TACC3/transforming acidic coiled-coil-containing protein 3
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].

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].

---

KHS101 induces neuronal differentiation of cultured rat hippocampal neural progenitor cells (NPCs) in a dose-dependent manner with an EC₅₀ of approximately 1 μM, as assessed by NeuroD mRNA expression and TuJ1 immunostaining. [1]
At concentrations of 1.5–5 μM, KHS101 treatment resulted in 40–60% of cells becoming TuJ1 positive under neurosphere-forming conditions. [1]
KHS101 (5 μM) suppressed bone morphogenetic protein 4 (BMP4)-induced astrocyte differentiation by more than 4-fold in NPC cultures, while simultaneously increasing neuronal differentiation. [1]
KHS101 treatment led to cell cycle exit and suppressed NPC proliferation, as evidenced by decreased Ki67 and phospho-histone H3 (P-HH3) positive cells, and loss of SOX2 expression within 72 hours. [1]
Microarray and qRT-PCR analysis showed that KHS101 upregulates the negative cell cycle regulator Cdkn1 (p21) by approximately 5-fold at 1.7 μM. [1]
KHS101 treatment increased nuclear localization of the transcription factor ARNT2 in NPCs. [1]
Knockdown of Tacc3 using shRNA recapitulated the KHS101-induced neuronal differentiation phenotype and suppression of BMP4-induced astrogenesis. [1]
KHS101 decreased the proliferation of rat oligodendrocyte precursor cells but did not induce their differentiation. [1]

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].

---

Subcutaneous administration of KHS101 (6 mg/kg, twice daily for 14 days) to adult rats significantly increased neuronal differentiation of bromodeoxyuridine (BrdU)-labeled cells in the hippocampal dentate gyrus. [1]
The percentage of BrdU/NeuN double-positive cells (indicative of new neurons) increased from approximately 20% in vehicle-treated animals to approximately 40% in KHS101-treated animals. [1]
KHS101 treatment significantly reduced the number of Ki67-positive and BrdU-positive cells in the subgranular layer, indicating decreased NPC proliferation. [1]
No significant change was observed in the percentage of BrdU/GFAP double-positive cells or in apoptosis (cleaved caspase 3 staining) within the dentate gyrus. [1]
KHS101 administration did not alter Ki67 immunostaining in non-neural tissues such as spleen and gut. [1]
No signs of lethargy, weight loss, or other indicators of sickness were observed in treated animals during the study. [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.

---

An affinity-based target identification strategy was employed to identify proteins that physically interact with KHS101. A photoreactive derivative of KHS101, containing a benzophenone crosslinking moiety and an alkyne handle (KHS101-BP), was synthesized. Rat NPC lysate was incubated with KHS101-BP (5 μM) with or without a 50-fold excess of unlabeled KHS101 for competition. Following incubation, the mixture was irradiated with UV light (365 nm) to induce covalent crosslinking between the compound and its target protein(s). A copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction was then performed to attach a biotin tag to the alkyne handle on the crosslinked complex. Proteins were separated by two-dimensional SDS-PAGE, and labeled proteins were detected by Western blot using streptavidin-HRP. A distinct labeled protein band was identified, and its intensity was reduced in the presence of excess free KHS101. Mass spectrometry analysis identified this protein as TACC3, which was confirmed by Western blot with a TACC3-specific antibody. Direct physical interaction was further confirmed in independent pulldown experiments using purified recombinant rat TACC3 protein and a biotinylated KHS101 derivative. [1]

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].

---

Neuronal Differentiation Assay: Rat hippocampal NPCs were cultured adherently on polyornithine/laminin-coated dishes in N2 medium. Cells were treated with KHS101 (0.5–5 μM), inactive analogs, DMSO (vehicle control), retinoic acid (RA, 1-2 μM), or brain-derived neurotrophic factor (BDNF, 100 ng/mL) for 4 days. Neuronal differentiation was assessed by quantitative RT-PCR for NeuroD mRNA and by immunocytochemistry for the neuronal marker βIII-tubulin (TuJ1). [1]
Astrocyte Differentiation Assay: NPCs were treated with the astrocyte-inducing cytokine BMP4 (50 ng/mL) in the presence or absence of KHS101 (5 μM) or RA (2 μM) for 4 days. Astrocyte and neuron formation were assessed by co-immunostaining for glial fibrillary acidic protein (GFAP) and TuJ1. [1]
Proliferation/Cell Cycle Analysis: NPCs were treated with KHS101 or DMSO. Proliferation was assessed by immunostaining for Ki67 and phospho-histone H3 (P-HH3) at various time points. Cell cycle regulator gene expression was analyzed by microarray and confirmed by qRT-PCR for Cdkn1. [1]
Gene Expression Analysis: Total RNA was extracted from treated NPCs using a commercial kit. cDNA was synthesized using a reverse transcription kit. Gene expression levels (e.g., NeuroD, Tacc3, Cdkn1) were quantified by real-time RT-PCR using TaqMan probes, with Gapdh as a reference gene. [1]
Immunocytochemistry: Treated cells were fixed with formalin, permeabilized with Triton X-100, and blocked with serum and BSA. Cells were incubated with primary antibodies (e.g., TuJ1, GFAP, Ki67, P-HH3, SOX2, ARNT2, TACC3) overnight at 4°C, followed by appropriate fluorescent secondary antibodies. Nuclei were visualized with DAPI. [1]
shRNA Knockdown: NPCs were electroporated with plasmids encoding Tacc3-specific shRNAs or a non-targeting control shRNA using a commercial nucleofector kit. Puromycin selection was applied to enrich transfected cells. Phenotypic effects on differentiation (TuJ1), proliferation (Ki67), and response to BMP4 were assessed as described above. [1]
Subcellular Localization: Nuclear and cytoplasmic fractions were prepared from KHS101-treated NPCs or 293T cells overexpressing TACC3 and ARNT2. ARNT2 levels in each fraction were analyzed by Western blot. Nuclear localization of endogenous ARNT2 in NPCs was also quantified by confocal microscopy and image analysis following immunostaining. [1]

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.

---

Pharmacokinetic Study: Male Sprague-Dawley rats were administered a single dose of KHS101 intravenously (3 mg/kg) or subcutaneously (6 mg/kg). For the 3 mg/kg i.v. study, one rat was euthanized per time point (5 min, 40 min, 1 h, 3 h) for collection of blood and whole brain. For the 6 mg/kg study, serial blood samples were collected via a jugular vein catheter at specified times up to 24 hours. Plasma and homogenized brain samples were analyzed by LC-MS/MS to determine compound concentrations. [1]
In Vivo Neurogenesis Study: Adult Fisher 344 rats (approximately 10 weeks old) received subcutaneous injections of KHS101 (6 mg/kg) or vehicle control (5% ethanol in 15% Captisol) twice daily (BID) for 14 consecutive days. All rats received a daily intraperitoneal injection of bromodeoxyuridine (BrdU, 200 mg/kg) for the first 7 days to label dividing cells. After 14 days, animals were perfused transcardially with fixative. Brains were removed, sectioned, and processed for immunohistochemistry to analyze BrdU/NeuN (neuronal fate), BrdU/GFAP (astrocyte fate), Ki67 (proliferation), and cleaved caspase 3 (apoptosis) in the dentate gyrus. [1]

Following a single intravenous injection (3 mg/kg) of KHS101 in rats, the drug was widely distributed in the brain, with a brain/plasma AUC (0–3 h) ratio of approximately 8. [1]
The plasma half-life (t₁/₂) of KHS101 was 1.1–1.4 hours. [1]
Subcutaneous injection (6 mg/kg) achieved reasonable plasma concentrations (>1.5 μM), with a relative bioavailability of 69% compared to intravenous injection. [1]
Oral administration resulted in extremely low systemic exposure. [1]

In a 14-day in vivo study, rats treated with KHS101 did not exhibit lethargy, weight loss, or other signs of disease. [1]
Compared to the control group, apoptosis in the dentate gyrus of the treated animals, as assessed by caspase 3 staining, was not altered. [1]
KHS101 administration did not affect the proliferation of TACC3-expressing cells in non-neural tissues such as the spleen and gastrointestinal tract (Ki67 staining). [1]

[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 sustained neuroplasticity in the brain. However, little is known about the molecular mechanisms regulating hippocampal neural progenitor cells (NPCs), and it is unclear whether their fate can be modulated by drugs to improve neuroplasticity and regenerative capacity. This article reports the properties of a small molecule (KHS101) that can selectively induce a neuronal differentiation phenotype. Mechanism of action studies have shown that KHS101 is associated with cell cycle exit and specific binding to the TACC3 protein, and knockdown of the TACC3 protein in NPCs can reproduce the KHS101-induced phenotype. After systemic administration, KHS101 is distributed to the brain and significantly promotes neuronal differentiation in vivo. Our results suggest that KHS101 accelerates neuronal differentiation by interacting with TACC3, which may provide a basis for drug interventions targeting endogenous neural progenitor cells (NPCs). [1]

---

Using small molecule inhibitors to inhibit uncontrolled cell growth is a potential strategy for treating glioblastoma multiforme (GBM, the most malignant primary brain cancer). We found that the synthetic small molecule KHS101 promoted tumor cell death in multiple GBM cell models regardless of tumor subtype and did not affect the viability of non-cancerous brain cell lines. KHS101 exerts its cytotoxic effect by disrupting mitochondrial molecular chaperone heat shock protein family member 1 (HSPD1). In GBM cells, KHS101 promoted the aggregation of proteins that regulate mitochondrial integrity and energy metabolism. The mitochondrial bioenergetic capacity and glycolytic activity of GBM cells treated with KHS101 were selectively impaired. In two mouse intracranial xenograft tumor models derived from patients, systemic administration of KHS101 inhibited tumor growth and prolonged survival without significant side effects. These findings suggest that targeting the HSPD1-dependent metabolic pathway may be an effective strategy for treating glioblastoma (GBM). [2] KHS101 is a synthetic small molecule belonging to the 4-aminothiazole class of compounds and is an analog of previously reported "neurothiazole" compounds. [1]
It exerts its pharmacological effect of inducing neuronal differentiation through physical interaction with the centrosome/spindle-associated protein TACC3. [1]
This mechanism involves KHS101-mediated interference with TACC3 function, leading to increased nuclear localization of the neuron-specific transcription factor ARNT2, promoting cell cycle exit (via Cdkn1/p21 upregulation), and activating neuronal differentiation programs in neural progenitor cells. [1]
It can inhibit glial cell-inducing signals (e.g., BMP4), thereby promoting neuronal fate. [1]
This compound is considered a potential tool for studying neurogenesis and a candidate for developing therapies aimed at enhancing endogenous neural repair or targeting brain cancer with progenitor-like cells. [1]

C18H22CLN5S

375.91878080368

375.128

C, 57.51; H, 5.90; Cl, 9.43; N, 18.63; S, 8.53

1784282-12-7

KHS101;1262770-73-9

90488983

White to off-white solid powder

3

6

7

25

361

0

Cl.S1C=C(CNC2=NC=CC(=N2)NCC(C)C)N=C1C1C=CC=CC=1

INVQHPQJFRKGIO-UHFFFAOYSA-N

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

2934.99.9001

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.

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

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.
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 corn oil and mix evenly.

---

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