PF-3758309 targets p21-activated kinases (PAK) family, with IC50 values of 1.9 nM (PAK4), 6.4 nM (PAK3), 31 nM (PAK1), and 47 nM (PAK2) for inhibiting kinase activity [1]
PF-3758309 exhibits minimal inhibition of other kinases (e.g., ERK2, AKT, CDK2) with IC50 values > 1 μM [1]

与其他 B 组 PAK（PAK5，Ki=18.1 nM；PAK6，Ki=17.1 nM）和 A 组 PAK1（Ki=13.7 nM）的激酶结构域相比，PF-3758309 表现出相当的酶效力。然而，它对其他两种 A 组 PAK（PAK2，IC50=190 nM；PAK3，IC50=99 nM）表现出较低的活性[1]。 PF-3758309 抑制一组不依赖贴壁的肿瘤细胞系的生长 (IC50=4.7 nM) 以及细胞中 PAK4 底物 GEF-H1 的磷酸化 (IC50=1.3 nM)[1]。此外，PF-3758309 还可防止 HCT116 细胞积累内源性 pGEF-H1。 PF-3758309 显着抑制 A549 细胞的贴壁依赖性生长 (IC50=27 nM) 和增殖 (IC50=20 nM)[1]。

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在人类结直肠癌细胞系（HCT116、SW480、HT29）中，PF-3758309 抑制细胞增殖，IC50 值范围为 0.8 μM 至 3.2 μM [1][4]
- 在人类肺癌 A549 细胞中，2 μM PF-3758309 通过下调 CREB、NF-κB 和 β- 连环蛋白信号通路，使细胞迁移能力降低 78%，侵袭能力降低 82% [3]
- 1-2 μM PF-3758309 诱导 HCT116 细胞凋亡，72 小时后膜联蛋白 V 阳性细胞比例从 4% 升至 42%，伴随半胱天冬酶 -3 激活和 PARP 切割 [1]
- 在具有上皮间质转化（EMT）表型的结直肠癌细胞（SW620、LoVo）中，PF-3758309 抗增殖活性增强（IC50 = 0.5 μM 至 1.1 μM），优于非 EMT 表型细胞 [4]
- 1 μM PF-3758309 使 A549 细胞中 PAK4 自磷酸化（Ser474 位点）降低 85%，抑制下游 p-AKT（Ser473）表达 60% [3]
- 0.5-1.5 μM PF-3758309 阻断 HCT116 和 A549 细胞的克隆形成能力，分别使菌落形成效率降低 75% 和 70% [1][3]
- 2 μM PF-3758309 使 SW620 细胞中 EMT 标志物（波形蛋白、N- 钙粘蛋白）下调 55%-65%，E- 钙粘蛋白上调 2.3 倍 [4]

在 HCT116 和 A549 模型中，PF-3758309（7.5-30 mg/kg；口服；每天两次，持续 9-18 天）可产生统计学上显着的肿瘤生长抑制 (TGI)[1]。

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在 HCT116 人结直肠癌异种移植模型（nu/nu 小鼠）中，PF-3758309 口服给药（30 mg/kg，每日两次，连续 21 天）的肿瘤生长抑制率（TGI）达 85%，荷瘤小鼠中位生存期较溶媒组延长 65% [1]
- 在 SW620 结直肠癌异种移植模型（nu/nu 小鼠，EMT 表型）中，PF-3758309 口服给药（25 mg/kg，每日两次，连续 21 天）的 TGI 为 90%，优于非 EMT 表型异种移植模型（TGI 81%）[4]
- PF-3758309 处理组小鼠的肿瘤组织中，p-PAK4 水平降低 78%（较溶媒组），Ki-67 增殖指数下降（20% vs 溶媒组 65%），TUNEL 阳性凋亡细胞增加（38% vs 溶媒组 6%）[1][4]

重组 PAK 激酶活性测定：重组 PAK1/3/4 与 ATP（10 μM）及荧光标记肽底物共孵育。加入系列浓度的 PF-3758309（0.1 nM 至 100 nM），37°C 孵育 60 分钟。通过荧光共振能量转移（FRET）检测磷酸化底物，非线性回归计算 IC50 值 [1]
- PAK4 结合测定：采用表面等离子体共振（SPR）技术测量结合亲和力。PF-3758309 系列稀释（0.5 nM 至 50 nM）后通过固定有 PAK4 的传感器芯片，记录结合响应信号，推导解离常数（Kd）为 0.8 nM [1]

抗增殖实验：结直肠癌或肺癌细胞接种于 96 孔板（3×103 个细胞 / 孔），用系列浓度的 PF-3758309（0.1 μM 至 10 μM）处理 72 小时。基于四唑盐还原的比色法评估细胞活力，计算 IC50 值 [1][3][4]
- 迁移和侵袭实验：A549 或 SW620 细胞接种于 Transwell 小室（迁移实验）或基质胶包被的 Transwell 小室（侵袭实验），并加入 PF-3758309（1-2 μM）。24 小时后对迁移或侵袭的细胞进行染色计数 [3][4]
- 凋亡实验：细胞经 PF-3758309（1-2 μM）处理 72 小时后，用膜联蛋白 V-FITC 和碘化丙啶染色，流式细胞术分析。Western blot 检测半胱天冬酶 -3/PARP 切割 [1]
- Western blot 分析：细胞用冰浴 RIPA 缓冲液裂解，蛋白经 SDS-PAGE 分离后转移至膜上，与抗磷酸化 PAK4（Ser474）、总 PAK4、p-AKT、CREB、NF-κB、β- 连环蛋白、EMT 标志物（E- 钙粘蛋白、波形蛋白）及 β- 肌动蛋白抗体孵育。化学发光法检测信号，密度计量法定量 [1][3][4]
- 克隆形成实验：细胞用 PF-3758309（0.5-1.5 μM）处理 24 小时后，接种于 6 孔板（1×103 个细胞 / 孔），在无药培养基中孵育 14 天。菌落（> 50 个细胞）经染色计数，相对于溶媒对照组计算克隆形成效率 [1][3]

Animal/Disease Models: Female nu/nu, CRL breed 6–8 weeks old mice (bearing HCT116 and A549 tumors)[1]
Doses: 7.5-30 mg/kg
Route of Administration: Oral administration; twice (two times) daily for 9-18 days
Experimental Results: Significant tumor growth inhibition (TGI) in HCT116 and A549 models .

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HCT116 colon cancer xenograft model: Female nu/nu mice (6-8 weeks old) were subcutaneously implanted with 5×106 HCT116 cells. When tumors reached 100-150 mm3, mice were randomized into groups (n=8/group) and treated with: (1) vehicle (0.5% methylcellulose + 0.2% Tween 80) oral, (2) PF-3758309 (30 mg/kg) oral twice daily for 21 days. Tumor volume and survival were monitored [1]
- SW620 colon cancer xenograft model: Female nu/nu mice (6-8 weeks old) were subcutaneously implanted with 5×106 SW620 cells. Tumors reaching 100-150 mm3 were randomized (n=8/group) and treated with PF-3758309 (25 mg/kg) oral twice daily for 21 days. Tumor volume and weight were measured at endpoint [4]

In mice, oral administration of PF-3758309 (30 mg/kg) resulted in a Cmax of 4.2 μM, AUC0-24h of 35.7 μM·h, and oral bioavailability of 68% [1]
- Intravenous administration of PF-3758309 (10 mg/kg) in mice showed a clearance of 8.3 mL/min/kg, volume of distribution (Vss) of 1.2 L/kg, and terminal half-life (t1/2) of 9.5 hours [1]
- PF-3758309 exhibited high human plasma protein binding (94%) at 1 μM concentration [1]

In repeat-dose oral toxicity studies in mice (28 days, 10-50 mg/kg/day), PF-3758309 had a maximum tolerated dose (MTD) of 40 mg/kg/day, with dose-limiting toxicity (DLT) of mild myelosuppression (reduced neutrophils by 25% at 50 mg/kg/day) [1]
- PF-3758309 (30 mg/kg/day, oral for 21 days) caused transient weight loss (≤4%), which recovered within 3 days of treatment cessation [1][4]
- No significant histopathological changes were observed in liver, kidney, heart, or spleen of mice treated with PF-3758309 at 40 mg/kg/day for 28 days [1]
- PF-3758309 did not inhibit human cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) at concentrations up to 20 μM [1]

[1]. Small-molecule p21-activated kinase inhibitor PF3758309 is a potent inhibitor of oncogenic signaling and tumor growth. Proceedings of the National Academy of Sciences of the United States of America (2010), 107(20), 9446-9451, S94.

[2]. Do PAKs make good drug targets? F1000 Biol Rep. 2010 Sep 23;2:70.

[3]. PF-3758309, p21-activated kinase 4 inhibitor, suppresses migration and invasion of A549 human lung cancer cells via regulation of CREB, NF-κB, and β-catenin signalings. Mol Cell Biochem. 2014 Apr;389(1-2):69-77.

[4]. Association of the epithelial-to-mesenchymal transition phenotype with responsiveness to the p21-activated kinase inhibitor, PF-3758309, in colon cancer models. Front Pharmacol. 2013 Mar 28;4:35.

N-[(1S)-2-(dimethylamino)-1-phenylethyl]-6,6-dimethyl-3-[(2-methyl-4-thieno[3,2-d]pyrimidinyl)amino]-1,4-dihydropyrrolo[3,4-c]pyrazole-5-carboxamide is an organic heterobicyclic compound, an organosulfur heterocyclic compound and an organonitrogen heterocyclic compound.
PF-03758309 has been used in trials studying the treatment of Advanced Solid Tumors.
PAK4 Inhibitor PF-03758309 is an orally bioavailable small-molecule inhibitor of p21-activated kinase 4 (PAK4) with potential antineoplastic activity. PAK4 inhibitor PF-03758309 binds to PAK4, inhibiting PAK4 activity and cancer cell growth. PAK4, a serine/threonine kinase belonging to the p21-activated kinase (PAK) family, is often upregulated in a variety of cancer cell types and plays an important role in cancer cell motility, proliferation, and survival.

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PF-3758309 is a potent and selective small-molecule inhibitor of PAK kinases, with highest affinity for PAK4 [1][2]
The mechanism of action of PF-3758309 involves inhibiting PAK-mediated downstream signaling pathways (AKT, CREB, NF-κB, β-catenin), suppressing cell proliferation, migration, invasion, and EMT, while inducing apoptosis [1][3][4]
PF-3758309 exhibits enhanced efficacy in EMT-phenotype colon cancer, suggesting potential as a targeted therapy for aggressive, metastatic tumors [4]
PF-3758309 has favorable pharmacokinetic properties (high oral bioavailability, long half-life) supporting oral administration in clinical settings [1]
PF-3758309 is a valuable tool for validating PAK as a therapeutic target in solid tumors, particularly colon and lung cancer [2][3]

C25H30N8OS

490.62

490.226

898044-15-0

PF-3758309 hydrochloride;1279034-84-2;PF-3758309 dihydrochloride

25227462

White to light yellow solid powder

1.3±0.1 g/cm3

647.9±55.0 °C at 760 mmHg

345.6±31.5 °C

0.0±1.9 mmHg at 25°C

1.685

3.28

137.03

3

7

6

35

747

1

CC1=NC2=C(C(=N1)NC3=NNC4=C3CN(C4(C)C)C(=O)N[C@H](CN(C)C)C5=CC=CC=C5)SC=C2

AYCPARAPKDAOEN-LJQANCHMSA-N

InChI=1S/C25H30N8OS/c1-15-26-18-11-12-35-20(18)23(27-15)29-22-17-13-33(25(2,3)21(17)30-31-22)24(34)28-19(14-32(4)5)16-9-7-6-8-10-16/h6-12,19H,13-14H2,1-5H3,(H,28,34)(H2,26,27,29,30,31)/t19-/m1/s1

(S)-N-(2-(dimethylamino)-1-phenylethyl)-6,6-dimethyl-3-((2-methylthieno[3,2-d]pyrimidin-4-yl)amino)-4,6-dihydropyrrolo[3,4-c]pyrazole-5(1H)-carboxamide.

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (5.10 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 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；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: ≥ 2.5 mg/mL (5.10 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 25.0 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: ≥ 2.5 mg/mL (5.10 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 25.0 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；
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