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| Targets |
PF-07208254 targets branched-chain ketoacid dehydrogenase kinase (BDK, BCKDK), an enzyme that phosphorylates and inactivates the branched-chain alpha-keto acid dehydrogenase complex (BCKDH). BCKDH is the rate-limiting enzyme in BCAA catabolism. BDK is a serine/threonine kinase that negatively regulates BCAA oxidation. By allosterically inhibiting BDK with high potency (IC₅0 = 110 nM in biochemical assays), PF-07208254 prevents the phosphorylation of BCKDH, maintaining it in its active (dephosphorylated) state, thereby promoting BCAA breakdown and utilization. This mechanism reduces circulating BCAAs and improves metabolic health.
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| ln Vitro |
In vitro, PF-07208254 is a potent inhibitor of BDK with an IC₅0 of 110 nM in biochemical enzyme assays and a Ki of 54 nM. In human skeletal muscle cells, PF-07208254 inhibits BDK activity with an IC₅0 of 540 nM, demonstrating cellular potency. Treatment with PF-07208254 (0.1-10 uM) in cultured primary human myotubes increases BCAA oxidation and reduces BCAA accumulation. The compound is 10-fold more potent than the benchmark BDK inhibitor BT2 (IC₅0 1,100 nM). It does not show significant off-target activity against other protein kinases. In addition, PF-07208254 improves lipid metabolism and increases insulin sensitivity in cell-based metabolic assays.
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| ln Vivo |
In vivo, PF-07208254 has demonstrated significant efficacy in improving cardiometabolic parameters in mouse models. In diet-induced obese mice, treatment with PF-07208254 (10-50 mg/kg, oral) leads to reductions in lipid accumulation, enhanced insulin sensitivity, and improved cardiac function. The compound increases BCAA oxidation in peripheral tissues, lowers plasma BCAA levels, and improves metabolic flexibility (the ability to switch between fuel sources). In heart failure models, PF-07208254 improves cardiac function as measured by echocardiography (increased ejection fraction, reduced left ventricular end-diastolic volume). The compound also reduces markers of cardiac hypertrophy and fibrosis. PF-07208254 is orally bioavailable and exhibits good pharmacokinetic properties in rodents.
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| Enzyme Assay |
For non-cellular assays (BDK enzyme inhibition), recombinant human BDK (10 nM) is incubated with PF-07208254 (0.1-1000 nM) in kinase assay buffer (50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.01% Triton X-100) for 30 minutes at 25degC. The reaction is initiated by adding ATP (100 uM) and a biotinylated BCKDH peptide substrate (1 uM). After 60 minutes at 30degC, the reaction is stopped with EDTA. Phosphorylation of the substrate is quantified using a time-resolved fluorescence resonance energy transfer (TR-FRET) assay or by Western blot with an anti-phospho-BCKDH antibody. The IC₅0 (110 nM) and Ki (54 nM) are calculated. Alternatively, a radioactive kinase assay using 32P-ATP can be used. For selectivity screening, PF-07208254 is tested against a panel of 50+ protein kinases at 1 uM to assess off-target activity.
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| Cell Assay |
For cell-based assays, human primary myotubes or skeletal muscle cells (e.g., differentiated from human myoblasts) are seeded in 6-well plates (1×10⁶ cells/well) in DMEM with 10% FBS. After differentiation into myotubes, cells are treated with PF-07208254 (0.1-10 uM) for 24-48 hours. BCAA oxidation is measured by adding 13C-labeled leucine (100 uM) and quantifying 13CO2 production by IRMS. Cellular BCAA levels are measured by LC-MS/MS. BDK activity is measured in cell lysates by an immunoprecipitation kinase assay using an anti-BDK antibody and BCKDH peptide as substrate; phosphorylation is detected by anti-phospho-BCKDH Western blot. Insulin signaling is assessed by measuring AKT and AMPK phosphorylation by Western blot. Cell viability is assessed by MTT assay.
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| Animal Protocol |
For in vivo animal experiments, diet-induced obese (DIO) C57BL/6J mice or leptin-deficient ob/ob mice (8-12 weeks old) are used. PF-07208254 is formulated in 0.5% methylcellulose or 20% PEG300/80% water and administered orally once daily at doses of 10-50 mg/kg for 4-8 weeks. Body weight and food intake are monitored daily. An oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) are performed at weeks 4 and 6. Blood samples are collected for measurement of plasma BCAA levels (LC-MS/MS), glucose, insulin (ELISA), triglycerides, and cholesterol. Echocardiography is performed to assess cardiac function (ejection fraction, fractional shortening, left ventricular mass). At the end of the study, tissues (skeletal muscle, liver, heart, adipose tissue) are harvested for measurement of BCAA oxidation (using 13C-labeled leucine ex vivo), gene expression (qPCR for BCAA catabolic enzymes), and histology (H&E staining for lipid accumulation, Picrosirius Red for fibrosis). In heart failure models (e.g., transverse aortic constriction, TAC), PF-07208254 is administered at 10-30 mg/kg for 4 weeks.
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| ADME/Pharmacokinetics |
PF-07208254 has a molecular weight of 236.67 and a molecular formula of C7H2ClFO2S2. The compound is a solid powder with a melting point of 210-215degC. It is soluble in DMSO (≥10 mg/mL), slightly soluble in ethanol, and practically insoluble in water. For in vivo formulation, 10% DMSO + 40% PEG300 + 5% Tween 80 + 45% saline is recommended. The compound should be stored as a powder at -20degC for up to 3 years, and in solution at -80degC for up to 6 months or at -20degC for up to 1 month. It is stable under recommended storage conditions and should be protected from light. The compound is an allosteric BDK inhibitor that is 10-fold more potent than the existing tool compound BT2.
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| Toxicity/Toxicokinetics |
PF-07208254 is a tool compound intended for research use only. No formal toxicity studies have been published. In preclinical animal studies at doses up to 50 mg/kg/day for 8 weeks, the compound is well-tolerated with no reported overt toxicity. Common observations at higher doses may include gastrointestinal disturbances or mild weight loss. No genotoxicity, carcinogenicity, or reproductive toxicity studies have been reported. Standard laboratory safety precautions for handling organic chemicals should be followed. The compound is not approved for human use. It is recommended to handle PF-07208254 in a fume hood with appropriate PPE (gloves, lab coat, safety glasses). Avoid inhalation, ingestion, and skin contact.
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| References | |
| Additional Infomation |
PF-07208254 is a research compound and is not an approved drug. It has not undergone clinical trials for therapeutic use in humans. It is currently in preclinical development for the treatment of cardiometabolic diseases, including heart failure and type 2 diabetes. The compound is an allosteric BDK inhibitor with 10-fold higher potency than the existing tool compound BT2. It enhances BCAA oxidation and improves cardiac function and metabolic flexibility in mouse models. PF-07208254 is used as a tool compound for studying the role of the BCAA catabolic pathway in metabolic and cardiovascular diseases. It is NOT intended for human therapeutic use. Available for research use only. Refer to the literature for detailed in vivo and in vitro data: Roth Flach RJ et al.
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| Molecular Formula |
C7H2CLFO2S2
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| Molecular Weight |
236.670981884003
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| Exact Mass |
235.916
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| CAS # |
2573122-40-2
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| PubChem CID |
155435321
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| Appearance |
White to off-white solid powder
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| LogP |
3.6
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
1
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| Heavy Atom Count |
13
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| Complexity |
241
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1=C(SC2=C1SC(=C2Cl)C(=O)O)F
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| InChi Key |
UXMBSHHPPVOPJU-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C7H2ClFO2S2/c8-4-5-2(1-3(9)13-5)12-6(4)7(10)11/h1H,(H,10,11)
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| Chemical Name |
6-chloro-2-fluorothieno[3,2-b]thiophene-5-carboxylic acid
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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 | 4.2253 mL | 21.1265 mL | 42.2529 mL | |
| 5 mM | 0.8451 mL | 4.2253 mL | 8.4506 mL | |
| 10 mM | 0.4225 mL | 2.1126 mL | 4.2253 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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