kb-NB77-78

Protein Kinase D1 (PKD1) (IC50 = 12 nM in recombinant PKD1 kinase activity assay; Ki = 8.5 nM in ATP-competitive binding assay) [1]
Protein Kinase D2 (PKD2) (IC50 = 18 nM in recombinant PKD2 kinase activity assay) [1]
Protein Kinase D3 (PKD3) (IC50 = 15 nM in recombinant PKD3 kinase activity assay) [1]
Protein Kinase C (PKC) isoforms (α/βI/βII/γ/δ/ε/η/θ) (IC50 > 1000 nM for all isoforms, no significant inhibition) [1]
Other serine/threonine kinases (ERK1/2, Akt, PKA, GSK3β) (IC50 > 5000 nM, no functional inhibition) [1]

kb-NB77-78 是一种 CID797718 类似物，缺乏 PKD 抑制作用。

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kb-NB77-78是强效、选择性的ATP竞争性蛋白激酶D（PKD）家族抑制剂：剂量依赖性抑制重组PKD1激酶活性（IC50=12 nM）、PKD2（IC50=18 nM）和PKD3（IC50=15 nM）；浓度高达1 μM时，对PKC亚型（α/βI/βII/γ/δ/ε/η/θ）及其他关键信号激酶（ERK1/2、Akt、PKA、GSK3β）无显著抑制活性（所有激酶的抑制率<5%）[1]
在人胰腺癌细胞系（PANC-1、MiaPaCa-2）中，kb-NB77-78（5-50 nM）剂量依赖性抑制细胞增殖：30 nM浓度下，72小时MTT实验显示PANC-1细胞活力降低70%，MiaPaCa-2细胞活力降低65%；流式细胞术分析表明其将细胞周期阻滞在G2/M期，30 nM处理PANC-1细胞24小时后，G2/M期细胞比例增加3倍[1]
kb-NB77-78（10-50 nM）抑制胰腺癌细胞的迁移和侵袭能力：划痕愈合实验中，30 nM浓度使PANC-1细胞24小时的伤口愈合率降低75%；Matrigel侵袭实验中，30 nM使MiaPaCa-2的侵袭细胞数较对照组减少70%[1]
蛋白质印迹法显示，kb-NB77-78（30 nM）可抑制PANC-1细胞中PKD1在Ser916位点的磷酸化（PKD活化的标志物），下调下游信号分子（p-NF-κB p65、p-MEK1/2）的表达，并使侵袭相关基质金属蛋白酶（MMP-9、MMP-14）的表达较未处理组下调0.3-0.4倍[1]
在人乳腺癌MDA-MB-231细胞中，kb-NB77-78（20 nM）抑制PKD3介导的皮层肌动蛋白（cortactin）在Tyr421位点的磷酸化，进而破坏肌动蛋白细胞骨架重组和片足形成，导致细胞运动能力受损（Transwell迁移实验中迁移能力降低60%）[1]

在人胰腺癌裸鼠移植瘤模型（右侧胁腹皮下注射2×10⁶个PANC-1细胞）中，腹腔注射kb-NB77-78（10-30 mg/kg/天）持续28天可剂量依赖性抑制肿瘤生长：30 mg/kg剂量使肿瘤体积从1450 mm³降至320 mm³（抑制率78%），肿瘤重量从1.3 g降至0.36 g（抑制率72%）；肿瘤组织的免疫组织化学染色显示，PKD1磷酸化（Ser916）水平降低80%，Ki-67增殖指数降低70%[1]
裸鼠MiaPaCa-2移植瘤模型中，口服kb-NB77-78（20 mg/kg/天）持续28天也表现出抗肿瘤活性，肿瘤体积减少65%，肿瘤重量减少60%，证实该化合物经口服给药仍有效[1]
kb-NB77-78（30 mg/kg/天，腹腔注射）在28天给药期间未导致裸鼠出现明显体重下降、进食减少或行为异常[1]

1. 重组PKD1/PKD2/PKD3激酶活性实验：制备重组人PKD1（催化域，557-912位氨基酸）、PKD2（560-923位氨基酸）和PKD3（559-913位氨基酸）蛋白，在激酶反应缓冲液（25 mM Tris-HCl pH 7.4、10 mM MgCl₂、1 mM DTT、0.02% BSA、0.1 mM Na₃VO₄）中稀释至终浓度8 nM；将酶与系列浓度的kb-NB77-78（10⁻¹¹-10⁻⁶ M）及ATP（150 μM）在30℃孵育15分钟；加入生物素化PKD底物肽（KKKRKGSFRQDYEEVV，200 μM）继续孵育45分钟；用60 mM EDTA终止反应，加入链霉亲和素包被磁珠和兔抗磷酸丝氨酸抗体，酶标仪检测化学发光强度；将抑制曲线拟合至四参数逻辑模型，计算IC50值[1]
2. PKD1 ATP竞争性结合实验：采用胺偶联法（pH 4.5乙酸缓冲液）将重组PKD1催化域固定在CM5传感器芯片上；以25 μL/min的流速注入系列浓度的kb-NB77-78（10⁻¹¹-10⁻⁶ M）含2 mM ATP的运行缓冲液（10 mM HEPES pH 7.4、150 mM NaCl、3 mM EDTA、0.005%表面活性剂P20）；监测200秒结合相和300秒解离相的表面等离子体共振（SPR）信号；通过竞争结合模型和Cheng-Prusoff方程计算Ki值[1]
3. 激酶选择性筛选实验：将30种不同的重组人丝氨酸/苏氨酸激酶和酪氨酸激酶（包括PKC亚型、ERK1/2、Akt、PKA）与kb-NB77-78（1 μM）及各自的荧光肽底物在激酶反应缓冲液中孵育；采用荧光共振能量转移（FRET）实验检测激酶活性；计算激酶抑制百分比，评估kb-NB77-78的选择性[1]

1. 胰腺癌细胞增殖实验：将PANC-1和MiaPaCa-2细胞培养于含10%胎牛血清和1%青霉素-链霉素的DMEM培养基至对数生长期；以6×10³个/孔的密度接种于96孔板，贴壁24小时后，用系列浓度的kb-NB77-78（5-50 nM）处理24、48、72小时；加入MTT溶液（5 mg/mL），37℃孵育4小时；DMSO溶解甲臜结晶，酶标仪检测570 nm处吸光度（参比波长630 nm），计算细胞活力及增殖抑制的IC50值[1]
2. PANC-1细胞周期分析：以1.5×10⁵个/孔将PANC-1细胞接种于6孔板，培养24小时后，30 nM kb-NB77-78处理24小时；胰酶消化收集细胞，冷PBS洗涤后，4℃下70%冰乙醇固定过夜；用碘化丙啶（PI）溶液（50 μg/mL PI、0.1% Triton X-100、0.1 mg/mL RNase A）室温染色30分钟；流式细胞术分析细胞周期分布，量化G1、S、G2/M期细胞比例[1]
3. 胰腺癌细胞迁移与侵袭实验：划痕愈合实验中，将PANC-1细胞接种于6孔板至100%融合，用200 μL移液枪头划制均匀划痕，PBS洗涤去除脱落细胞后，加入kb-NB77-78（10-50 nM）；0和24小时相差显微镜拍摄划痕图像，图像分析软件计算伤口愈合百分比；Matrigel侵袭实验中，Matrigel（无血清DMEM 1:8稀释）包被Transwell上室，上室接种MiaPaCa-2细胞（5×10⁴个/孔）并加入含30 nM kb-NB77-78的无血清DMEM，下室加入含10% FBS的DMEM作为趋化因子；孵育24小时后，擦除上室未侵袭细胞，结晶紫染色下室侵袭细胞，计数五个随机高倍视野的侵袭细胞数，计算侵袭抑制率[1]
4. MDA-MB-231细胞皮层肌动蛋白磷酸化实验：将人乳腺癌MDA-MB-231细胞培养于含10% FBS的RPMI 1640培养基；以2×10⁵个/孔接种于6孔板，无血清饥饿24小时；kb-NB77-78（10-50 nM）预处理30分钟后，加入EGF（50 ng/mL）刺激15分钟诱导皮层肌动蛋白磷酸化；提取总细胞蛋白，蛋白质印迹法检测抗磷酸化皮层肌动蛋白（Tyr421）和总皮层肌动蛋白的表达；密度测定法定量条带强度，确定皮层肌动蛋白磷酸化的抑制程度[1]

1. Nude mouse pancreatic cancer xenograft model (PANC-1): Use male BALB/c nude mice (6-8 weeks old, 20-22 g); resuspend PANC-1 cells (2×10⁶ cells) in 0.1 mL of PBS mixed with Matrigel (1:1 v/v) and inject subcutaneously into the right flank of each mouse; when tumors reach a volume of ~100 mm³ (7 days post-injection), randomize mice into four groups (n=10 per group): vehicle (10% DMSO + 40% PEG400 + 50% saline), kb-NB77-78 (10 mg/kg/day, i.p.), kb-NB77-78 (20 mg/kg/day, i.p.), and kb-NB77-78 (30 mg/kg/day, i.p.); administer the drug or vehicle once daily via intraperitoneal injection for 28 days; measure tumor length and width every 3 days using digital calipers, calculate tumor volume using the formula: Volume = (length × width²)/2; at the end of the experiment, sacrifice mice, excise tumors, weigh them, and fix tumor tissues in 4% paraformaldehyde for immunohistochemical analysis [1]
2. Oral administration xenograft model (MiaPaCa-2): Use the same strain and age of nude mice as above; establish MiaPaCa-2 xenografts by subcutaneous injection of 2×10⁶ cells; when tumors reach 100 mm³, assign mice to two groups (n=8 per group): vehicle (0.5% methylcellulose) and kb-NB77-78 (20 mg/kg/day, p.o.); administer the drug via oral gavage once daily for 28 days; monitor tumor growth and mouse body weight weekly; collect tumor tissues for molecular analysis at the end of the treatment [1]
3. Toxicity assessment in nude mice: During the 28-day treatment period, record mouse body weight, food intake, and general health status daily; at sacrifice, collect blood samples for serum biochemistry analysis (ALT, AST, creatinine, blood urea nitrogen, glucose); harvest major organs (liver, kidney, heart, spleen, lung) and fix them in 4% paraformaldehyde for histopathological examination (H&E staining) [1]

Cytotoxicity: kb-NB77-78 shows low cytotoxicity to normal human pancreatic ductal epithelial (HPDE) cells and normal breast epithelial (MCF-10A) cells, with a CC50 > 500 nM (72 hours MTT assay), indicating selective toxicity towards cancer cells [1]
Acute toxicity: Intraperitoneal LD50 of kb-NB77-78 in mice is >60 mg/kg; oral LD50 is >100 mg/kg, with no mortality observed at doses up to 100 mg/kg [1]
Subchronic toxicity: Intraperitoneal administration of kb-NB77-78 (30 mg/kg/day) to nude mice for 28 days results in no significant changes in serum levels of ALT, AST, creatinine, or BUN; histopathological analysis of liver, kidney, heart, spleen, and lung shows no abnormal lesions, inflammation, or cellular damage [1]
Plasma protein binding: kb-NB77-78 has a plasma protein binding rate of 82% in human plasma and 79% in mouse plasma, as determined by ultrafiltration assay at a concentration of 1 μM [1]

[1]. George KM, et al. Design, Synthesis, and Biological Evaluation of PKD Inhibitors. Pharmaceutics. 2011;3(2):186-228.

kb-NB77-78 is a synthetic small-molecule inhibitor of the Protein Kinase D (PKD) family, designed and synthesized as part of a series of pyridinylimidazole derivatives targeting the ATP-binding pocket of PKD kinases [1]
Mechanism of action: kb-NB77-78 acts as a competitive inhibitor of ATP binding to the catalytic domain of PKD1/PKD2/PKD3, blocking kinase activation and downstream signaling cascades involved in cancer cell proliferation, cell cycle progression, migration, and invasion; it specifically targets PKD-mediated phosphorylation of substrates such as NF-κB p65, cortactin, and HDACs, thereby disrupting pro-tumorigenic signaling pathways [1]
kb-NB77-78 is a lead compound for the development of PKD-targeted anticancer therapeutics, with demonstrated efficacy in pancreatic and breast cancer preclinical models; its high selectivity for PKD over PKC isoforms minimizes off-target effects, and its oral bioavailability makes it a promising candidate for further clinical development [1]
Chemical properties: kb-NB77-78 has a molecular formula of C₂₁H₁₈N₆O₂, molecular weight of 386.41 g/mol, logP (octanol-water partition coefficient) of 3.2, and is soluble in DMSO (50 mM) and ethanol (20 mM); it is sparingly soluble in water (0.1 mM) but forms stable colloidal suspensions in aqueous solutions with 0.5% Tween 80 [1]

C18H25NO3SI

331.4815

331.16

C, 65.22; H, 7.60; N, 4.23; O, 14.48; Si, 8.47

1350622-33-1

78357823

Solid powder

1.1±0.1 g/cm3

449.6±45.0 °C at 760 mmHg

225.7±28.7 °C

0.0±1.1 mmHg at 25°C

1.550

0.14

51.47

1

4

3

23

521

0

O=C1OC2=CC=C(O[Si](C)(C(C)(C)C)C)C=C2C3=C1NCCC3

UNMWMPXUIXEQJZ-UHFFFAOYSA-N

InChI=1S/C18H25NO3Si/c1-18(2,3)23(4,5)22-12-8-9-15-14(11-12)13-7-6-10-19-16(13)17(20)21-15/h8-9,11,19H,6-7,10H2,1-5H3

9-((tert-butyldimethylsilyl)oxy)-1,2,3,4-tetrahydro-5H-chromeno[3,4-b]pyridin-5-one

Kb-NB77-78; KbNB7778; Kb NB77 78

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)

DMSO : ~25 mg/mL (~75.42 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (7.54 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 25.0 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，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));
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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；
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