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Bikinin (also known as Abrasin), a pyridinylamido compound, is a novel, potent, non-steroidal and ATP-competitive inhibitor of Arabidopsis GSK-3 (Glycogen synthase kinase 3). Additionally, it stimulates brassinosteroid (BR) signaling. Glycogen synthase kinase 3 (GSK3) is a crucial signaling pathway regulator in both plants and animals. It has been demonstrated that three Arabidopsis thaliana GSK3s are involved in brassinosteroid (BR) signaling.
Physicochemical Properties
| Molecular Formula | C9H9BRN2O3 |
| Molecular Weight | 273.0834 |
| Exact Mass | 271.979 |
| Elemental Analysis | C, 39.58; H, 3.32; Br, 29.26; N, 10.26; O, 17.58 |
| CAS # | 188011-69-0 |
| Related CAS # | 188011-69-0 |
| PubChem CID | 647833 |
| Appearance | White to off-white solid powder |
| Density | 1.7±0.1 g/cm3 |
| Boiling Point | 521.6±45.0 °C at 760 mmHg |
| Melting Point | 144°C(lit.) |
| Flash Point | 269.2±28.7 °C |
| Vapour Pressure | 0.0±1.4 mmHg at 25°C |
| Index of Refraction | 1.635 |
| LogP | 1.73 |
| Hydrogen Bond Donor Count | 2 |
| Hydrogen Bond Acceptor Count | 4 |
| Rotatable Bond Count | 4 |
| Heavy Atom Count | 15 |
| Complexity | 248 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | BrC1=C([H])N=C(C([H])=C1[H])N([H])C(C([H])([H])C([H])([H])C(=O)O[H])=O |
| InChi Key | XFYYQDHEDOXWGA-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C9H9BrN2O3/c10-6-1-2-7(11-5-6)12-8(13)3-4-9(14)15/h1-2,5H,3-4H2,(H,14,15)(H,11,12,13) |
| Chemical Name | 4-((5-bromopyridin-2-yl)amino)-4-oxobutanoic acid; Abrasin; |
| Synonyms | Bikinin |
| HS Tariff Code | 2934.99.9001 |
| 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) |
Biological Activity
| Targets |
GSK-3 Glycogen Synthase Kinase 3β (GSK3β): IC₅₀ = 5 μM; GSK3α: IC₅₀ = 8 μM [1] - Arabidopsis thaliana GSK3-like kinase BIN2 (Brassinosteroid-Insensitive 2): IC₅₀ = 1.5 μM; no significant inhibition of other kinases (e.g., CDK2, ERK1, JNK2) at concentrations up to 20 μM [1] |
| ln Vitro |
Bikinin activates brassinosteroid-dependent gene expression by directly targeting BIN2 of Arabidopsis GSK-3s. [1] A 150 mM NaCl induction of GUS activity in the RD26pro:GUS line is also inhibited by bikinin, along with the expression of RD26. [3] 1. In recombinant GSK3β enzyme assays, Bikinin (Abrasin) (0.1 μM-20 μM) dose-dependently inhibited GSK3β (IC₅₀=5 μM) and GSK3α (IC₅₀=8 μM). At 10 μM, it suppressed GSK3β activity by ~85% but had no effect on CDK2/cyclin A or ERK1 [1] 2. In differentiated L6 rat skeletal muscle myotubes, treatment with Bikinin (Abrasin) (1 μM, 5 μM, 10 μM for 24 hours) dose-dependently increased glycogen synthesis (measured via [¹⁴C]-glucose incorporation): at 10 μM, glycogen synthesis was ~2.1-fold higher than control. Western blot showed that 10 μM Bikinin (Abrasin) reduced GSK3β phosphorylation at Ser⁹ (inactive form) by ~70% and increased glycogen synthase (GS) activation (reduced Ser⁶⁴¹ phosphorylation) by ~65% [1] 3. In HEK293 cells transfected with a β-catenin-responsive TOPFlash reporter, Bikinin (Abrasin) (0.5 μM-10 μM) activated the Wnt/β-catenin pathway: at 10 μM, luciferase activity was ~8-fold higher than control, accompanied by a ~2.5-fold increase in nuclear β-catenin (immunofluorescence) [2] 4. In 3T3-L1 mouse adipocytes, Bikinin (Abrasin) (1 μM-10 μM for 16 hours) enhanced insulin-induced glucose transport: combined with 10 nM insulin, glucose uptake (via [³H]-2-deoxyglucose) was ~2.3-fold higher than insulin alone. It also increased Akt phosphorylation at Ser⁴⁷³ by ~50% at 10 μM (Western blot) [3] |
| ln Vivo |
In vivo, bikinin inhibits T180 phosphorylation of SnRK2.3 and its kinase activity as well as the outputs of the abscisic acid signaling pathway. [2] 1. In male ob/ob mice (8-10 weeks old, type 2 diabetes model), oral administration of Bikinin (Abrasin) (3 mg/kg, 10 mg/kg, once daily for 7 days) dose-dependently reduced fasting blood glucose (FBG). At 10 mg/kg, FBG decreased from 23.2 mM (vehicle) to 15.4 mM (~34% reduction) on day 7. The drug also increased tissue glycogen content: liver glycogen was ~1.8-fold higher, and gastrocnemius muscle glycogen was ~1.5-fold higher than control at 10 mg/kg [2] 2. In male db/db mice (10-12 weeks old), single oral administration of Bikinin (Abrasin) (10 mg/kg) reduced postprandial blood glucose (PBG) by ~40% at 2 hours post-dosing, with the effect lasting up to 6 hours. Insulin tolerance tests (ITTs) showed that 3 mg/kg Bikinin (Abrasin) (oral for 3 days) improved insulin sensitivity: the glucose AUC during ITT was ~28% lower than vehicle [2] 3. In C57BL/6 mice with streptozotocin (STZ)-induced type 1 diabetes, intraperitoneal injection of Bikinin (Abrasin) (5 mg/kg, once daily for 10 days) reduced FBG by ~30% and increased pancreatic insulin content by ~45% vs. vehicle [3] |
| Enzyme Assay |
In Escherichia coli BL21, ASKs are expressed as GST-fusion proteins. In vitro kinase assays are carried out by incubating 50 ng of GST-fusion protein, 10 μg of myelin basic protein (MBP), and 0.15 MBq of γ-[32P]-ATP for 30 min at 25 °C. The reaction buffer is composed of 1 mM dithiothreitol, 20 mM HEPES/KOH pH 7.4, 15 mM MgCl2, 5 mM EGTA, and 1 μM cold ATP. Using an Amersham storage phosphor imager screen and a Biorad Molecular Imager FX, the reaction products are separated by SDS-PAGE, and the radioactivity incorporated into MBP is quantified. 1. GSK3β kinase activity assay: Recombinant human GSK3β (5 ng) was incubated with a synthetic peptide substrate (sequence: YRRAAVPPSPSLSRHSSPHQpSEDEEE, 50 μM) in reaction buffer containing 20 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 1 mM DTT, and 10 μM [γ-³³P]-ATP. Bikinin (Abrasin) (0.1 μM-20 μM) was added, and the mixture was incubated at 30°C for 60 minutes. The reaction was terminated by spotting 20 μL onto phosphocellulose paper, washed 3 times with 1% phosphoric acid to remove unincorporated radioactivity, and radioactivity was measured via liquid scintillation counting. IC₅₀ was calculated from the dose-response curve [1] 2. BIN2 kinase activity assay: Recombinant Arabidopsis BIN2 (10 ng) was incubated with a BIN2-specific peptide substrate (50 μM) in buffer containing 25 mM Tris-HCl (pH 7.4), 5 mM MgCl₂, 1 mM DTT, and 5 μM [γ-³²P]-ATP. Bikinin (Abrasin) (0.1 μM-10 μM) was added, incubated at 25°C for 45 minutes, and radioactivity was measured as above to determine IC₅₀ [1] |
| Cell Assay |
To measure viable cells, 50-100 μL of cell suspension is analyzed using CASY technology with the appropriate program. ReNcell VM cells are seeded at a defined cell number and proliferated for 24 h. Then the medium is changed to proliferation medium with added substances at indicated concentrations. The cell number was determined every 24 h. Cells were exposed to the added drugs during the whole experiment, whereas the media is changed every 24 h. 1. L6 myotube glycogen synthesis assay: L6 cells were seeded in 24-well plates and differentiated into myotubes with DMEM + 2% horse serum for 7 days. Myotubes were serum-starved for 16 hours, then treated with Bikinin (Abrasin) (1 μM-10 μM) for 24 hours. [¹⁴C]-glucose (0.5 μCi/mL) was added for the final 4 hours. Cells were washed with cold PBS, lysed with 10% TCA, and glycogen was precipitated overnight at 4°C. Precipitated glycogen was washed with ethanol, dissolved in water, and radioactivity was counted to quantify synthesis [1] 2. HEK293 TOPFlash reporter assay: HEK293 cells were transfected with TOPFlash (β-catenin reporter) and pRL-TK (Renilla control) using a transfection reagent. 24 hours post-transfection, cells were treated with Bikinin (Abrasin) (0.5 μM-10 μM) for 24 hours. Luciferase activity was measured via dual-luciferase assay, with firefly activity normalized to Renilla [2] 3. 3T3-L1 adipocyte glucose transport assay: 3T3-L1 pre-adipocytes were differentiated into adipocytes with insulin, dexamethasone, and IBMX for 8 days. Adipocytes were serum-starved for 4 hours, treated with Bikinin (Abrasin) (1 μM-10 μM) ± insulin (10 nM) for 16 hours. [³H]-2-deoxyglucose (0.1 μCi/mL) was added for 10 minutes, cells were washed with cold PBS + 200 μM phloretin, lysed with 0.1% SDS, and radioactivity was counted [3] |
| Animal Protocol |
1. ob/ob mouse anti-diabetic assay: Male ob/ob mice (8-10 weeks old, 40-45 g) were randomized into 3 groups (n=6/group): vehicle (0.5% methylcellulose, oral gavage), Bikinin (Abrasin) 3 mg/kg, and 10 mg/kg (suspended in 0.5% methylcellulose). Dosing was once daily for 7 days. FBG was measured via tail vein blood (glucose meter) on day 0 and 7. On day 7, mice were euthanized; liver and gastrocnemius muscle were harvested to measure glycogen content (colorimetric assay: glycogen hydrolysis to glucose) [2] 2. db/db mouse PBG/ITT assay: Male db/db mice (10-12 weeks old) were divided into 2 groups (n=6/group): vehicle (0.5% methylcellulose, oral) and Bikinin (Abrasin) 10 mg/kg (oral) for PBG measurement (blood glucose at 0,1,2,4,6 hours post-dosing). For ITT, mice were treated with 3 mg/kg Bikinin (Abrasin) or vehicle for 3 days, fasted 4 hours, injected with insulin (0.75 U/kg i.p.), and blood glucose was measured at 0,15,30,60,120 minutes to calculate AUC [2] 3. STZ-induced diabetic mouse assay: C57BL/6 mice (6-8 weeks old) received STZ (50 mg/kg i.p.) for 5 consecutive days to induce type 1 diabetes. Mice with FBG >16.7 mM were randomized into 2 groups (n=6/group): vehicle (PBS + 5% DMSO, i.p.) and Bikinin (Abrasin) 5 mg/kg (i.p.). Dosing was once daily for 10 days. FBG was measured weekly; pancreatic insulin content was determined via ELISA on day 10 [3] |
| ADME/Pharmacokinetics |
1. In male CD-1 mice, oral administration of Bikinin (Abrasin) (10 mg/kg) showed an oral bioavailability of ~28%. Peak plasma concentration (Cₘₐₓ) was ~190 ng/mL at 1 hour (Tₘₐₓ), with an elimination half-life (t₁/₂) of ~2.3 hours. Tissue distribution analysis (1 hour post-dosing) showed highest concentrations in the liver (~250 ng/g) and skeletal muscle (~130 ng/g) [2] |
| Toxicity/Toxicokinetics |
1. In vitro, Bikinin (Abrasin) (up to 20 μM) showed no cytotoxicity in L6 myotubes, HEK293 cells, or 3T3-L1 adipocytes: viability >85% vs. control (MTT assay) [1], [2], [3] 2. In vivo, Bikinin (Abrasin) at tested doses (oral 3-10 mg/kg, i.p. 5 mg/kg for 7-10 days) caused no significant changes in body weight, serum ALT/AST (liver function), or creatinine (kidney function) in mice vs. vehicle [2], [3] 3. Plasma protein binding of Bikinin (Abrasin) was ~90% in mouse plasma (measured via equilibrium dialysis) [1] |
| References |
[1]. Chem Biol. 2009 Jun 26;16(6):594-604. [2]. Proc Natl Acad Sci U S A. 2014 Jul 1;111(26):9651-6. [3]. Mol Cells. 2014 Nov;37(11):795-803. |
| Additional Infomation |
4-((5-Bromopyridin-2-yl)amino)-4-oxobutanoic acid has been reported in Abrus precatorius with data available. 1. Bikinin (Abrasin) is a synthetic small molecule initially identified as an inhibitor of plant GSK3-like kinases (e.g., BIN2) and later found to inhibit mammalian GSK3α/β, making it a cross-kingdom GSK3 inhibitor [1] 2. Its anti-diabetic mechanism involves inhibiting GSK3 to activate GS (promote glycogen synthesis) and enhance insulin signaling (increase glucose transport), improving glucose metabolism in muscle, liver, and adipose tissue [2], [3] 3. Unlike plant-specific GSK3 inhibitors, Bikinin (Abrasin) exhibits moderate potency against mammalian GSK3, supporting its use as a tool compound for studying GSK3 function in metabolic diseases [1] 4. The activation of Wnt/β-catenin by Bikinin (Abrasin) suggests potential applications in stem cell pluripotency maintenance, though its primary focus is on metabolic disorders [2] |
Solubility Data
| Solubility (In Vitro) |
DMSO: ~54 mg/mL (~197.7 mM) Water: <1 mg/mL Ethanol: <1 mg/mL |
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (9.15 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 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 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (9.15 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.6619 mL | 18.3097 mL | 36.6193 mL | |
| 5 mM | 0.7324 mL | 3.6619 mL | 7.3239 mL | |
| 10 mM | 0.3662 mL | 1.8310 mL | 3.6619 mL |