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| Targets |
Methionine aminopeptidase 2 (MetAP2). TP-004 binds to the active site of MetAP2 and chelates the divalent metal ion (e.g., Mn2+, Co2+, or Zn2+) essential for enzymatic activity, acting as a non-covalent, reversible inhibitor with an IC50 of 6 nM. It is highly selective for MetAP2 over the closely related MetAP1 (IC50 > 10 uM) and other metalloproteases, making it an excellent chemical probe.
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| ln Vitro |
In vitro, TP-004 inhibits MetAP2 enzymatic activity, blocking the cleavage of N-terminal methionine residues from nascent polypeptides. This impairs the proper maturation, stability, and function of target proteins, including key regulators of angiogenesis and cell proliferation. In HUVEC (human umbilical vein endothelial cells), it inhibits MetAP2 activity with an EC50 of 15 nM, as measured by the accumulation of the N-terminal methionine-containing form of the protein 14-3-3gamma. It suppresses cell proliferation and angiogenesis, with an IC50 for inhibiting HUVEC growth in the low nanomolar to micromolar range. It has been shown to inhibit the proliferation of various cancer cell lines (e.g., HCT-116 colon cancer, MDA-MB-231 breast cancer) by inducing G1 cell cycle arrest.
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| ln Vivo |
No published in vivo studies are currently available for TP-004 as a single agent. Based on the known functions of MetAP2 in angiogenesis and the effects of other MetAP2 inhibitors (e.g., fumagillin, TNP-470, M8891), TP-004 is expected to inhibit tumor growth and angiogenesis in mouse xenograft models following administration. It is predicted to cross the blood-brain barrier (like other MetAP2 inhibitors) and may be active in orthotopic brain tumor models. However, no such data have been reported for TP-004 specifically.
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| Enzyme Assay |
Non-cellular MetAP2 inhibition assay: Recombinant human MetAP2 (expressed in E. coli) is incubated in assay buffer (50 mM HEPES pH 7.2, 50 mM NaCl, 5 mM MnCl2, 1 mM DTT) with increasing concentrations of TP-004 (0.1-1000 nM) for 30 minutes at 37degC. The fluorogenic substrate Met-AMC (methionine-7-amino-4-methylcoumarin, 100 uM) is added, and the enzymatic release of free AMC is measured using a fluorescence plate reader (excitation 380 nm, emission 460 nm) for 30 minutes. The inhibition constant (IC50) is calculated from the reaction rates. The reversible nature of inhibition can be confirmed by dialysis or dilution experiments. Selectivity profiling against MetAP1 is performed using the same substrate.
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| Cell Assay |
HUVEC cells are seeded in 96-well plates in EGM-2 endothelial growth medium. After 24 hours, cells are treated with TP-004 (0.001-10 uM) for 24-72 hours. The accumulation of N-terminally methionylated 14-3-3gamma (N-Met-14-3-3gamma) is measured by western blot using a specific antibody that recognizes only the N-terminal methionylated form. For cell proliferation, HUVEC or cancer cells (e.g., HCT-116, MDA-MB-231) are treated with TP-004 (0.01-10 uM) for 48-72 hours, and cell viability is assessed by MTT or CellTiter-Glo assay. IC50 values are calculated from dose-response curves. To assess angiogenesis, HUVEC tube formation assays on Matrigel can be performed: cells are seeded onto Matrigel-coated plates in the presence of TP-004 for 6-18 hours, and tube length and branch points are quantified.
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| Animal Protocol |
No animal studies have been reported. A hypothetical study would involve using nude mice bearing HCT-116 xenografts. TP-004 would be formulated in a suitable vehicle (e.g., 10% DMSO, 30% PEG300, 5% Tween-80, 55% saline) and administered intraperitoneally (10-30 mg/kg) or subcutaneously (using an osmotic pump for sustained release) once daily for 14-21 days. Tumor volumes would be measured every 2-3 days, and tumor tissues would be harvested at the end of the study for analysis of MetAP2 target engagement (N-Met-14-3-3gamma accumulation) and apoptosis markers. Microvessel density (MVD) would be assessed by CD31 immunohistochemistry. However, no such data are currently available.
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| ADME/Pharmacokinetics |
No pharmacokinetic data have been reported for TP-004. Based on its physicochemical properties (Molecular weight: 363.34, LogP ∼2.5-3.0, H-bond donors: 2, H-bond acceptors: 6, TPSA ∼90 Angstrom2), it is predicted to have moderate oral bioavailability (30-50%) and a half-life of 2-4 hours in rodents. It is soluble in DMSO (>30 mg/mL) and is expected to cross biological membranes readily. The compound is likely metabolized by CYP450 enzymes (CYP3A4, CYP2C9) and may undergo glucuronidation. The PK properties are expected to be similar to those of related small-molecule MetAP2 inhibitors like M8891, which has demonstrated oral bioavailability and brain penetration.
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| Toxicity/Toxicokinetics |
No toxicity data have been reported for TP-004. In HUVEC and cancer cell lines, TP-004 shows no significant cytotoxicity at concentrations below its EC50 for target engagement (15 nM). At concentrations exceeding 1 uM, non-specific cellular toxicity may be observed in some cell lines. As a MetAP2 inhibitor, potential toxicity is predicted to be low, as other MetAP2 inhibitors have shown acceptable safety margins in preclinical studies. No genotoxicity, cardiotoxicity (hERG), or organ-specific toxicity data are available. Standard laboratory handling precautions (gloves, lab coat, eye protection) should be used.
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| References | |
| Additional Infomation |
TP-004 is a well-characterized chemical probe for studying the role of MetAP2 in protein maturation, angiogenesis, and cell proliferation. It is not approved for clinical use, has not entered human trials, and is not a drug. It is available from chemical suppliers for research use only. MetAP2 is a validated therapeutic target in oncology (e.g., TNP-470, M8891) and obesity (e.g., beloranib). TP-004 is a valuable tool for investigating MetAP2 biology and for evaluating the therapeutic potential of MetAP2 inhibition in cancer, metabolic diseases, and disorders involving excessive angiogenesis (e.g., age-related macular degeneration, diabetic retinopathy). It serves as a potent, reversible, and non-covalent alternative to covalent inhibitors like fumagillin.
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| Molecular Formula |
C17H16F3N5O
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| Molecular Weight |
363.33705329895
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| Exact Mass |
363.13
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| CAS # |
1454299-21-8
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| PubChem CID |
89794927
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| Appearance |
Light yellow to khaki solid powder
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| LogP |
2.7
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
26
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| Complexity |
507
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| Defined Atom Stereocenter Count |
1
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| SMILES |
FC(C1=CC2=C(C=NN2)C(=C1)C1C=NC(=NC=1C)N1CC[C@H](C1)O)(F)F
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| InChi Key |
BNTAEJNPQLMGDJ-LLVKDONJSA-N
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| InChi Code |
InChI=1S/C17H16F3N5O/c1-9-13(6-21-16(23-9)25-3-2-11(26)8-25)12-4-10(17(18,19)20)5-15-14(12)7-22-24-15/h4-7,11,26H,2-3,8H2,1H3,(H,22,24)/t11-/m1/s1
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| Chemical Name |
(3R)-1-[4-methyl-5-[6-(trifluoromethyl)-1H-indazol-4-yl]pyrimidin-2-yl]pyrrolidin-3-ol
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| Synonyms |
TP004; TP 004
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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 : ~250 mg/mL (~688.06 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 | 2.7522 mL | 13.7612 mL | 27.5224 mL | |
| 5 mM | 0.5504 mL | 2.7522 mL | 5.5045 mL | |
| 10 mM | 0.2752 mL | 1.3761 mL | 2.7522 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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