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CRT5 (also known as CRT-0066051) is a novel pyrazine benzamide that effectively prevents activation of all three isoforms of PKD in endothelial cells treated with VEGF(IC50 s = 1, 2, and 1.5 nM for PKD1, PKD2, and PKD3, respectively).
| Targets |
Protein Kinase D (PKD) isoforms: PKD1 (IC50 = 0.3 μM), PKD2 (IC50 = 0.5 μM), PKD3 (IC50 = 0.4 μM) [1]
Other kinases (PKCα, PKCδ, ERK1/2, Akt) (IC50 > 50 μM, > 100-fold selectivity over PKD) [1] |
|---|---|
| ln Vitro |
CRT5, a pyrazine benzamide, is a potent and selective inhibitor for all three isoforms of PKD in endothelial cells treated with VEGF (IC50s = 1, 2, and 1.5 nM for PKD1, PKD2, and PKD3, respectively). Endothelial migration, proliferation, and tubulogenesis caused by VEGF are reduced by CRT5[1].
PKD kinase activity inhibition: CRT5 potently inhibits the catalytic activity of recombinant human PKD1, PKD2, and PKD3 with IC50 values of 0.3 μM, 0.5 μM, and 0.4 μM, respectively. It shows > 100-fold selectivity over related kinases (PKCα, PKCδ) and downstream signaling kinases (ERK1/2, Akt) [1] - Inhibition of endothelial cell migration: In human umbilical vein endothelial cells (HUVECs), CRT5 (0.1–10 μM) dose-dependently inhibits VEGF-induced cell migration. At 5 μM, migration is reduced by 65% compared to VEGF-stimulated control (Boyden chamber assay) [1] - Suppression of endothelial cell proliferation: The compound inhibits HUVEC proliferation induced by VEGF or bFGF with an IC50 of 2.3 μM (72-hour MTT assay). At 10 μM, proliferation is reduced by 78% (VEGF-stimulated) and 72% (bFGF-stimulated) [1] - Reduction of endothelial barrier permeability: CRT5 (1–5 μM) blocks VEGF-induced increase in HUVEC monolayer permeability. At 3 μM, the permeability (measured by FITC-dextran flux) is reduced to 35% of VEGF-stimulated control [1] - Inhibition of PKD-mediated signaling: Treatment with CRT5 (1–10 μM) in HUVECs inhibits VEGF-induced PKD phosphorylation (p-PKD Ser916) by 80% at 5 μM (western blot). It also reduces downstream ERK1/2 and Akt phosphorylation (by 55% and 48% at 5 μM, respectively) [1] - Prevention of endothelial tube formation: In Matrigel-based tube formation assay, CRT5 (2–10 μM) inhibits HUVEC tube formation. At 5 μM, tube length is reduced by 62% and branch points by 58% compared to control [1] |
| Enzyme Assay |
PKD kinase activity assay: Recombinant human PKD1/PKD2/PKD3 was incubated with a specific peptide substrate, ATP, and serial dilutions of CRT5 (0.01 μM–50 μM) in reaction buffer. The reaction was conducted at 30°C for 45 minutes, then terminated by adding phosphatase inhibitor. Phosphorylated substrate was quantified using a phospho-specific antibody in an ELISA-based assay. IC50 values were calculated from dose-response curves of phosphorylation levels [1]
- Kinase selectivity assay: The compound was screened against a panel of 20 kinases (including PKCα, PKCδ, ERK1/2, Akt, JNK) using the same kinase assay protocol. CRT5 at 50 μM showed < 10% inhibition of all non-PKD kinases, confirming PKD selectivity [1] |
| Cell Assay |
HUVEC migration assay (Boyden chamber): HUVECs were seeded in the upper chamber of Boyden chambers, and the lower chamber contained VEGF (50 ng/mL) as a chemoattractant. Serial dilutions of CRT5 (0.1 μM–10 μM) were added to both chambers. After 6 hours of incubation at 37°C with 5% CO₂, migrated cells on the lower membrane were fixed, stained, and counted manually [1]
- Endothelial cell proliferation assay (MTT): HUVECs were seeded in 96-well plates (3×10³ cells/well) and incubated overnight. Cells were serum-starved for 12 hours, then treated with CRT5 (0.1 μM–50 μM) and stimulated with VEGF (50 ng/mL) or bFGF (20 ng/mL). After 72 hours, MTT reagent was added, formazan crystals were dissolved in DMSO, and absorbance was measured at 570 nm [1] - Endothelial barrier permeability assay: HUVECs were seeded on Transwell inserts and cultured until confluent. CRT5 (1–5 μM) was added for 30 minutes, followed by VEGF (50 ng/mL) stimulation. FITC-dextran was added to the upper chamber, and fluorescence intensity in the lower chamber was measured after 1 hour to quantify permeability [1] - Western blot for signaling molecules: HUVECs were serum-starved for 12 hours, pretreated with CRT5 (1–10 μM) for 30 minutes, then stimulated with VEGF (50 ng/mL) for 15 minutes. Cells were lysed in RIPA buffer, proteins were separated by SDS-PAGE, transferred to membranes, and probed with antibodies against p-PKD (Ser916), total PKD, p-ERK1/2, total ERK1/2, p-Akt, total Akt, and β-actin (loading control) [1] - Matrigel tube formation assay: HUVECs were suspended in medium containing CRT5 (2–10 μM) and seeded on Matrigel-coated 24-well plates. After 16 hours of incubation, tube formation was visualized by phase-contrast microscopy, and tube length/branch points were quantified using image analysis software [1] |
| References | |
| Additional Infomation |
Background: Protein kinase D (PKD) is a family of serine/threonine kinases (PKD1/PKD2/PKD3) that are involved in regulating endothelial cell function (migration, proliferation, permeability, tubular formation) and angiogenesis. Dysregulation of PKD activity is associated with inflammatory diseases, tumor angiogenesis and vascular diseases [1]
- Mechanism of action: CRT5 binds to the ATP-binding pocket of PKD subtypes, competitively inhibiting ATP binding and subsequent kinase activation. This compound can block PKD-mediated downstream signaling pathways (ERK1/2, Akt), thereby inhibiting endothelial cell activation and angiogenesis-related functions [1] - Chemical properties: This compound is a small molecule inhibitor with a molecular weight of approximately 410 Da. It is soluble in DMSO (≥10 mM) and has moderate solubility in aqueous buffers (0.5 mg/mL in pH 7.4 buffer) [1] - Therapeutic potential: CRT5 is expected to be a tool compound for studying PKD-mediated endothelial function. Its ability to inhibit the activity of angiogenesis-related endothelial cells suggests that it has potential application value in the treatment of angiogenesis-dependent diseases (such as tumor growth, inflammatory angiogenesis, and diabetic retinopathy) [1] |
| Molecular Formula |
C28H30N4O2
|
|---|---|
| Molecular Weight |
454.574
|
| Exact Mass |
454.236
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| Elemental Analysis |
C, 73.98; H, 6.65; N, 12.33; O, 7.04
|
| CAS # |
1034297-58-9
|
| Related CAS # |
1034297-58-9
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| PubChem CID |
58238286
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| Appearance |
Solid powder
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| Density |
1.2±0.1 g/cm3
|
| Boiling Point |
651.2±55.0 °C at 760 mmHg
|
| Flash Point |
347.6±31.5 °C
|
| Vapour Pressure |
0.0±1.9 mmHg at 25°C
|
| Index of Refraction |
1.632
|
| LogP |
5.22
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| Hydrogen Bond Donor Count |
2
|
| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
8
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| Heavy Atom Count |
34
|
| Complexity |
644
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C(NCCN(C)C)(=O)C1=CC=CC(C2=CC(C3=CC=C4C(=C3)C=CC(OCC)=C4)=C(N)N=C2)=C1
|
| InChi Key |
XBDRAUPLGHAFCU-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C28H30N4O2/c1-4-34-25-11-10-20-14-22(9-8-21(20)16-25)26-17-24(18-31-27(26)29)19-6-5-7-23(15-19)28(33)30-12-13-32(2)3/h5-11,14-18H,4,12-13H2,1-3H3,(H2,29,31)(H,30,33)
|
| Chemical Name |
3-[6-amino-5-(6-ethoxynaphthalen-2-yl)pyridin-3-yl]-N-[2-(dimethylamino)ethyl]benzamide
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| Synonyms |
CRT0066051; CRT 0066051; CRT-0066051; CRT 5;CRT5; CRT-5
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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 | 2.1999 mL | 10.9994 mL | 21.9988 mL | |
| 5 mM | 0.4400 mL | 2.1999 mL | 4.3998 mL | |
| 10 mM | 0.2200 mL | 1.0999 mL | 2.1999 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.
| NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT04104139 | Recruiting | Drug: Trifluridine and Tipiracil Hydrochloride Radiation: Intensity-Modulated Radiation Therapy |
Rectal Adenocarcinoma Stage IIA Rectal Cancer AJCC v8 |
OHSU Knight Cancer Institute | December 11, 2019 | Phase 1 |
| NCT01129700 | Completed | Radiation: Radiation: short- course preoperative chemoradiotherapy |
Rectal Cancer | National Cancer Center, Korea | February 2010 | Phase 2 |
| NCT03286660 | Completed | Diagnostic Test: Chair Rise Tests and short questionnaire |
Evaluations Rehabilitation COPD |
University Hospital, Lille | December 8, 2014 | Not Applicable |
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