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].

在贴壁培养的辅助 NPC 中，KHS101 以剂量依赖性方式增强神经元蒸发（EC50=~1 μM）。在神经球形成情况下，KHS101 (1.5–5 μM) 指导辅助海马和次级神经球中神经元的产生，这些神经球由含有 40–60% TuJ1+ 细胞的室下区 (SVZ) 产生。此外，与用载体[二甲基亚烷基（DMSO）]处理的细胞相比，用KHS101处理并附着在微电极上12天的海马NPC显示出神经元形态和自发的KHS101尖峰肿瘤细胞的增殖。已证明 TACC3（神经祖细胞 KHS101）会导致 TACC3 表达细胞不稳定，最终降低内源性 TACC3 蛋白水平 [2]。

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KHS101 以剂量依赖的方式诱导培养的大鼠海马神经祖细胞神经元分化，通过NeuroD mRNA表达和TuJ1免疫染色评估的EC₅₀约为1 μM。[1]
在1.5–5 μM浓度下，在神经球形成条件下，KHS101 处理导致40–60%的细胞变为TuJ1阳性。[1]
KHS101 (5 μM) 在NPC培养中抑制了骨形态发生蛋白4诱导的星形胶质细胞分化超过4倍，同时增加了神经元分化。[1]
KHS101 处理导致细胞周期退出并抑制NPC增殖，这通过Ki67和磷酸化组蛋白H3阳性细胞减少以及72小时内SOX2表达丢失来证明。[1]
微阵列和qRT-PCR分析表明，KHS101 在1.7 μM浓度下使负向细胞周期调节因子Cdkn1 (p21) 的表达上调约5倍。[1]
KHS101 处理增加了转录因子ARNT2在NPC中的核定位。[1]
使用shRNA敲低Tacc3重现了KHS101 诱导的神经元分化表型以及对BMP4诱导的星形胶质细胞生成的抑制作用。[1]
KHS101 降低了大鼠少突胶质前体细胞的增殖，但未诱导其分化。[1]

在 KHS101 处理的样品中，细胞生长显着减少（大致增加）肿瘤。与用肿瘤载体对照治疗的肿瘤相比，用 KHS101 治疗的肿瘤表现出更高水平的细胞死亡（细胞结构较低/固缩较高）。 KHS101 治疗显着抑制了波形蛋白诱导的 GBM1 细胞前部和胼胝体周围的肿瘤生长。研究还发现，为期 10 周的 KHS101 治疗方案显着提高了患有 GBMX1 肿瘤（治疗前 2 或 6 周形成）的大鼠的死亡率。由于治疗的副作用，没有动物被排除在研究之外。在另一项对携带 GBMX1 的小鼠采用连续 KHS101 治疗方案目标的实验中，也发现动物死亡率显着上升。根据用药物和载体治疗的动物的组织学终点检查，在 KHS101 治疗的小鼠中观察到肿瘤大小显着减小 [2]。

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给成年大鼠皮下注射KHS101 (6 mg/kg，每日两次，持续14天) 显著增加了海马齿状回中溴脱氧尿苷标记细胞的神经元分化。[1]
BrdU/NeuN双阳性细胞（指示新生神经元）的百分比从载体处理组动物的约20%增加到KHS101 处理组动物的约40%。[1]
KHS101 处理显著减少了颗粒下层中Ki67阳性和BrdU阳性细胞的数量，表明NPC增殖减少。[1]
在齿状回内，BrdU/GFAP双阳性细胞的百分比或细胞凋亡（裂解的caspase 3染色）未观察到显著变化。[1]
KHS101 给药未改变脾脏和肠道等非神经组织中的Ki67免疫染色。[1]
在研究期间，未在处理动物中观察到嗜睡、体重减轻或其他疾病迹象。[1]

基于亲和力的目标识别。[1]
通过在PBS中超声处理制备NPC裂解物，并以2 mg/mL的浓度制备蛋白质样品。将二苯甲酮-KHS101化合物（KHS101-BP，5μM；SI Text）加入到50μL的蛋白质组反应中，有和没有未标记的化合物（250μM）。使用长波长（365nm）的手持紫外灯照射1小时，随后进行铜催化的叠氮-炔烃环加成反应（SI Text）。在室温下孵育1小时后，使用三氯乙酸沉淀蛋白质，并将其重新悬浮在等电聚焦样品缓冲液中。按照制造商的方案，使用ReadyStripe IPG条进行2D SDS-PAGE。

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基于亲和力的目标识别[2]
GBM1细胞在有或没有未标记的KHS101（250μM）的情况下与KHS101-BP（5μM）一起孵育30分钟，并用紫外光（365nm）照射30分钟。使用0.5%Triton X-100和蛋白酶抑制剂混合物裂解细胞。细胞裂解物与25μM叠氮化生物素、1 mM TCEP、100 mM配体（TBTA）和1 mM硫酸铜水溶液在4°C下孵育过夜。随后，使用硫酸铵对蛋白质进行分级，并对20-40%的级分进行2D SDS-PAGE。使用Abcam通过蛋白质印迹检测生物素标记的蛋白质；ab1227）。在平行凝胶上用银染色显示与特定生物素标记的蛋白质相对应的蛋白质斑点。切除一个明显的斑点，并使用液相色谱-串联质谱法鉴定蛋白质。对于HSPD1相互作用确认试验，将总共1μg重组HSPD1稀释在1 mL PBS（含2 mM MgCl2、2 mM DDT和0.1%吐温20）中，并在有或没有未标记的KHS101的情况下，在4°C下与5μM生物素化的KHS101一起孵育过夜。将链霉抗生物素蛋白琼脂糖珠加入孵育混合物中，并在4°C下旋转2小时。然后沉淀珠粒，在PBS中洗涤三次。用2x SDS样品缓冲液洗脱结合的蛋白质，用SDS-PAGE分析，然后进行银染和蛋白质印迹。

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该研究采用基于亲和力的靶点识别策略来鉴定与KHS101 发生物理相互作用的蛋白。合成了KHS101 的光反应性衍生物，该衍生物含有苯丙酮交联部分和炔烃手柄（KHS101-BP）。将大鼠NPC裂解物与KHS101-BP (5 μM) 孵育，同时设置或不设置50倍过量未标记的KHS101 作为竞争对照。孵育后，用紫外线（365 nm）照射混合物，以诱导化合物与其靶蛋白之间形成共价交联。随后进行铜催化的叠氮-炔环加成反应，将生物素标签连接到交联复合物的炔烃手柄上。通过二维SDS-PAGE分离蛋白质，并使用链霉亲和素-HRP通过Western blot检测标记的蛋白质。鉴定出一个明显的标记蛋白条带，其在过量游离KHS101 存在下强度降低。质谱分析将该蛋白鉴定为TACC3，并通过TACC3特异性抗体的Western blot得以确认。使用纯化的重组大鼠TACC3蛋白和生物素化的KHS101 衍生物进行的独立下拉实验进一步证实了直接物理相互作用。[1]

对于分化试验，将细胞以每孔5000个细胞的密度接种到96孔板中，并用100ng/mL的重组人BMP4处理4天。随后，在100μL培养基中用DMSO（0.1%）或KHS101（1-20μM）处理细胞48小时，并根据制造商的说明进行CellTiter-Glo测定。2.

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对于集落形成试验，以125个细胞/孔的密度（在24孔板中）接种细胞并使其粘附。第二天，计数每孔的单细胞，并用DMSO或KHS101处理。10天后计数由>6个细胞组成的集落，并确定能够形成集落的细胞百分比。2.

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对于活细胞分析，在加入KHS101（7.5μM）或DMSO（0.1%）之前，让细胞生长2天，随后监测3天。使用IncuCyte ZOOM活细胞成像系统以45分钟的间隔采集图像。2.

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为了分析细胞活力和半胱天冬酶3/7活化，将细胞分别以10000和2500个细胞的密度接种到96孔板中。第二天，用载体（DMSO）、KHS101、KHS101/Z-VAD-FMK（20μM）或Staurosporine在100μL培养基中以指定浓度处理细胞。CellTiter Glo和Caspase Glo 3/7测定 根据制造商的说明在指定时间点进行。2.

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为了使用膜联蛋白V和碘化丙啶定量凋亡，GBM1细胞用KHS101（7.5μM）、巴非霉素A1（10 nM）或载体（DMSO，0.1%）处理48小时，然后用胰蛋白酶收获，用PBS洗涤，并根据制造商的方案在37°C下用膜联蛋白V-荧光素染色试剂盒用膜联素V和碘化丙啶染色15分钟。使用NC3000细胞仪通过象限门控定量标记的早期凋亡和晚期凋亡/坏死细胞[2]。

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神经元分化实验: 大鼠海马NPC在多聚鸟氨酸/层粘连蛋白包被的培养皿中于N2培养基中进行贴壁培养。用KHS101 (0.5–5 μM)、无活性类似物、DMSO（载体对照）、视黄酸（1-2 μM）或脑源性神经营养因子（100 ng/mL）处理细胞4天。通过NeuroD mRNA的定量RT-PCR和神经元标记物βIII-tubulin (TuJ1) 的免疫细胞化学评估神经元分化。[1]
星形胶质细胞分化实验: NPC在存在或不存在KHS101 (5 μM) 或RA (2 μM) 的情况下，用星形胶质细胞诱导细胞因子BMP4 (50 ng/mL) 处理4天。通过胶质纤维酸性蛋白和TuJ1的共免疫染色评估星形胶质细胞和神经元形成。[1]
增殖/细胞周期分析: NPC用KHS101 或DMSO处理。通过在不同时间点对Ki67和磷酸化组蛋白H3进行免疫染色来评估增殖。通过微阵列分析细胞周期调节因子基因表达，并通过Cdkn1的qRT-PCR确认。[1]
基因表达分析: 使用商业试剂盒从处理的NPC中提取总RNA。使用逆转录试剂盒合成cDNA。使用TaqMan探针通过实时RT-PCR定量基因表达水平（如NeuroD、Tacc3、Cdkn1），以Gapdh作为内参基因。[1]
免疫细胞化学: 处理的细胞用福尔马林固定，用Triton X-100透化，并用血清和BSA封闭。细胞在4°C与一抗（如TuJ1、GFAP、Ki67、P-HH3、SOX2、ARNT2、TACC3）孵育过夜，然后使用相应的荧光二抗。用DAPI显示细胞核。[1]
shRNA敲低: 使用商业核转染试剂盒，将编码Tacc3特异性shRNA或非靶向对照shRNA的质粒电转到NPC中。应用嘌呤霉素选择来富集转染细胞。如上述方法评估对分化、增殖和对BMP4反应的表型效应。[1]
亚细胞定位: 从KHS101 处理的NPC或过表达TACC3和ARNT2的293T细胞中制备核质组分。通过Western blot分析各组分中的ARNT2水平。免疫染色后，还通过共聚焦显微镜和图像分析定量了NPC中内源性ARNT2的核定位。[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.

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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.

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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 dose (3 mg/kg) in rats, KHS101 showed extensive distribution to the brain, with a brain-to-plasma AUC(0-3h) ratio of approximately 8. [1]
The plasma half-life (t₁/₂) of KHS101 was 1.1–1.4 hours. [1]
Subcutaneous administration (6 mg/kg) resulted in reasonable plasma concentrations (>1.5 μM) and a relative bioavailability of 69% compared to intravenous dosing. [1]
Oral administration resulted in very low systemic exposure. [1]

In the 14-day in vivo study, KHS101-treated rats showed no signs of lethargy, weight loss, or other indicators of sickness. [1]
Apoptosis, as assessed by cleaved caspase 3 staining, was not altered in the dentate gyrus of treated animals compared to controls. [1]
KHS101 administration did not affect proliferation (Ki67 staining) in non-neural TACC3-expressing tissues such as spleen and gastrointestinal tract. [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 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]

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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]

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KHS101 is a synthetic small molecule belonging to the 4-aminothiazole chemical class, developed as an analog of previously reported "neuropathiazol" compounds. [1]
It acts as a pharmacological inducer of neuronal differentiation by physically interacting with the centrosomal/spindle-associated protein TACC3. [1]
The proposed mechanism involves KHS101-mediated interference with TACC3 function, leading to increased nuclear localization of the neuron-specific transcription factor ARNT2, promotion of cell cycle exit (via Cdkn1/p21 upregulation), and activation of a neuronal differentiation program in neural progenitor cells. [1]
It can override glial-inducing signals (e.g., BMP4) to promote neuronal fate. [1]
The compound is proposed as a potential tool for studying neurogenesis and as a candidate for developing therapies aimed at enhancing endogenous neural repair or targeting brain cancers 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

91

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

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

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

配方 1 中的溶解度: ≥ 2.67 mg/mL (7.10 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 26.7 mg/mL的澄清DMSO储备液加入到400 μL PEG300中并混合均匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.67 mg/mL (7.10 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 26.7 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.67 mg/mL (7.10 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 26.7 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。