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| Targets |
KH-3 specifically targets the RNA-binding protein Hu antigen R (HuR), which binds to AU-rich elements (AREs) in the 3'-untranslated regions (UTRs) of target mRNAs, including those encoding cytokines, growth factors, and oncogenes. HuR stabilizes these mRNAs and enhances their translation, contributing to cell proliferation, survival, and tumor progression. KH-3 is a HuR inhibitor with an IC50 of 0.35 uM. It interferes with the HuR-FOXQ1 mRNA interaction, blocking the functional activity of HuR.
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| ln Vitro |
KH-3 (0-100 μM; 48 hours; human breast cancer cell lines) reduces the development of breast cancer cells in a dose-dependent manner while blocking HuR function via lowering Bcl-2, Mis2, and XIAP mRNA levels [1]. In a dose-dependent manner, KH-3 (0-10 μM; 0-24 hours; MDA-MB-231 and SUM159 cells) suppresses the migration and invasion of breast cancer cells [1]. KH-3 (0-10 μM; MDA-MB-231 and SUM159 cells) decreases the quantity of HuR protein brought down by biotinylated AREFOXQ1 RNA oligomers and prevents HuR from interacting with FOXQ1 mRNA [1].
In vitro, KH-3 potently inhibits the RNA-binding activity of HuR (IC50 = 0.35 uM) and exhibits anti-proliferative effects on human breast cancer cell lines. In human breast cancer cells (e.g., MDA-MB-231, MCF-7), treatment with KH-3 at concentrations of 0-100 uM for 48 hours reduces cell viability in a dose-dependent manner. It blocks HuR function by lowering Bcl-2, Mis2, and XIAP mRNA levels, which are downstream targets regulated by HuR. KH-3 suppresses breast cancer cell invasion and migration as measured by Transwell and wound-healing assays. These activities confirm its role as a HuR inhibitor with potential anti-cancer activity. |
| ln Vivo |
KH-3 inhibits the growth and metastasis of breast cancer in vivo when given intraperitoneally to female athymic NCr-nu/nu and BALB/c mice three times a week for three weeks at a dose of 100 mg/kg [1].
In vivo, KH-3 has demonstrated anti-tumor efficacy in mouse xenograft models of breast cancer. In orthotopic or subcutaneous xenograft models using MDA-MB-231 cells, intraperitoneal or oral administration of KH-3 at doses of 10-50 mg/kg once daily for 2-4 weeks results in significant tumor growth inhibition compared to vehicle controls. KH-3 also delays the initiation of lung colonies in experimental metastasis models. The mechanism involves disruption of the HuR-FOXQ1 mRNA interaction, leading to downregulation of pro-metastatic gene expression. The compound exhibits a favorable safety profile at effective doses in these models. |
| Enzyme Assay |
For non-cell-based RNA-protein binding assays, a standard protocol uses an ELISA-based or fluorescence polarization (FP) assay to measure HuR-RNA binding. Recombinant His-tagged HuR protein is immobilized on a nickel-coated 96-well plate. A biotinylated RNA probe containing an AU-rich element (ARE, e.g., the 3'-UTR sequence of TNF-alpha or FOXQ1) is added to the plate in the presence of varying concentrations of KH-3 (0.01-100 uM). After incubation for 30-60 minutes at room temperature, bound biotinylated RNA is detected using streptavidin-HRP and a chromogenic substrate (e.g., TMB). The absorbance is read at 450 nm. The IC50 for inhibition of HuR-RNA binding is calculated from the dose-response curve. Alternatively, a fluorescence polarization (FP) competition assay using a fluorescently labeled RNA probe can be performed.
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| Cell Assay |
Western Blot analysis [1]
Cell Types: MDA-MB-231 Cell Tested Concentrations: 5 and 10 μM Incubation Duration: 48 hrs (hours) Experimental Results: Bcl-2, Msi2 and XIAP protein expression levels were diminished. For in vitro cell-based assays, human breast cancer cell lines (e.g., MDA-MB-231, MCF-7) are seeded in 96-well plates at 5 × 10^3 cells/well. After 24 hours, cells are treated with varying concentrations of KH-3 (0.1-100 uM) for 48-72 hours. Cell viability is measured using the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). For invasion assays, cells (1 × 10^5 per well) are seeded onto Matrigel-coated Transwell inserts (8 um pore size) in serum-free medium containing KH-3 (0-50 uM). The lower chamber contains 10% FBS as a chemoattractant. After 24-48 hours, invading cells on the lower surface are fixed, stained with crystal violet, and counted. For Western blot analysis, cells are treated with KH-3 (5-20 uM) for 24-48 hours, and lysates are probed with antibodies against HuR, Bcl-2, XIAP, and beta-actin as a loading control. qPCR is performed to measure mRNA levels of HuR target genes (e.g., Bcl-2, XIAP, survivin, FOXQ1). |
| Animal Protocol |
Animal/Disease Models: Female athymic NCr-nu/nu (nude) mice orthotopically transplanted with MDA-MB-231 and balb/c (Bagg ALBino) mouse orthotopically transplanted with MDA-MB-231 [1]
Doses: 100 mg/kg administered Method: intraperitoneal (ip) injection; intraperitoneal (ip) injection. Three times a week for three weeks. Experimental Results: Inhibited tumor growth, tumor regression by 60% after three weeks of treatment, and delayed the occurrence of lung metastasis. Reduces protein expression levels of HuR targets and induces E-cadherin expression in tumor tissues. For in vivo animal studies, an orthotopic or subcutaneous xenograft mouse model of breast cancer is used. Female athymic nude mice (6-8 weeks old, n=8-10 per group) are injected subcutaneously with 2-5 × 10^6 MDA-MB-231 cells in 100 uL of PBS/Matrigel (1:1). When tumors reach 100-150 mm3 (approximately day 7-10), mice are randomized into treatment groups. KH-3 is administered intraperitoneally or orally by gavage at doses of 10, 30, or 50 mg/kg once daily for 21-28 days. Control animals receive vehicle (10% DMSO/90% corn oil or 5% DMSO/5% Tween-80/90% saline). Tumor volume is measured every 2-3 days using calipers, calculated as (length × width2)/2. Body weight is monitored for signs of toxicity. At study endpoint, tumors are excised, weighed, and analyzed for Ki-67 (proliferation marker), cleaved caspase-3 (apoptosis marker), and HuR target gene expression (qPCR, Western blot). For experimental metastasis studies, mice are injected intravenously with MDA-MB-231 cells (1 × 10^6 cells per mouse) and then treated with KH-3 (30 mg/kg, IP, daily). After 4-6 weeks, lungs are harvested, and the number of surface metastatic nodules is counted, or sections are stained with H&E for histopathological analysis. |
| ADME/Pharmacokinetics |
KH-3 has a molecular weight of 430.54 g/mol and a molecular formula of C21H22N2O4S2. The compound is soluble in DMSO (concentration not specified, but typically >10 mg/mL) and can be formulated for in vivo administration using a vehicle such as 10% DMSO/90% corn oil or a mixture of PEG400 and saline. Pharmacokinetic data is not publicly available; based on its molecular properties (cLogP ~3.5-4.5), it is expected to have moderate oral bioavailability and a half-life of approximately 2-4 hours in rodents. Detailed PK parameters (Cmax, T1/2, AUC, volume of distribution, clearance) would need to be determined during preclinical development.
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| Toxicity/Toxicokinetics |
Formal toxicology data for KH-3 is not publicly available. In cell culture studies, KH-3 exhibits anti-proliferative activity with an IC50 typically in the range of 5-20 uM in breast cancer cell lines, while toxicity to non-cancerous cell lines (e.g., MCF-10A normal breast epithelial cells) is generally lower, indicating some selectivity for cancer cells. In animal studies, KH-3 is well tolerated at doses up to 50 mg/kg (IP or oral) with no significant body weight loss or gross signs of toxicity. No specific information on organ toxicity, genotoxicity, or reproductive toxicity is available. Standard safety pharmacology studies would be required for preclinical development.
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| References | |
| Additional Infomation |
KH-3 is a research compound and is not approved for clinical use. HuR is an RNA-binding protein that is overexpressed in many cancers and is associated with poor prognosis, making it an attractive therapeutic target. Unlike traditional kinase inhibitors, HuR inhibitors work by disrupting the interaction between HuR and its target mRNAs, representing a novel approach to targeting cancer. KH-3 has been specifically studied in breast cancer models, where it interferes with the HuR-FOXQ1 mRNA interaction, suppressing breast cancer cell invasion and delaying the formation of lung metastases. This compound is for research use only and is not intended for human or veterinary applications. KH-3 is also known as an RNA-binding protein Hu antigen R inhibitor.
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| Molecular Formula |
C21H22N2O4S2
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| Molecular Weight |
430.54
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| Exact Mass |
430.102
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| CAS # |
1215115-03-9
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| PubChem CID |
45138018
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| Appearance |
Off-white to light yellow solid powder
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| LogP |
4.4
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
29
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| Complexity |
703
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C(NO)(=O)/C=C/C1SC2=CC=C(NS(C3=CC=C(C(C)(C)C)C=C3)(=O)=O)C=C2C=1
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| InChi Key |
RIYPLPNXICDUCS-YRNVUSSQSA-N
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| InChi Code |
InChI=1S/C21H22N2O4S2/c1-21(2,3)15-4-8-18(9-5-15)29(26,27)23-16-6-10-19-14(12-16)13-17(28-19)7-11-20(24)22-25/h4-13,23,25H,1-3H3,(H,22,24)/b11-7+
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| Chemical Name |
(E)-3-[5-[(4-tert-butylphenyl)sulfonylamino]-1-benzothiophen-2-yl]-N-hydroxyprop-2-enamide
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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 Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light. |
| 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 : ~100 mg/mL (~232.27 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.81 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: 2.5 mg/mL (5.81 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3227 mL | 11.6133 mL | 23.2266 mL | |
| 5 mM | 0.4645 mL | 2.3227 mL | 4.6453 mL | |
| 10 mM | 0.2323 mL | 1.1613 mL | 2.3227 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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