KHS101 is a novel inhibitor of transforming acidic coiled-coil protein 3 (TACC3). It is a selective inducer of neuronal differentiation.

KHS101能够依赖剂量增加贴壁培养大鼠神经前体细胞（NPCs）的神经分化（EC50约1μM）。使用KHS101诱导的神经元形成（在1.5-5μM KHS101条件下，40-60%的TuJ1+细胞）也在来源于成年大鼠海马和侧脑室（SVZ）的二代神经球体形成条件下观察到[2]。SMMC-7721和SK-Hep-1细胞在不同浓度的KHS101（分别为40μM和20μM）中培养，以确定IC50值。与对照（DMSO）处理相比，KHS101处理后球体的数量和大小显著减少。球体形成依赖于KHS101的浓度。在KHS101存在下，Bmi1、c-Myc和Nanog的表达均降低。与对照（DMSO）相比，KHS101降低了p-AKT、p-GSK3β和β-catenin的表达，以及下游标志物c-Myc和cyclin D1的表达[2]。

6 mg/kg KHS101（静脉注射和皮下注射）的剂量产生了合理的血浆浓度（>1.5 μM），血浆半衰期为1.1-1.4小时，皮下给药后的相对生物利用度为69%。最为重要的是，KHS101在大脑中的分布非常广泛，这一点通过脑-血浆AUC(0-3h)比率约为8（给药：3 mg/kg KHS101，静脉注射）[1]得到了证明。

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) 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. 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 [1].

Rat NPCs were derived and cultured as described previously by others. After hippocampal cell isolation, the number of dissociated cells was determined and ～5 × 10^5 cells were plated in 60-mm uncoated plates. After overnight incubation (37 °C, 5% CO2, and 95% humidity), the medium was changed and the cells were expanded and maintained in an undifferentiated state on polyornithine- (10 μg/mL in water) and laminin-coated (5 μg/mL in PBS;) dishes in DMEM/F12 supplemented with N2 and basic fibroblast growth factor (bFGF, 20 ng/mL;). For KHS101 and shRNA-induction experiments, early passage cells (passaged no more than six times after hippocampal isolation) were trypsinized and plated at a density of ～1,000 cells/cm2 into N2 medium (DMEM/F12 supplemented with N2) containing KHS analogs (e.g., KHS101, KHS92, and NP; SI Text) at different concentrations (0.5–5 μM) or DMSO (0.1%), RA (1–2 μM), BDNF (100 ng/mL), and/or BMP4 (50–100 ng/mL) for 4 d [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 [1].