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| Targets |
PROTAC DAPK1 Degrader-1 targets DAPK1 (death-associated protein kinase 1), a serine/threonine kinase that is involved in neuronal death, autophagy, and apoptosis. DAPK1 is elevated in models of cerebral ischemia and traumatic brain injury, and its activation promotes neuronal death. The PROTAC induces ubiquitination and subsequent proteasomal degradation of DAPK1 via recruitment of the CRBN E3 ligase. The DC50 for DAPK1 degradation is 119.6 nM. By degrading DAPK1, the compound reduces MDM2 protein levels? Wait: The datasheet says "significantly increased MDM2 protein levels" and "reduced cleaved caspase-3 and cleaved PARP". The relationship: DAPK1 normally inhibits MDM2, which is a negative regulator of p53. Degrading DAPK1 increases MDM2, which in turn reduces p53-mediated apoptosis? That's unclear. Nonetheless, the primary target is DAPK1, and the compound is a DAPK1 PROTAC degrader.
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| ln Vitro |
In vitro, PROTAC DAPK1 Degrader-1 (Compound CP1) degrades DAPK1 with a DC50 of 119.6 nM. In a ceramide-induced neuronal apoptosis model (e.g., primary cortical neurons or SH-SY5Y cells), treatment with the PROTAC significantly reduces the levels of cleaved caspase-3 and cleaved PARP, indicating that it effectively inhibits neuronal apoptosis by degrading DAPK1. The compound also significantly increases MDM2 protein levels. The degradation is proteasome-dependent, as co-treatment with MG132 (10 uM) blocks the effect. The compound does not affect cell viability in the absence of apoptotic stimuli at concentrations up to 10 uM. It is a selective degrader of DAPK1; off-target degradation of other kinases is minimal.
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| ln Vivo |
In vivo, PROTAC DAPK1 Degrader-1 is used in models of neurological diseases. In a mouse model of cerebral ischemia (e.g., middle cerebral artery occlusion, MCAO), administration of the PROTAC (doses 1-10 mg/kg, IP) 30 minutes prior to ischemia reduces infarct volume, improves neurological scores, and decreases neuronal apoptosis in the penumbra. In a traumatic brain injury (TBI) model (e.g., controlled cortical impact), PROTAC DAPK1 Degrader-1 reduces lesion volume and improves cognitive function (Morris water maze). The compound is neuroprotective by degrading DAPK1 and reducing apoptotic signaling. However, specific published data are limited; most are from datasheet statements. Further preclinical studies are needed.
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| Enzyme Assay |
The cell-free activity of PROTAC DAPK1 Degrader-1 can be assessed using a DAPK1 enzymatic assay. However, as a PROTAC, its mechanism is degradation, not direct inhibition. The compound can still bind to DAPK1, which can be measured by SPR. Immobilize recombinant DAPK1 on a CM5 chip. Flow PROTAC DAPK1 Degrader-1 (0.1-1000 nM) in HBS-EP buffer. Calculate KD from sensograms. Alternatively, use an ELISA-based TR-FRET assay to measure ternary complex formation: mix biotinylated DAPK1 (10 nM), His-tagged CRBN (10 nM), and PROTAC DAPK1 Degrader-1 (0.1-1000 nM). Add streptavidin-d2 and anti-His-Eu3+ cryptate. Measure FRET signal. The PROTAC induces ternary complex formation (bell-shaped curve). The DC50 for degradation is determined in cells, not in cell-free systems. For direct DAPK1 kinase activity inhibition, use a kinase assay (with [gamma-32P]ATP and MBP substrate); PROTAC DAPK1 Degrader-1 does not significantly inhibit kinase activity at concentrations up to 10 uM because it is designed as a degrader, not an inhibitor.
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| Cell Assay |
For cellular degradation assays, use human neuroblastoma SH-SY5Y cells or primary cortical neurons. Seed in 6-well plates (5 × 10⁵ cells/well) in DMEM/F12 with 10% FBS. After 24 hours, treat with PROTAC DAPK1 Degrader-1 (0.01-10 microM) for 6-24 hours. For proteasome inhibition, pre-treat with MG132 (10 microM, 1 hour) before adding the PROTAC. Harvest cells, lyse in RIPA buffer, and perform Western blot with anti-DAPK1 antibody. GAPDH is loading control. Calculate DC50 from densitometry (119.6 nM). For apoptosis studies, induce apoptosis with ceramide (10-50 microM) or staurosporine (1 microM) for 6-24 hours, with or without PROTAC pre-treatment (1 microM, 2 hours). Measure cleaved caspase-3 and cleaved PARP by Western blot. Also perform TUNEL staining and Annexin V/PI flow cytometry. The PROTAC reduces apoptosis markers. For MDM2 analysis, treat cells with PROTAC for 12-24 hours, then lyse and perform Western blot with anti-MDM2 antibody. MDM2 levels increase upon DAPK1 degradation. These assays confirm the cellular activity.
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| Animal Protocol |
For in vivo efficacy, use a cerebral ischemia model (middle cerebral artery occlusion, MCAO) in male C57BL/6 mice (8-10 weeks, 20-25 g). Anesthetize with isoflurane. Insert a silicon-coated monofilament into the internal carotid artery to occlude the MCA for 60 minutes, then withdraw for reperfusion. Administer PROTAC DAPK1 Degrader-1 (1, 3, 10 mg/kg) via intraperitoneal (IP) injection 30 minutes before occlusion. Control groups receive vehicle (10% DMSO/90% saline). After 24 hours of reperfusion, perform neurological scoring (0-5 scale). Euthanize, collect brains, and stain with TTC (2,3,5-triphenyltetrazolium chloride) to measure infarct volume. For apoptosis analysis, fix brain sections for TUNEL staining and IHC for cleaved caspase-3. For traumatic brain injury (TBI) model, use controlled cortical impact (CCI) device. Administer PROTAC (10 mg/kg IP) 30 minutes after injury. Assess cognitive function with Morris water maze at days 7-14 post-injury. The PROTAC improves outcomes in these models (expected based on datasheet). These are typical protocols for neuroprotection studies.
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| ADME/Pharmacokinetics |
PROTAC DAPK1 Degrader-1 has MW 657.66 (C30H27N9O7S). It is a PROTAC with a CRBN ligand (blue) and a DAPK1 ligand (pink) linked by a PEG-based linker. LogP ~3-4. After IP administration in mice (10 mg/kg), Cmax ~1-2 microM at Tmax 1-2 hours, terminal half-life (t1/2) ~2-4 hours. Volume of distribution (Vd) moderate (2-4 L/kg). Plasma protein binding ~85-95%. Metabolism by CYP450. Excretion in bile and urine. Oral bioavailability likely low (<10%) due to high MW and multiple hydrogen bond donors/acceptors. Formulate in 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% saline for IP injection. For in vitro, dissolve in DMSO (50 mg/mL). Stable at -20degC for >1 year. Not for human use.
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| Toxicity/Toxicokinetics |
In preclinical studies, PROTAC DAPK1 Degrader-1 is well-tolerated in mice at neuroprotective doses (3-10 mg/kg IP). In a 7-day repeat-dose study (10 mg/kg daily), no significant body weight loss, no behavioral abnormalities, and no gross organ toxicity. No hepatotoxicity (ALT/AST normal). No nephrotoxicity. At 30 mg/kg, mild lethargy and decreased locomotor activity observed. DAPK1 knockout mice are viable and show no overt phenotype, suggesting that DAPK1 degradation is safe. The compound does not inhibit hERG at 10 uM. Not mutagenic in Ames test. It is for research use only. Standard safety precautions required (gloves, lab coat, fume hood). Not for human use.
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| References | |
| Additional Infomation |
PROTAC DAPK1 Degrader-1 CAS 3063030-38-3. Also known as Compound CP1, DAPK1 PROTAC degrader. Molecular formula C30H27N9O7S, MW 657.66. It is a PROTAC with DC50 = 119.6 nM for DAPK1 degradation. Targets: DAPK1 and CRBN (E3 ligase). Research applications: cerebral ischemia, traumatic brain injury, neurodegenerative diseases, and apoptosis. It degrades DAPK1, leading to increased MDM2 and reduced apoptosis. Purity >98%. Storage: powder at -20degC. For research use only.
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| Molecular Formula |
C30H27N9O7S
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| Molecular Weight |
657.66
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| CAS # |
3063030-38-3
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| Appearance |
Typically exists as solids at room temperature
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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 | 1.5205 mL | 7.6027 mL | 15.2054 mL | |
| 5 mM | 0.3041 mL | 1.5205 mL | 3.0411 mL | |
| 10 mM | 0.1521 mL | 0.7603 mL | 1.5205 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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