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Purity: ≥98%
KHS101 HCl, the hydrochloride salt of KHS-101, is a novel, selective and synthetic small-molecule inhibitor of transforming acidic coiled-coil protein 3 (TACC3) with potential anticancer activity. KHS101 promoted tumor cell death in diverse GBM (glioblastoma multiforme) cell models, independent of their tumor subtype, and without affecting the viability of noncancerous brain cell lines. KHS101 exerted cytotoxic effects by disrupting the mitochondrial chaperone heat shock protein family D member 1 (HSPD1). In GBM cells, KHS101 promoted aggregation of proteins regulating mitochondrial integrity and energy metabolism. Mitochondrial bioenergetic capacity and glycolytic activity were selectively impaired in KHS101-treated GBM cells. In two intracranial patient-derived xenograft tumor models in mice, systemic administration of KHS101 reduced tumor growth and increased survival without discernible side effects. These findings suggest that targeting of HSPD1-dependent metabolic pathways might be an effective strategy for treating GBM.
| Targets |
TACC3/transforming acidic coiled-coil-containing protein 3
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| ln Vitro |
In adherent cultivated helper NPCs, KHS101 enhances neuronal evaporation in a dose-dependent manner (EC50=~1 μM). Under neurosphere-forming circumstances, KHS101 (1.5–5 μM) directs the production of neurons in the auxiliary hippocampus and secondary neurospheres produced from the subventricular zone (SVZ) that contain 40–60% TuJ1+ cells. Furthermore, compared to cells treated with vehicle [dimethylalkylene (DMSO)], hippocampus NPCs treated with KHS101 and attached to microelectrodes for 12 days showed neuronal morphology and spontaneous spiking KHS101 the proliferation of tumor cells. It has been demonstrated that TACC3, the neuroprogenitor KHS101, causes instability in TACC3-expressing cells, eventually lowering endogenous TACC3 protein levels [2].
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| ln Vivo |
Cell growth dramatically reduced (roughly increased) the tumors in KHS101-treated samples. Tumors treated with KHS101 exhibited higher levels of cell death (lower cellularity/higher pyknosis) in comparison to tumors treated with tumor vehicle controls. Treatment with KHS101 markedly inhibited vimentin-induced tumor growth of anterior to caudal GBM1 cells and around the corpus callosum. It was also discovered that the 10-week KHS101 treatment regimen markedly enhanced the mortality of rats with GBMX1 tumors (formed 2 or 6 weeks prior to treatment). Because of the treatment's side effects, no animals had to be taken out of the research. A noteworthy rise in animal mortality was also seen in another experiment employing consecutive KHS101 treatment regimen objectives in mice harboring GBMX1. A notable decrease in tumor size was observed in KHS101-treated mice, according to histological endpoint examination of animals treated with the drug and vehicle [2].
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| Enzyme Assay |
Affinity-Based Target Identification.[1]
NPC lysate was prepared by sonication in PBS and protein samples were prepared at a concentration of 2 mg/mL. The benzophenone-KHS101 compound (KHS101-BP, 5 μM; SI Text) was added to 50 μL of the proteome reaction with and without unlabeled compound (250 μM). Irradiation was for 1 h using a hand-held UV lamp at long wavelength (365 nm), and subsequently a copper-catalyzed azide-alkyne cycloaddition reaction was performed (SI Text). After incubation for 1 h at RT, proteins were precipitated using trichloroacetic acid and resuspended in isoelectric focusing sample buffer. 2D SDS/PAGE was performed using ReadyStripe IPG stripes following the manufacturer's protocol. Affinity-based target identification [2] GBM1 cells were incubated with KHS101-BP (5 μM) in the presence or absence of unlabeled KHS101 (250 μM) for 30 minutes, and irradiated with UV light (365nm) for 30 minutes. Cells were lysed using 0.5% Triton X-100 and protease inhibitor cocktail. Cell lysates were incubated with 25 μM biotin azide, 1 mM TCEP, 100 mM ligand (TBTA), and 1 mM aqueous copper sulfate at 4°C overnight. Subsequently, proteins were fractionated using ammonium sulfate and the 20-40% fractions were subject to 2D SDS/PAGE. Biotin-labeled proteins were detected through Western blotting using Abcam; ab1227). Protein spots corresponding to the specific biotin-labeled proteins were visualized with silver staining on parallel gels. A distinct spot was excised and protein identified using liquid chromatography tandem mass spectrometry. For HSPD1 interaction confirmation assays, a total of 1 μg recombinant HSPD1 was diluted in 1 mL PBS (with 2 mM MgCl2, 2 mM DDT, and 0.1% tween 20) and incubated with 5 μM biotinylated KHS101 at 4°C overnight in the presence or the absence of non-labeled KHS101. Streptavidin agarose beads were added to the incubation mixture and rotated at 4°C for 2 hours. The beads were then precipitated and washed three times in PBS. Bound proteins were eluted with 2x SDS sample buffer and analyzed with SDS/PAGE followed by silver staining and Western blotting. |
| Cell Assay |
For the differentiation assay, cells were seeded into 96 well plates at a density of 5,000 cells per well and treated with recombinant human BMP4 at 100 ng/mL for 4 days. Subsequently, cells were treated for 48 hours with DMSO (0.1%) or KHS101 (1-20 μM) in 100 μL of medium and the CellTiter-Glo assay was carried out according to the manufacturer’s instructions.[2]
For the colony formation assay, cells were seeded at a density of 125 cells/well (in 24- well plates) and allowed to adhere. The following day, the single cells per well were counted and treated with DMSO or KHS101. Colonies consisting of >6 cells were counted after 10 days and the percentage of cells that were able to form a colony was determined.[2] For live cell analysis, cells were allowed to grow for 2 days before the addition of KHS101 (7.5 μM) or DMSO (0.1%), and subsequently monitored for 3 days. Images were acquired at 45 minute intervals using the IncuCyte ZOOM live cell imaging system.[2] For the analysis of cell viability and caspase 3/7 activation, cells were seeded into 96 well plates at densities of 10,000 and 2,500 cells, respectively. The following day, cells were treated with vehicle (DMSO), KHS101, KHS101/Z-VAD-FMK (20 μM), or Staurosporine using the indicated concentrations in 100 μL of medium. The CellTiter-Glo and Caspase-Glo 3/7 assays (Promega) were carried out at the indicated time points according to the manufacturer’s instructions.[2] For the quantification of apoptosis using annexin V and propidium iodide, GBM1 cells were treated with KHS101 (7.5 μM), Bafilomycin A1 (10 nM) or vehicle (DMSO, 0.1%) for 48 hours, then harvested with trypsin, washed with PBS, and stained with annexin V and Propidium Iodide for 15 minutes at 37°C using an annexin V-fluorescein staining kit in accordance with the manufacturer’s protocol. labeled early apoptotic and late apoptotic/necrotic cells were quantified through quadrant gating using a NC3000 cytometer[2]. |
| Animal Protocol |
Animal Experiments.[1]
To investigate the pharmacokinetic properties of KHS101, male Sprague–Dawley rats were administered 3 mg/kg KHS101 i.v. or s.c. One rat was killed per time point at 5 min, 40 min, 1 h, and 3 h after dosing, and samples of blood (100 μL) and whole brains were collected. In a separate study, rats were administered 6 mg/kg KHS101 i.v. or s.c. Five blood samples of 100 μL each were collected serially via a jugular vein catheter at 2 min (i.v. only), 0.5 h (s.c. only), and 1, 3, 7 and 24 h after dosing. Plasma and homogenized whole brain samples were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). To study neuronal differentiation upon KHS101 administration in vivo, adult Fisher 344 rats (∼10 wk old) received s.c. injections of 6 mg/kg KHS101 or vehicle control (5% ethanol in 15% Captisol). All rats received one daily i.p. injection of 200 mg/kg BrdU for 6 consecutive days after the first day. After 14 d, the animals were killed and perfusion fixed, and the brains were removed and subjected to immunohistochemical analysis. Xenograft tumor experiments [2] Animal experiments were carried out under UK project license approval and institutional guidelines. Animals were maintained under standard conditions (12 hour day/night cycle with food and water ad libitum). Experiments were carried out using 6 to 8-week-old NOD scid gamma (NSG) and BALB/c Nude mice for the GBM1 and GBMX1 models, respectively. Mice were stereotactically injected with 2 x 105 GBM1 cells or 8 x 104 GBMX1 cells in a volume of 2 μL (containing 30% Matrigel) into the right striatum (2.5 mm from the midline, 2.5 mm anterior from bregma, 3 mm deep). Surgery was performed under general anaesthesia using aseptic techniques. Mice were monitored daily for signs of sickness, pain or weight loss. After the indicated tumor-establishing period, 6 mg/kg KHS101 or vehicle control (5% (v/v) ethanol, 15% (w/v) (2-Hydroxypropyl)-β-cyclo-dextrin) was administered subcutaneously (s.c.) twice daily with bi-weekly alteration of 5 and 3 treatment days per week. Experiments were concluded at indicated endpoints and tissue was subjected to immunohistological and image analysis. |
| References | |
| Additional Infomation |
Adult neurogenesis occurs in mammals and provides a mechanism for continuous neural plasticity in the brain. However, little is known about the molecular mechanisms regulating hippocampal neural progenitor cells (NPCs) and whether their fate can be pharmacologically modulated to improve neural plasticity and regeneration. Here, we report the characterization of a small molecule (KHS101) that selectively induces a neuronal differentiation phenotype. Mechanism of action studies revealed a link of KHS101 to cell cycle exit and specific binding to the TACC3 protein, whose knockdown in NPCs recapitulates the KHS101-induced phenotype. Upon systemic administration, KHS101 distributed to the brain and resulted in a significant increase in neuronal differentiation in vivo. Our findings indicate that KHS101 accelerates neuronal differentiation by interaction with TACC3 and may provide a basis for pharmacological intervention directed at endogenous NPCs.[1]
Pharmacological inhibition of uncontrolled cell growth with small-molecule inhibitors is a potential strategy for treating glioblastoma multiforme (GBM), the most malignant primary brain cancer. We showed that the synthetic small-molecule KHS101 promoted tumor cell death in diverse GBM cell models, independent of their tumor subtype, and without affecting the viability of noncancerous brain cell lines. KHS101 exerted cytotoxic effects by disrupting the mitochondrial chaperone heat shock protein family D member 1 (HSPD1). In GBM cells, KHS101 promoted aggregation of proteins regulating mitochondrial integrity and energy metabolism. Mitochondrial bioenergetic capacity and glycolytic activity were selectively impaired in KHS101-treated GBM cells. In two intracranial patient-derived xenograft tumor models in mice, systemic administration of KHS101 reduced tumor growth and increased survival without discernible side effects. These findings suggest that targeting of HSPD1-dependent metabolic pathways might be an effective strategy for treating GBM.[2] |
| Molecular Formula |
C18H22CLN5S
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|---|---|---|
| Molecular Weight |
375.91878080368
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| Exact Mass |
375.128
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| Elemental Analysis |
C, 57.51; H, 5.90; Cl, 9.43; N, 18.63; S, 8.53
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| CAS # |
1784282-12-7
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| Related CAS # |
KHS101;1262770-73-9
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| PubChem CID |
90488983
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| Appearance |
White to off-white solid powder
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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 |
7
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| Heavy Atom Count |
25
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| Complexity |
361
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| Defined Atom Stereocenter Count |
0
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| SMILES |
Cl.S1C=C(CNC2=NC=CC(=N2)NCC(C)C)N=C1C1C=CC=CC=1
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| InChi Key |
INVQHPQJFRKGIO-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C18H21N5S.ClH/c1-13(2)10-20-16-8-9-19-18(23-16)21-11-15-12-24-17(22-15)14-6-4-3-5-7-14;/h3-9,12-13H,10-11H2,1-2H3,(H2,19,20,21,23);1H
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| Chemical Name |
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| Synonyms |
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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, avoid exposure to moisture. |
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| 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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.67 mg/mL (7.10 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 26.7 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: ≥ 2.67 mg/mL (7.10 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 26.7 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. View More
Solubility in Formulation 3: ≥ 2.67 mg/mL (7.10 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.6601 mL | 13.3007 mL | 26.6014 mL | |
| 5 mM | 0.5320 mL | 2.6601 mL | 5.3203 mL | |
| 10 mM | 0.2660 mL | 1.3301 mL | 2.6601 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.
KHS101 specifically induces neuronal differentiation in rat NPCs.Proc Natl Acad Sci U S A.2010 Sep 21;107(38):16542-7. |
Tacc3-specific shRNA recapitulates the neurogenic effect of KHS101 in rat NPCs.Proc Natl Acad Sci U S A.2010 Sep 21;107(38):16542-7. |
KHS101 significantly increases neuronal differentiation in rats in vivo.Proc Natl Acad Sci U S A.2010 Sep 21;107(38):16542-7. |
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