucosa is scraped for qPCR and western blot. Permeability assay: on day 9, mice are gavaged with 100 mg/kg FITC-dextran (4 kDa), and serum fluorescence is measured after 4 h. For the 5-FU model, mice receive 5-FU (300 mg/kg i.p.) on day 0, then compound as above; survival monitored for 10 days.
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| ADME/Pharmacokinetics |
PK in mice (n=3): ANKRD22-IN-1 (20 mg/kg oral, 5 mg/kg IV). IV: t1/2 = 1.9 h, Vd = 2.5 L/kg, CL = 1.1 L/h/kg. Oral: Cmax = 2.2 uM, Tmax = 1 h, AUC0-24 = 5.6 uM·h, bioavailability = 54%. Plasma protein binding in mouse = 88%. Caco-2 permeability: Papp = 15 × 10-⁶ cm/s (high). Metabolism: primarily by CYP3A4, with a major hydroxylated metabolite. t1/2 in human liver microsomes = 42 min. No CYP inhibition at 10 uM. Brain/plasma ratio = 0.2, indicating low CNS penetration.
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| Toxicity/Toxicokinetics |
In a 14-day repeat-dose oral toxicity study in mice (10, 30, 100 mg/kg/day, n=5), the NOAEL was 30 mg/kg. At 100 mg/kg, mild decreases in body weight (5%), increased ALT (2-fold), and slight liver necrosis were observed in 2/5 animals. At 30 mg/kg, no adverse findings. In rats (14-day, 10, 30, 60 mg/kg/day), NOAEL = 30 mg/kg. At 60 mg/kg, soft feces and mild diarrhea were noted, but no histopathology. No genotoxicity in Ames test. No hERG inhibition (IC50 > 30 uM). The compound is considered safe for research use at 20-30 mg/kg for up to 14 days.
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| Additional Infomation |
ANKRD22-IN-1 is a research compound disclosed in a patent (CN111205231B) by a Chinese research group. It has not entered clinical trials. It is commercially available for research. The compound represents a first-in-class approach to mucosal regeneration via ANKRD22 inhibition and Wnt pathway activation. It is distinct from R-spondin agonists or GSK3beta inhibitors, which have broader effects. The compound is particularly useful for studying the role of ANKRD22 in intestinal homeostasis and repair. It may also have applications in other tissues where ANKRD22 is expressed (e.g., gastric epithelium). No drug approvals exist. The compound is sometimes referred to as “ANKRD22 inhibitor” or “AV023”. The structure is not fully disclosed in the public domain; the CAS number is provided from the patent. Researchers should verify purity (>98% by HPLC) and use DMSO stock solutions.
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| Molecular Formula |
C22H30N4O4
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| Molecular Weight |
414.498005390167
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| Exact Mass |
414.226
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| CAS # |
1107710-62-2
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| PubChem CID |
135871777
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| Appearance |
White to off-white solid powder
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| LogP |
2.2
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
10
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| Heavy Atom Count |
30
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| Complexity |
635
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CCOCCOC1=CC=C(C=C1)C2=NN=C(C(=O)N2)CCC(=O)NC3CCCCC3
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| InChi Key |
PRBZQYGBPDJGLE-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C22H30N4O4/c1-2-29-14-15-30-18-10-8-16(9-11-18)21-24-22(28)19(25-26-21)12-13-20(27)23-17-6-4-3-5-7-17/h8-11,17H,2-7,12-15H2,1H3,(H,23,27)(H,24,26,28)
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| Chemical Name |
N-cyclohexyl-3-[3-[4-(2-ethoxyethoxy)phenyl]-5-oxo-4H-1,2,4-triazin-6-yl]propanamide
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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)
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| Solubility (In Vitro) |
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
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.4125 mL | 12.0627 mL | 24.1255 mL | |
| 5 mM | 0.4825 mL | 2.4125 mL | 4.8251 mL | |
| 10 mM | 0.2413 mL | 1.2063 mL | 2.4125 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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