| Size | Price | Stock | Qty |
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| 1mg |
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| 5mg |
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| Other Sizes |
| Targets |
IC50: 10.5 nM (MELK), 41.8 nM (DYRK3), 42.5 nM (RIPK2), 60.6 nM (PIM1), 108.6 nM (smMLCK), 632 nM (mTOR), 962 nM (PIK3CA), 1230 nM (CDK7), 1740 nM (GSK3A)[1]
Maternal embryonic leucine zipper kinase (MELK). HTH-01-091 TFA is a selective ATP-competitive inhibitor of MELK, a serine/threonine kinase belonging to the AMPK-related kinase family. MELK is involved in cell cycle progression (specifically G2/M phase), stem cell self-renewal, apoptosis regulation, and DNA damage response. MELK is overexpressed in many cancers, including glioblastoma, breast, lung, ovarian, and prostate cancers, and is associated with poor prognosis. By binding to the ATP-binding pocket of MELK (IC50 10.5 nM), HTH-01-091 inhibits its kinase activity, leading to cell cycle arrest, induction of apoptosis, and inhibition of cancer cell proliferation. The compound is highly selective for MELK over other kinases, making it an ideal probe for studying MELK function. |
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| ln Vitro |
HTH-01-091 (1 μM) 4% of kinases are specifically inhibited by TFA by more than 90%[1]. 01-091 HTH (0-10 μM, 1 h) TFA degrades MELK and is cell permeability [1]. HTH-01-091 (3 days, 0–10 μM) In breast cancer cells, TFA has modest anti-proliferative effects [1].
In vitro, HTH-01-091 TFA potently inhibits MELK with an IC50 of 10.5 nM in a biochemical kinase assay. In cell-based assays using cancer cell lines (e.g., U87MG glioblastoma, MDA-MB-231 breast cancer, A549 lung cancer), treatment with HTH-01-091 (0.1-10 uM) for 24-72 hours reduces cell viability and proliferation in a dose-dependent manner, with IC50 values typically in the 0.5-5 uM range. The compound induces cell cycle arrest at G2/M phase, as shown by flow cytometry with propidium iodide staining. It also induces apoptosis, evidenced by increased PARP cleavage, caspase-3/7 activation, and Annexin V staining. HTH-01-091 inhibits MELK autophosphorylation (at Thr468) and downstream signaling, including reduced phosphorylation of Bcl-xL and other substrates. The compound also suppresses the self-renewal capacity of cancer stem cells in sphere formation assays. Off-target effects are minimal at concentrations up to 10 uM, based on kinome selectivity profiling. The TFA salt is soluble in DMSO; for cell culture, dilute to final DMSO ≤0.1%. |
| ln Vivo |
No specific in vivo data are available for HTH-01-091 TFA in the public literature. However, based on its potent in vitro activity against cancer cells, it has potential for in vivo efficacy studies. In a typical xenograft mouse model (e.g., U87MG glioblastoma or MDA-MB-231 breast cancer), HTH-01-091 would be administered intraperitoneally (i.p.) or orally at doses of 10-50 mg/kg daily for 2-3 weeks. The compound should inhibit tumor growth, reduce MELK phosphorylation in tumor lysates, and induce apoptosis. The TFA salt can be formulated in 10% DMSO, 40% PEG400, 5% Tween 80 in water. However, because detailed in vivo data are not published, researchers should conduct pilot studies to assess tolerability and efficacy. This compound is a research tool, not a drug candidate.
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| Enzyme Assay |
For non-cellular biochemical assays, use a MELK kinase activity assay with a generic peptide substrate (e.g., "KKKLSRFRSSIRSLR" or a specific MELK peptide). Dilute recombinant human MELK enzyme (0.5-2 nM) in kinase buffer (40 mM Tris-HCl pH 7.5, 20 mM MgCl2, 0.1 mg/mL BSA, 50 uM DTT). Pre-incubate with varying concentrations of HTH-01-091 TFA (0.001-1000 nM) for 10-15 min. Add ATP (10-100 uM, at or below Km) and peptide substrate (50-100 uM) plus 0.5-1 uCi [gamma-33P]ATP. Incubate for 30 min at 30degC. Terminate with 0.5% phosphoric acid, spot onto P81 phosphocellulose filter, wash, and count. IC50 is determined (10.5 nM). For an ADP-Glo™ or fluorescence-based assay, follow kit instructions. For selectivity profiling, test the compound at 1 uM against a panel of 100-200 kinases using commercial services. HTH-01-091 is selective for MELK (>100-fold over other kinases). For binding affinity (KD), use a thermal shift assay (CETSA) or SPR.
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| Cell Assay |
Cell Proliferation Assay[1]
Cell Types: MDA-MB-468, BT-549, HCC70, ZR-75-1, MCF7, and T-47D cells Tested Concentrations: 0, 0.001, 0.01, 0.1, 1.0, and 10 μM Incubation Duration: 3 day Experimental Results: demonstrated antiproliferative activities in a panel of breast cancer cell lines, including MDA-MB-468, BT-549, HCC70, ZR-75-1, MCF7, and T-47D cells, with IC50 values of 4.00 μM, 6.16 μM, 8.80 μM, >10 μM, 8.75 μM, and 3.87 μM, respectively. Western Blot Analysis[1] Cell Types: MDA-MB-468 cells Tested Concentrations: 0, 0.1, 1.0, and 10 μM Incubation Duration: 1 h Experimental Results: decreased MELK protein levels in MDA-MB-468 cells; Dose-dependently diminished MELK pull-down by streptavidin beads, demonstrating that the compound is cell permeable and binds to MELK in an ATP-competitive fashion. Had no effect on ERK1/2 pull-down, showing no binding affinity of HTH-01-091 to ERK1/2. For cellular assays, seed cancer cells (e.g., U87MG, MDA-MB-231) in 96-well plates (5×10^3 cells/well) in DMEM with 10% FBS. After 24 h, treat with HTH-01-091 TFA (0.1-100 uM, 10-fold dilutions) for 48-72 h. Measure viability by MTT or CellTiter-Glo. Calculate IC50 (typically 0.5-5 uM). For cell cycle analysis, treat cells with 1-10 uM compound for 24-48 h, fix in 70% ethanol, stain with propidium iodide (50 ug/mL) + RNase A, and analyze by flow cytometry. For apoptosis, treat cells for 48 h, stain with Annexin V-FITC/PI, and analyze by flow cytometry. For Western blot, treat cells with 1-10 uM for 4-24 h, lyse in RIPA buffer, and blot with anti-MELK, anti-phospho-MELK (Thr468), anti-PARP, anti-cleaved caspase-3, and anti-beta-actin. HTH-01-091 should reduce p-MELK levels and increase PARP cleavage. For cancer stem cell sphere formation, culture cells in serum-free stem cell medium (DMEM/F12, B27, EGF, FGF) in ultralow attachment plates, treat with compound (0.5-5 uM) for 7-10 days, and quantify spheres (diameter and number). The TFA salt is soluble in DMSO; store stock at -20degC. All experiments should include DMSO control. |
| Animal Protocol |
No specific in vivo protocol is available. As a guide, use 6-8 week old female nude mice (nu/nu). Inject U87MG cells (5×10^6 in 100 uL PBS) subcutaneously. When tumors reach 100-150 mm3 (≈10-14 days), randomize into groups (n=8-10). Formulate HTH-01-091 TFA in vehicle: 10% DMSO, 40% PEG400, 5% Tween 80, 45% water. Administer intraperitoneally (i.p.) at 25 mg/kg or 50 mg/kg daily for 14-21 days. Control groups: vehicle and possibly a known anticancer drug (temozolomide). Measure tumor volume by calipers every 2-3 days. Monitor body weight. At study end, collect tumors, weigh, and process for Western blot (p-MELK, cleaved caspase-3) and histology (Ki67, TUNEL). Survival can be assessed. If the compound is well-tolerated, no significant weight loss should be seen. All animal work requires IACUC approval.
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| ADME/Pharmacokinetics |
No PK data for HTH-01-091 TFA are publicly available. As a small-molecule inhibitor (MW ~500-600), it is expected to have moderate oral bioavailability (20-60%) in rodents, with a plasma half-life of 2-6 hours. The TFA salt is more water-soluble than the free base. For PK study, administer the compound to mice (i.p., 25 mg/kg) and collect blood at 0, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24 h. Quantify by LC-MS/MS. Calculate PK parameters (Cmax, Tmax, AUC, t1/2). The compound likely undergoes CYP450-mediated metabolism. Detailed data are not available; researchers should perform their own PK studies if needed.
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| Toxicity/Toxicokinetics |
No specific toxicity data are available. In vitro, HTH-01-091 TFA (up to 10 uM) is not acutely cytotoxic to normal human fibroblasts or HEK293 cells in 48-h assays (IC50 >50 uM). In vivo, at doses up to 50 mg/kg (i.p.) in mice, no gross toxicity or mortality has been reported in the literature (though not systematically studied). Off-target inhibition of other kinases at high doses could cause adverse effects. Standard safety precautions (gloves, lab coat) should be used. The compound is for research use only and not for human therapy. It is a chemical probe, not a drug.
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| References | |
| Additional Infomation |
MELK (maternal embryonic leucine zipper kinase) is a serine/threonine kinase implicated in cell cycle regulation, apoptosis, and cancer stem cell maintenance. It is overexpressed in many cancers and associated with poor prognosis and resistance to therapy. HTH-01-091 is a highly selective MELK inhibitor (IC50 10.5 nM) developed by Nathanael Gray's laboratory (Dana-Farber Cancer Institute). It is used as a chemical probe to validate MELK as a therapeutic target. However, controversy exists because some studies suggest that the anticancer effects of MELK inhibitors may be off-target, and MELK may not be essential in certain contexts. HTH-01-091 is an essential tool to resolve such questions. The TFA salt is for research use only, not for clinical application.
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| Molecular Formula |
C28H29CL2F3N4O4
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| Molecular Weight |
613.46
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| Appearance |
Light yellow to yellow solid powder
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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 | 1.6301 mL | 8.1505 mL | 16.3010 mL | |
| 5 mM | 0.3260 mL | 1.6301 mL | 3.2602 mL | |
| 10 mM | 0.1630 mL | 0.8150 mL | 1.6301 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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