The mechanism of action of DB0662 involves binding to a specific kinase of interest (target protein) and to an E3 ubiquitin ligase, bringing them into close proximity. This leads to the ubiquitination of the kinase and its subsequent degradation by the 26S proteasome. The compound is designed for kinase-targeted protein degradation and can be used in studies of diseases mediated by abnormal kinase activity. It may also be used for the identification of degradable kinases (i.e., kinases that can be effectively degraded by this compound or related PROTACs) and optimal kinases (i.e., those that are most effectively degraded by this molecular design). The specific kinase(s) targeted by DB0662 are not specified in the search results; the compound is described as a general tool for studying kinase degradation. It likely targets a panel of kinases or a specific kinase family, depending on the warhead used. DB0662 is a research tool for validating the utility of PROTACs in targeting previously "undruggable" kinases or for overcoming resistance to kinase inhibitors.

In vitro studies demonstrate that DB0662 is a compound for kinase-targeted protein degradation. It is likely a PROTAC (proteolysis-targeting chimera) that facilitates the degradation of specific kinases. The compound can be used in cell-based assays to evaluate its ability to reduce the levels of target kinases. Standard experimental protocols involve treating cells (e.g., cancer cell lines) with DB0662 at varying concentrations (0.01-10 uM) for 6-48 hours. The levels of target kinases and downstream signaling proteins are then assessed by Western blotting or targeted proteomics (e.g., NanoString, mass spectrometry). The compound's efficacy (DC50, the concentration for 50% degradation) and maximal degradation (Dmax, the percentage of target degraded at saturation) are calculated. The compound's effect on cellular proliferation (MTT assay) and viability (CellTiter-Glo) can also be assessed. DB0662 may have a different pharmacological profile compared to traditional kinase inhibitors, as it eliminates the target protein rather than just inhibiting its catalytic activity. This may lead to sustained pathway suppression and potentially overcome resistance caused by mutations in the kinase that interfere with inhibitor binding. The specificity of DB0662 can be assessed by proteome-wide mass spectrometry to identify off-target degraded proteins. The compound is likely used as a probe to discover which kinases are "degradable" (i.e., can be effectively degraded by a PROTAC design) versus those that are not.

In vivo activity data for DB0662 is not publicly available. As a PROTAC, its in vivo efficacy would depend on its pharmacokinetic properties (e.g., oral bioavailability, half-life, tissue distribution) and its ability to engage both the target kinase and the E3 ligase in vivo. Typical animal studies for PROTACs involve subcutaneous xenograft mouse models of cancer. DB0662 would be administered via intraperitoneal (i.p.) or intravenous (i.v.) injection at doses of 10-100 mg/kg, daily or every other day for 2-4 weeks. Tumor growth inhibition would be measured, and tumor tissues would be collected at endpoint to assess target kinase degradation by Western blotting and IHC. The compound's ability to induce protein degradation in vivo would be confirmed. No specific data is available in the search results.

Non-cell-based assays for DB0662 could include surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) to measure the binding affinity of the compound for the target kinase and for the E3 ligase (e.g., CRBN or VHL). A standard SPR assay would immobilize the purified kinase or E3 ligase on a sensor chip, and increasing concentrations of DB0662 (1-1000 nM) would be injected to determine the binding kinetics (ka, kd) and affinity (KD). A microscale thermophoresis (MST) assay could also be used. To assess ternary complex formation (the simultaneous binding of the PROTAC to both the kinase and the E3 ligase), a time-resolved FRET (TR-FRET) assay or a surface plasmon resonance (SPR) with sequential injections (kinase, followed by PROTAC, followed by E3 ligase) can be performed. For example, biotinylated target kinase is immobilized on a streptavidin sensor chip. DB0662 (0.1-10 uM) is injected, followed by a solution containing the E3 ligase (e.g., CRBN or VHL). An increase in response units upon the E3 ligase injection indicates formation of the ternary complex. The kinetics of ternary complex formation and the half-life of the complex can be determined. For a homogenous TR-FRET assay, the target kinase is labeled with a donor fluorophore (e.g., terbium) and the E3 ligase is labeled with an acceptor fluorophore (e.g., d2). The addition of DB0662 brings the two proteins into close proximity, resulting in increased FRET signal. The EC50 for ternary complex formation is calculated. For ubiquitination assays, purified components (kinase, E3 ligase, E2 ubiquitin-conjugating enzyme, ubiquitin, ATP) are incubated with DB0662 in a test tube for 1-2 hours at 37degC. The reaction is then subjected to SDS-PAGE and immunoblotted with an anti-ubiquitin antibody to detect the formation of polyubiquitin chains on the kinase. An increase in high molecular weight smears indicates ubiquitination. A cell-free degradation assay using purified 26S proteasome and ubiquitinated kinase can be used to assess the final step of degradation.

For cell-based studies, cancer cell lines expressing the target kinase (e.g., HEK293, HeLa, or specific cancer cell lines) are cultured in DMEM with 10% FBS and 1% penicillin-streptomycin at 37degC in a 5% CO2 incubator. Cells are seeded in 6-well plates (3-5×10^5 cells/well) and treated with DB0662 at varying concentrations (0.001-10 uM) for 6-48 hours. Positive control: a known PROTAC or an inhibitor of the target kinase. Negative control: DMSO vehicle. After treatment, cells are washed with PBS and lysed in RIPA buffer with protease and phosphatase inhibitors. Protein lysates (20-50 ug) are separated by SDS-PAGE and immunoblotted with antibodies against the target kinase, downstream signaling proteins (e.g., phospho-ERK, total ERK), and a loading control (e.g., GAPDH, beta-actin). The intensity of the bands is quantified by densitometry, and the DC50 and Dmax are calculated by fitting the data to a four-parameter logistic equation. To determine if degradation is mediated by the proteasome, cells are co-treated with DB0662 and a proteasome inhibitor (e.g., MG132, 10 uM) or a NEDD8-activating enzyme inhibitor (MLN4924, which prevents ubiquitination). The rescue of target protein levels by these inhibitors confirms on-target degradation. To assess the role of the E3 ligase, cells are pre-treated with a competitive ligand for the E3 ligase (e.g., lenalidomide for CRBN, or VHL ligand for VHL) to block the binding of DB0662 to the E3 ligase. A rescue of target protein levels indicates that the degradation is mediated by that specific E3 ligase. For cell proliferation assays, cells are seeded in 96-well plates (5×10^3 cells/well) and treated with DB0662 (0.001-10 uM) for 48-96 hours, and viability is measured by MTT or CellTiter-Glo. The effect on colony formation (soft agar assay) and cell migration (scratch wound assay) can also be assessed. For cell cycle analysis, cells are treated with DB0662 for 24-48 hours, fixed in 70% ethanol, stained with propidium iodide (PI) and RNase, and analyzed by flow cytometry. Apoptosis can be assessed by Annexin V-FITC/PI staining and flow cytometry, or by measuring caspase-3/7 activity. For proteomics analysis, cells are treated with DB0662 or DMSO for 6-24 hours, proteins are extracted, digested with trypsin, and analyzed by LC-MS/MS. The fold change of thousands of proteins is quantified, and the selectivity of degradation is assessed by the number of significantly downregulated proteins (target degraders) versus non-targets. The compound may also be used to study the kinetics of target degradation using a time-course (e.g., 0, 2, 4, 8, 12, 24, 48 hours) and to evaluate the "hook effect" (lower degradation at high concentrations due to formation of binary complexes).

In vivo protocols for DB0662 are not established in the public domain. For a typical PROTAC, efficacy studies are performed in xenograft mouse models. Female athymic nude mice (6-8 weeks old) are injected subcutaneously with cancer cells (e.g., 5×10^6 cells) in the flank. When tumors reach 100-200 mm3, mice are randomized (n=8-10 per group). DB0662 is formulated in a vehicle such as 10% DMSO, 40% PEG300, 5% Tween-80, and 45% saline, or a more complex vehicle for PROTACs (e.g., 10% DMSO, 30% PEG400, 10% Solutol HS15, 50% water). The compound is administered via intraperitoneal (i.p.) or intravenous (i.v.) injection at doses of 10, 30, 100 mg/kg, once daily or every other day for 14-28 days. Control groups receive vehicle alone, and a positive control group receives a standard-of-care drug or a kinase inhibitor. Tumor volumes are measured every 2-3 days with calipers, and body weight is monitored. At the endpoint, tumors are excised, weighed, and processed for Western blotting to assess target protein degradation and downstream signaling. Immunohistochemistry (IHC) for the target protein, Ki-67 (proliferation), and cleaved caspase-3 (apoptosis) is performed. Blood samples are collected at the end of the study for serum chemistry (ALT, AST, BUN, creatinine) and hematology. For pharmacokinetic (PK) studies, separate cohorts of mice (n=3 per time point) receive a single i.v. or i.p. dose of DB0662 (e.g., 10 mg/kg), and plasma is collected at 0, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24 hours. Compound concentrations are measured by LC-MS/MS. The plasma half-life (t1/2), maximum concentration (Cmax), area under the curve (AUC), and clearance (CL) are calculated. For pharmacodynamic (PD) studies, tumors are collected at various time points after a single dose to assess target degradation kinetics in vivo. The relationship between PK (exposure) and PD (degradation) can be modeled.

Detailed PK data for DB0662 is not available. As a PROTAC (MW 932 Da, LogP likely 3-5), it may have low water solubility and moderate to good permeability. PROTACs often have poor oral bioavailability due to their high molecular weight and polarity, and are typically administered by intraperitoneal (i.p.) or intravenous (i.v.) injection in preclinical studies. The plasma half-life may be short (e.g., 1-4 hours), and the volume of distribution (Vd) may be moderate to high. The compound is likely metabolized by CYP450 enzymes and may undergo phase II conjugation. For in vitro assays, stock solutions are prepared in DMSO (e.g., 10-50 mM) and diluted in cell culture medium, keeping the final DMSO concentration below 0.1%. The compound should be stored as a powder at -20degC, protected from light and moisture. In solution, it should be stored at -80degC in aliquots and used within 3-6 months.

Preclinical toxicity data for DB0662 is not available. As a PROTAC, its safety profile may differ from that of a conventional kinase inhibitor. On-target toxicity due to degradation of the target kinase in normal tissues, as well as off-target degradation of other proteins, could lead to adverse effects. In cell viability assays, the compound's IC50 is likely in the low to sub-micromolar range in sensitive cancer cell lines. In animal models, dose-limiting toxicities might include liver toxicity, gastrointestinal disturbances, or hematological effects. No genotoxicity, hERG inhibition, or reproductive toxicity data is available. Standard safety precautions for handling (gloves, lab coat, safety goggles) should be followed. The compound is for research use only and is not for human use.

[1]. Nathanael S. Gray, et al. Compounds for targeted protein degradation of kinases. WO2022093742.

DB0662 is a research compound for kinase-targeted protein degradation. It is classified as a PROTAC (proteolysis-targeting chimera) and is used to study the biological consequences of targeted protein degradation of kinases. It is not a drug and is not FDA-approved. It is supplied as a solid powder and should be stored at -20degC, protected from light and moisture. In solution (DMSO), it should be stored at -80degC. The compound is a valuable tool for chemical biology research, enabling the validation of novel kinase targets and the development of new therapeutic strategies for diseases driven by aberrant kinase activity. It is part of a growing class of molecules that exploit the ubiquitin-proteasome system to eliminate disease-causing proteins, offering advantages over traditional inhibitors, including the potential to overcome drug resistance and target non-enzymatic functions of kinases.

C47H44F3N11O7

931.916979789734

931.337

2769753-51-5

165412591

Light yellow to yellow solid powder

4.4

3

16

12

68

1870

0

CC1=C(C=C(C=C1)NC(=O)C2=CC(=CC=C2)C(F)(F)F)N3CC4=CN=C(N=C4N(C3=O)C)NC5=CN=C(C=C5)N6CCN(CC6)CCCOC7=CC=CC8=C7C(=O)N(C8=O)C9CCC(=O)NC9=O

PAYXBIAZSMSRHO-UHFFFAOYSA-N

InChI=1S/C47H44F3N11O7/c1-27-10-11-31(53-41(63)28-6-3-7-30(22-28)47(48,49)50)23-35(27)60-26-29-24-52-45(56-40(29)57(2)46(60)67)54-32-12-14-37(51-25-32)59-19-17-58(18-20-59)16-5-21-68-36-9-4-8-33-39(36)44(66)61(43(33)65)34-13-15-38(62)55-42(34)64/h3-4,6-12,14,22-25,34H,5,13,15-21,26H2,1-2H3,(H,53,63)(H,52,54,56)(H,55,62,64)

N-[3-[7-[[6-[4-[3-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]oxypropyl]piperazin-1-yl]pyridin-3-yl]amino]-1-methyl-2-oxo-4H-pyrimido[4,5-d]pyrimidin-3-yl]-4-methylphenyl]-3-(trifluoromethyl)benzamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 50 mg/mL (53.65 mM)

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)