TACC3/transforming acidic coiled-coil-containing protein 3

NPC neurons in adherent culture are adsorbable to KHS101 in a dose-dependent manner (EC50 ~ 1 μM)[1]. KHS101 (5 μM) stimulates the production of NPC astrocytes [1]. The effects of KHS101 (5 μM) and KHS101 (0-15 μM; 24 h) on the loading and advancement of the NPC cell cycle are unfavorable [1]. Protein potential response of KHS101 and TACC3 [1]. KHS101 (0-15 μM; 24 h) Regulator ARNT2 nuclear localization [RT-PCR[1]

KHS101 (6 mg/kg; sc; BID for 14 days) is distributed in the brain in vivo and significantly promotes neuronal secretion [1].

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.

RT-PCR[1]
Cell Types: rat NPC
Tested Concentrations: 0.6, 1.7 and 5 μM
Incubation Duration: 24 h
Experimental Results: Shows dose-dependent induction of Cdkn1 mRNA expression. 1].

---

Cell proliferation assay [1]
Cell Types: rat NPC
Tested Concentrations: 5 μM
Incubation Duration: 24, 48 and 72 h
Experimental Results: The vast majority of NPC stopped proliferating within 72 h and became non-mitotic.

---

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/Disease Models: Adult Fisher 344 rats (∼10 weeks old) [1] Usage and
Doses: 6 mg/kg
Route of Administration: SC, BID, for 14 days
Experimental Results: Increased neuronal differentiation. NPC proliferation diminished.

---

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. 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.

[1]. A small molecule accelerates neuronal differentiation in the adult rat. Proc Natl Acad Sci U S A. 2010 Sep 21;107(38):16542-7.

[2]. KHS101 disrupts energy metabolism in human glioblastoma cells and reduces tumor growth in mice. Sci Transl Med. 2018 Aug 15;10(454). pii: eaar2718.

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]

C18H21N5S

339.461

339.152

C, 63.69; H, 6.24; N, 20.63; S, 9.44

1262770-73-9

KHS101 hydrochloride;1784282-12-7

71304818

Solid powder

3.775

2

6

7

24

361

0

CC(C)CN=C1C=CN=C(NCC2=CSC(=N2)C3=CC=CC=C3)N1

DGRJOOOHPBSAHD-UHFFFAOYSA-N

InChI=1S/C18H21N5S/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)

N4-isobutyl-N2-((2-phenylthiazol-4-yl)methyl)pyrimidine-2,4-diamine

KHS-101; KHS 101; N4-isobutyl-N2-((2-phenylthiazol-4-yl)methyl)pyrimidine-2,4-diamine; KHS-101; N4-(2-Methylpropyl)-N2-[(2-phenyl-1,3-thiazol-4-yl)methyl]pyrimidine-2,4-diamine; 4-N-(2-methylpropyl)-2-N-[(2-phenyl-1,3-thiazol-4-yl)methyl]pyrimidine-2,4-diamine; MLS006010727; CHEMBL3186037; KHS101;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

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

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 Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

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
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

 (Please use freshly prepared in vivo formulations for optimal results.)