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Purity: ≥98%
IN04, also know as Methyl 3-(2-((5-(3,5-dimethoxyphenyl)-1,3,4-oxadiazol-2-yl)thio)acetamido)benzoate, is a novel and potent inhibitor of Leucine rich repeat kinase 1 (LRRK1), suppressing osteoclasts (OC) function with no effect on OC formation. IN04 inhibits LRRK1 by binding to the active site of the kinase domain but not the ROC domain. LRRK1 plays a critical role in regulating OC function and peak bone mass. Therefore, LRRK1 is a novel drug target for alternative anti-resorptive drugs to treat osteoporosis and osteoporotic fractures.
Targets |
LRRK1/Leucine rich repeat kinase 1
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ln Vitro |
IN04 was chosen for evaluation of biological effects on osteoclasts (OCs) in vitro. INO4 at 16 nM completely blocked ATP binding to hLRRK1 KD in an in vitro pulldown assay. In differentiation and pit assays, while the number of OCs on bone slices were comparable for OCs treated with IN04 and DMSO, IN04 treatment of OCs significantly impaired their ability to resorb bone. The area of pits on bone slices was reduced by 43% at 5 μM and 83% at 10 μM as compared to DMSO. Individual pits appeared smaller and shallower. F-actin staining revealed that DMSO-treated OCs displayed clear actin rings, and F-actin forms a peripheral sealing zone. By contrast, IN04-treated OCs showed disarranged F-actin in the cytoplasm, and F-actin failed to form a sealing zone on bone slices. IN04 treatment had no effects on OC-derived coupling factor production nor on osteoblast nodule formation. These data indicate IN04 is a potent inhibitor of LRRK1, suppressing OC function with no effect on OC formation [1].
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Enzyme Assay |
In vitro inhibition of ATP binding assay [1]
IN04inhibition of ATP binding to hLRRK1 KD was assessed by a pulldown assay with ATP-agarose according to the manufacturer's instructions (ATP Affpur kit III). Briefly, 50 ng recombinant hLRRK1 KD was first incubated with various concentrations of IN04 inhibitor (3.2 nM, 16, nM, 80 nM, 0.4 μM, and 5.0 μM) or the same volume of DMSO in 500 μl of 1x binding buffer on ice for 15 minutes. After the incubation, 50 μl of the equilibrated ATP-agarose was added into the reaction and the reactions were incubated by end-over-end mixing for 1 hour at 4°C after which beads were washed 4 times in 1x washing buffer. Proteins were eluted using 2x LDS sample buffer, heated at 90°C for 5 minutes and analyzed with 10% NuPage Bis-tris gel by western blot with anti-6xHis antibody as described previously |
Cell Assay |
In vitro OC formation [1]
Primary CD11b+ monocytes derived from the spleen of 5-week old C57BL/6J mice were positively isolated with magnetic CD11b microbeads according to the manufacturer’s instructions. Briefly, 108 splenocytes in 1 ml MACS buffer were incubated with 150 μl of CD11b microbeads at 4°C for 15 minutes after removing red blood cells with RBC lysis buffer. The cells were then washed with 15 ml PBS washing buffer containing 0.5% BSA and 2 mM EDTA, spun down, and re-suspended in 500 μl of washing buffer. The cell suspension was then loaded onto a MACS® Column placed in the magnetic field of a MACS Separator. The magnetically labeled CD11b+ monocytes were eluted and flushed out with the plunger after 3 times washing and removing from the separator. The isolated CD11b+ monocytes were maintained in α-MEM supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/ml), streptomycin (100 μg/ml), and M-CSF (20 ng/ml) at 37°C in 5% CO2 for 3 days to stimulate monocyte proliferation. The cells were then induced to differentiate in a medium containing the LRRK1 inhibitor IN04. The medium was changed every 2 days. Osteoclastogenesis was evaluated by counting TRAP staining positive, multinucleated cells having at least three nuclei. Mature OC size was measured by the Lieca Application Suite X software equipped with Lieca STP6000 microscope and Image J. |
Animal Protocol |
Bone resorption pit and actin ring formation assays [1]
Slices from bovine cortical bone were placed in 48-well plates and cells were differentiated on top of the bone slices as described previously. Cells on bone slices were digested with trypsin at 37°C overnight. Multinucleated cells were further removed by 5-minute sonication in 1M ammonia. Air-dried bone slices were stained with hematoxylin. The entire surface of each bone slice was examined and the total resorbed area per bone slice was quantified using ImageJ software. Resorption pits were also visualized by nano-CT at a 0.66 μM voxel dimension. The pit area and pit depth on bone slices were quantified with ImageJ software and TXM Reconstructor, respectively. Cells on bone slices were also stained with Alexa Fluor 488-conjugated phalloidin for F-actin and DAPI for nuclei staining. Actin ring formation, sealing zone, and 3D OC images were visualized by Olympus Fluoview 3000 confocal microscopy. Nodule assay[1] Bone marrow stromal cells isolated from the femurs and the tibias of the mice were grown to 80% confluence. The cells were then treated with a mineralization medium containing 10 mM β-glycerophosphate, 50 μg/ml ascorbic acid, and 10% FBS for 24 days. The cells were washed, fixed, and stained with 40 mM alizarin red (pH 4.2). The mineralized area was measured as described previously [1]. |
References | |
Additional Infomation |
Since the 3D structure of Lrrk1 is not available, we used TAK1 as a template to build 3D structures of hLRRK1 KD for drug screening. Thus, it is possible that IN04 may inhibit enzymatic activity of TAK1 or other MAPKKK family members. Although we minimized off-target effects by eliminating the compounds that also interact with other known kinases (e.g., p38 MAP kinase, PKC, GSK, c-Src, and B-RAF kinase) and focused on compounds that suppress OC activity by disrupting cytoskeleton rearrangement, but not through affecting OC differentiation, further characterization of the kinase selectivity of IN04 are still needed with experimental high-throughput approaches such as kinase binding assays by the KINOMEscanTM methodology and activity-based enzymatic assays by the KiNativTM technology. In vivo testing in ovariectomized osteoporotic mice or inflammatory bone mouse models with high bone turnover is also needed to confirm that IN04 inhibits bone resorption with no effect on bone formation and to determine whether IN04 has better pharmacologic efficacy than bisphosphonates in treating osteoporosis. It will be our future direction to conjugate IN04 inhibitor with penetrating oligopeptide (DSS)6 to target active OCs on bone surface. Lastly, a crystallographic structure of LRRK1 needs to be resolved for refinement of potential LRRK1 inhibitors and for high throughput screening of more specific and selective inhibitors. [1]
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Molecular Formula |
C20H19N3O6S
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Molecular Weight |
429.4464
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Exact Mass |
429.099
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CAS # |
838816-57-2
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PubChem CID |
1259183
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Appearance |
Typically exists as solid at room temperature
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Density |
1.39±0.1 g/cm3(Predicted)
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Index of Refraction |
1.628
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LogP |
4.59
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Hydrogen Bond Donor Count |
1
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Hydrogen Bond Acceptor Count |
9
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Rotatable Bond Count |
9
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Heavy Atom Count |
30
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Complexity |
571
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Defined Atom Stereocenter Count |
0
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SMILES |
ClC1C=CC=CC=1C(C1C=CC=CC=1)N1CCN(CCOCC(=O)O)CC1
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InChi Key |
MCQJCSNFOKHYLJ-UHFFFAOYSA-N
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InChi Code |
InChI=1S/C20H19N3O6S/c1-26-15-8-13(9-16(10-15)27-2)18-22-23-20(29-18)30-11-17(24)21-14-6-4-5-12(7-14)19(25)28-3/h4-10H,11H2,1-3H3,(H,21,24)
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Chemical Name |
methyl 3-[[2-[[5-(3,5-dimethoxyphenyl)-1,3,4-oxadiazol-2-yl]sulfanyl]acetyl]amino]benzoate
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Synonyms |
IN04; IN 04; 838816-57-2; Methyl 3-(2-((5-(3,5-dimethoxyphenyl)-1,3,4-oxadiazol-2-yl)thio)acetamido)benzoate; Methyl3-(2-((5-(3,5-dimethoxyphenyl)-1,3,4-oxadiazol-2-yl)thio)acetamido)benzoate; CBKinase1_012394; CBKinase1_024794; IN-04
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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 | 2.3286 mL | 11.6428 mL | 23.2856 mL | |
5 mM | 0.4657 mL | 2.3286 mL | 4.6571 mL | |
10 mM | 0.2329 mL | 1.1643 mL | 2.3286 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.