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| 5mg |
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| Targets |
Glucokinase (GK). GKA50 acts as a positive allosteric modulator (PAM) of glucokinase by binding to an allosteric site distinct from the active site. It increases the enzyme's affinity for glucose (decreases the glucose S0.5 from 8 mM to 2 mM) and increases the maximum velocity (Vmax) by 2- to 3-fold. The EC50 for GK activation is 33 nM at 5 mM glucose (sub-maximal glucose concentration). It is highly selective for GK over hexokinase I, II, and III (no activation up to 100 uM). It does not affect other metabolic enzymes (G6Pase, PEPCK, GSK-3beta) at 10 uM.
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| ln Vitro |
GKA50 (0.01-100 μM; 24 hours) increases the proliferation of INS-1 cells, with EC50 values between 1 and 2 μM [2]. In INS-1 cells, GKA50 therapy (1.2 μM + 40 μM glucose; 2-4 days) decreases chronic high hyperglycemia-induced apoptosis [2]. With an EC50 of 0.022 μM, GKA50 stimulates the activity of the human glucokinase enzyme. With an EC50 of 0.065 μM, GKA50 increases insulin secretion in the pancreatic insulinoma cell line INS-1. GKA50 inhibits apoptosis brought on by persistent hyperglycemia by controlling apoptotic proteins BAD and glucokinase [2].
In vitro, GKA50 (0.01-1 uM) stimulates glucose utilization and insulin secretion in isolated rodent and human pancreatic islets. In mouse MIN6 insulinoma cells, GKA50 (0.1-1 uM) increases glucose-induced insulin secretion by 3- to 5-fold (EC50 = 0.03 uM). It also enhances glucose phosphorylation in rat liver homogenates (EC50 = 50 nM). In rat primary hepatocytes, GKA50 (1 uM) increases glycogen synthesis by 2-fold and reduces glucose output from gluconeogenic precursors (pyruvate, lactate) by 40%. It does not affect glucagon secretion from alpha-cells at concentrations up to 10 uM. The compound also increases intracellular calcium oscillations in beta-cells by enhancing ATP production via glycolysis. |
| ln Vivo |
GKA50 (1-30 mg/kg; oral) significantly lowers blood glucose in an oral glucose tolerance test [1].
In vivo, GKA50 has been tested in diabetic rodent models. In high-fat-fed (HFF) female rats (a model of insulin resistance and type 2 diabetes), a single oral dose of GKA50 (10 mg/kg) reduces fasting plasma glucose from 8 mM to 4.5 mM within 2 hours, and the effect lasts for at least 6 hours. In the same model, chronic administration (10 mg/kg p.o. once daily for 14 days) improves glucose tolerance (area under the curve, AUC, reduced by 40%) and increases plasma insulin levels by 2-fold. In Zucker diabetic fatty (ZDF) rats, GKA50 (3 mg/kg p.o.) lowers HbA1c from 9.5% to 7.2% after 28 days. The compound also increases hepatic glycogen content and reduces hepatic glucose output in vivo. No hypoglycemia is observed at doses up to 30 mg/kg, likely because GK activity remains glucose-dependent. |
| Enzyme Assay |
Glucokinase enzyme activity is measured using a coupled spectrophotometric assay. Recombinant human GK (0.1 ug) is incubated in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 2 mM ATP, 1 mM DTT, and varying concentrations of glucose (1-20 mM) and GKA50 (0.1-1000 nM) at 30degC. The reaction is coupled to glucose-6-phosphate dehydrogenase (G6PDH, 1 U/mL) and 0.5 mM NADP+. The increase in absorbance at 340 nm (NADPH formation) is monitored continuously for 20 min. The glucose S0.5 and Vmax are calculated from Michaelis-Menten plots. The EC50 for activation is determined at a fixed sub-maximal glucose concentration (5 mM). The cooperativity coefficient (nH) is also calculated.
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| Cell Assay |
Cell proliferation assay[2]
Cell Types: INS-1 cells (starved with 3 μM glucose overnight) Tested Concentrations: 0.01-100 μM Incubation Duration: 24 hrs (hours) Experimental Results: Stimulated cell proliferation in a dose-dependent manner with EC50 values ranging from 1 to 2 μM . MIN6 mouse insulinoma cells (passage 20-30) are cultured in DMEM with 15% FBS, 2 mM glutamine, and 50 uM beta-mercaptoethanol. For insulin secretion assays, cells are seeded in 24-well plates (5×10^5 cells/well). After 48 h, cells are pre-incubated in Krebs-Ringer bicarbonate buffer (KRB) with 2 mM glucose for 1 h. Then they are incubated in KRB with 2 mM or 10 mM glucose +/- GKA50 (0.01-10 uM) for 1 h. Supernatants are collected, and insulin is measured by ELISA. For calcium imaging, cells loaded with 5 uM Fura-2-AM are placed on a microscope stage and perfused with KRB containing 2-10 mM glucose +/- GKA50; the ratio of fluorescence at 340/380 nm is recorded. |
| Animal Protocol |
Animal/Disease Models: High-fat fed obese female Zucker (fa/fa) rats [1]
Doses: 1, 3, 10, 30 mg/kg Route of Administration: Oral Experimental Results: Significant reduction in blood glucose percentage. High-fat-fed (HFF) female rats: Female Sprague-Dawley rats (6 weeks old) are fed a high-fat diet (60% kcal from fat) for 4 weeks to induce insulin resistance. On the study day, rats are fasted for 6 h (morning) and then given GKA50 (3, 10, or 30 mg/kg) or vehicle (0.5% carboxymethylcellulose + 0.1% Tween 80) by oral gavage. Blood glucose is measured at baseline and at 0.5, 1, 2, 4, 6, 8, and 24 h using a glucometer. For chronic study, rats receive GKA50 (10 mg/kg) once daily for 14 days, and an oral glucose tolerance test (OGTT, 2 g/kg glucose) is performed on day 13. Blood samples are also collected for insulin measurement. At the end of the study, livers are collected for glycogen measurement (enzyme method) and for assessment of GK activity. |
| ADME/Pharmacokinetics |
Pharmacokinetics of GKA50 in rats: After oral administration (10 mg/kg), Cmax is 1.2 ug/mL (≈2.5 uM) at Tmax = 1 h; t½ = 2.3 h; oral bioavailability = 65% (due to high solubility and moderate logP). After IV (2 mg/kg), clearance = 18 mL/min/kg, Vd = 1.1 L/kg. Plasma protein binding is 92% (primarily albumin). The compound is metabolized by CYP2C9 and CYP3A4 in rat liver microsomes, with major metabolites being hydroxylated and glucuronidated forms. In humans (data from related GKAs), half-life is typically 2-4 hours. No published PK data for GKA50 specifically in humans because it is a preclinical tool.
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| Toxicity/Toxicokinetics |
Acute toxicity in mice: oral LD50 > 500 mg/kg; no deaths at 200 mg/kg. In a 14-day repeat-dose study in rats (10, 30, 100 mg/kg/day p.o.), the highest dose causes mild hepatosteatosis and elevated liver enzymes (ALT, AST) by 2- to 3-fold, which reverse after washout. The NOAEL is 30 mg/kg/day. In a 28-day study in dogs (5, 15, 45 mg/kg/day), no significant toxicity is observed at 5 mg/kg; at 45 mg/kg, mild hypoglycemia (blood glucose 40-50 mg/dL) occurs, along with lethargy and reduced food intake. No genotoxicity (Ames test negative). The compound does not inhibit hERG (IC50 > 30 uM). The hepatosteatosis is attributed to increased lipogenesis secondary to GK activation in the liver, a class effect of GKAs.
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| References |
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| Additional Infomation |
GKA50 is a research tool for studying glucokinase activation in diabetes and metabolic syndrome. It is not approved for clinical use and has not entered human trials. However, several other GKAs (e.g., dorzagliatin, MK-0941) have advanced to clinical trials, with dorzagliatin recently approved in China for T2DM (as HuaTangNing). GKA50 is often used as a positive control in preclinical studies of GK activation. It is available from chemical suppliers for research only. The compound is also known as "GKA50" (no generic name). This compound is a key tool for understanding the pharmacology of GKAs, including their potential for causing hypoglycemia and hepatic steatosis, which have been challenges in clinical development.
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| Molecular Formula |
C₂₆H₂₈N₂O₆
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| Molecular Weight |
464.51
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| Exact Mass |
464.195
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| CAS # |
851884-87-2
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| Related CAS # |
GKA50 quarterhydrate
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| PubChem CID |
11340230
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| Appearance |
White to off-white solid powder
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| LogP |
4.839
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
11
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| Heavy Atom Count |
34
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| Complexity |
641
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| Defined Atom Stereocenter Count |
2
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| SMILES |
C[C@@H](CC1=CC=CC=C1)OC2=CC(=CC(=C2)C(=O)NC3=NC=C(C=C3)C(=O)O)O[C@@H](C)COC
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| InChi Key |
OCBMECSFDVUYQN-ROUUACIJSA-N
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| InChi Code |
InChI=1S/C26H28N2O6/c1-17(11-19-7-5-4-6-8-19)33-22-12-21(13-23(14-22)34-18(2)16-32-3)25(29)28-24-10-9-20(15-27-24)26(30)31/h4-10,12-15,17-18H,11,16H2,1-3H3,(H,30,31)(H,27,28,29)/t17-,18-/m0/s1
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| Chemical Name |
6-[[3-[(2S)-1-methoxypropan-2-yl]oxy-5-[(2S)-1-phenylpropan-2-yl]oxybenzoyl]amino]pyridine-3-carboxylic acid
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| Synonyms |
GKA50; GKA-50
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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) |
DMSO : ~46 mg/mL (~99.03 mM)
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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 | 2.1528 mL | 10.7640 mL | 21.5281 mL | |
| 5 mM | 0.4306 mL | 2.1528 mL | 4.3056 mL | |
| 10 mM | 0.2153 mL | 1.0764 mL | 2.1528 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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