TP-3654

Pim-1 (Ki=5 nM); Pim-2 (Ki=239 nM); Pim-3 (Ki=42 nM)
PIM kinases (PIM-1, PIM-2, PIM-3) (Ki values of TP-3654 for PIM kinases were comparable to IC50 determinations; PIM-1–specific cellular EC50 of TP-3654 was 67 nM on average in HEK-293 cells transfected with BAD and PIM-1 (phospho-BAD (S112) Surefire assay)) [1]

在 PIM-1/BAD 过表达系统中，TP-3654 表现出强烈的 PIM-1 特异性细胞作用，平均 EC50 为 67 nM。 TP-3654 疗法可在体外使用 UM-UC-3 膀胱癌细胞系降低磷酸化 BAD 水平。 TP-3654 抑制 T24 和 UM-UC3 细胞集落生长，表明这两种细胞系的增殖需要 PIM-1[1]。

---

1. TP-3654在人膀胱癌UM-UC-3细胞系的药效学生物标志物调控、细胞增殖实验和集落形成实验中表现出亚微摩尔级活性 [1]
2. 在转染空载体、PIM1、PIM1激酶失活突变体（PIM1KD，K67M）（单独或与BAD共转染）的HEK293T细胞中，PIM-1过表达可升高细胞内Ser¹¹²位点磷酸化BAD（pBAD）水平，而TP-3654处理可剂量依赖性降低pBAD水平 [1]
3. UM-UC3细胞经TP-3654（0.03、0.3、1、3 μM）处理12小时后，S112位点磷酸化BAD和Th37/46位点磷酸化4EBP1的水平降低，而总BAD和总4EBP1水平无显著变化 [1]
4. 二维集落形成实验中，TP-3654抑制人膀胱癌T24细胞生长的平均EC50为1.1 ± 0.4 μM（n=4），抑制UM-UC3细胞生长的平均EC50为2.2 ± 0.2 μM（n=2）[1]
5. 与第一代PIM抑制剂SGI-1776相比，TP-3654对人ether-à-go-go相关基因（hERG）和细胞色素P450酶的抑制谱更优 [1]

在体重或总体不良毒性没有明显变化的情况下，口服剂量 200 mg/kg TP-3654 可显着降低 UM-UC-3 和 PC-3 肿瘤的生长（以体积（厚度）和最终肿瘤重量衡量）[1] 。

---

1. 将5×10⁶个UM-UC-3膀胱癌细胞接种至裸鼠（Nu/Nu）体内（每组12只）；每日口服给予TP-3654 200 mg/kg，连续给药3周（给药5天，停药2天），可显著抑制肿瘤生长（卡尺测量：P = .0028），并降低实验结束时的肿瘤重量（P = .02），且小鼠体重无显著变化 [1]
2. 将7.5×10⁶个PC-3前列腺腺癌细胞接种至雄性裸鼠体内（每组12只）；每日口服给予TP-3654 200 mg/kg，连续3周（给药5天，停药2天），可显著抑制肿瘤生长（卡尺测量：P = .007）和降低肿瘤重量（P = .0002），小鼠体重无显著变化 [1]
3. 将5×10⁶个亲本22RV1或22RV1/PIM-1细胞接种至裸鼠体内（每组10只）；腹腔注射SGI-9481（25 mg/kg，每日一次，连续3周，给药5天停药2天）可抑制肿瘤生长（P = .023） [1]
4. 将5×10⁶个亲本NIH-3 T3或NIH-3 T3/PIM-2细胞接种至裸鼠体内（每组10只）；腹腔注射SGI-9481（25 mg/kg，每日一次，连续3周，给药5天停药2天）可抑制肿瘤生长（P = .002） [1]

1. TP-3654激酶选择性实验：测定TP-3654对多种激酶的IC50值，重点检测其对PIM激酶的Ki值（Ki值与IC50值相当）；实验过程为将TP-3654与不同激酶共孵育，检测激酶活性并计算抑制浓度 [1]
2. PIM-1特异性细胞EC50实验：将HEK-293细胞转染BAD和PIM-1；转染后用不同浓度TP-3654处理细胞，通过Surefire实验检测phospho-BAD (S112)水平；采用GraphPad Prism软件计算EC50值（四次独立实验平均值为67 nM）[1]

TP-3654 在 10 μM ATP 浓度下针对 336 种激酶进行了测试。 PI3K 和所有受抑制激酶的 IC50 测定均使用 TP-3654 的 10 剂量、三倍系列稀释液进行，每种激酶的 Km ATP 浓度从 10 μM 开始。[1]。

---

1. UM-UC-3细胞PIM-1 shRNA转染实验：将UM-UC-3膀胱癌细胞用慢病毒颗粒过夜转染PIM-1 shRNA或对照shRNA（非靶向）；更换培养基后，在转染后48小时收集细胞，提取RNA检测PIM-1 mRNA水平，提取蛋白检测PIM-1蛋白水平；取部分细胞以500个/孔接种至6孔板，培养10天后固定、染色，进行二维集落形成实验（PIM-1 mRNA：P = .0028；集落形成：P = .0002）[1]
2. HEK293T细胞pBAD和4EBP1 Western blot实验：采用转染试剂将空载体、PIM1、PIM1KD（K67M）（单独或与BAD共转染）导入HEK293T细胞；转染24小时后，细胞饥饿处理过夜并裂解；提取可溶性蛋白，通过Western blot结合荧光扫描仪检测Ser¹¹²位点磷酸化BAD水平 [1]
3. UM-UC3细胞pBAD和4EBP1 Western blot实验：将UM-UC3细胞用3、1、0.3、0.03 μM的TP-3654处理12小时；裂解细胞后，将蛋白样品分别进行Western blot，用对应抗体检测S112磷酸化BAD、总BAD、Th37/46磷酸化4EBP1和总4EBP1的水平 [1]
4. T24和UM-UC3细胞集落形成实验：将T24膀胱癌细胞以300个/孔、UM-UC3细胞以500个/孔接种至12孔板；次日用梯度浓度的TP-3654处理细胞；T24细胞培养10天、UM-UC3细胞培养6天后，染色成像并裂解细胞，通过560 nm吸光度定量细胞生长，计算EC50值 [1]

The study involved mice with tumors measuring 100-200 mm3 and randomized to receive oral dosing of TP-3654 or vehicle control every day for 5 days, with 2 days off for 18-21 days, and tumor volumes and body weights determined weekly[1].

---

1. UM-UC-3 bladder carcinoma xenograft model: Nu/Nu mice were implanted with 5 × 10⁶ UM-UC-3 cells per mouse (12 mice per group); TP-3654 was administered orally at 200 mg/kg QD for 3 weeks (5 days of dosing followed by 2 days off per week); tumor measurements were obtained by caliper, and tumor weights were recorded at the end of the study; body weights of mice were monitored throughout the experiment [1]
2. PC-3 prostate adenocarcinoma xenograft model: Male Nu/Nu mice were implanted with 7.5 × 10⁶ PC-3 cells per mouse (12 mice per group); TP-3654 was administered orally at 200 mg/kg QD for 3 weeks (5 days on, 2 days off); tumor measurements (caliper) and tumor weights were assessed, and body weights were monitored [1]
3. 22RV1/PIM-1 xenograft model: Nu/Nu mice were implanted with 5 × 10⁶ parental 22RV1 or 22RV1/PIM-1 cells per mouse (10 mice per group); SGI-9481 (25 mg/kg) was administered by intraperitoneal injection QD for 3 weeks (5 days on, 2 days off); tumor measurements and body weights were recorded (no TP-3654 dosing data) [1]
4. NIH-3 T3/PIM-2 xenograft model: Nu/Nu mice were implanted with 5 × 10⁶ parental NIH-3 T3 or NIH-3 T3/PIM-2 cells per mouse (10 mice per group); SGI-9481 (25 mg/kg) was administered by intraperitoneal injection QD for 3 weeks (5 days on, 2 days off); tumor measurements and body weights were recorded (no TP-3654 dosing data) [1]
5. Oral pharmacokinetic assay in rats: Female Sprague-Dawley (SD) rats were dosed with TP-3654 either by intravenous (IV) injection at 2 mg/kg or orally at 40 mg/kg; the drug was formulated with 10% polysorbate 20 to achieve maximum exposure; plasma levels of TP-3654 were determined using liquid chromatography-mass spectrometry (LC-MS) [1]

1. TP-3654 exhibited oral bioavailability in female SD rats; when formulated with 10% polysorbate 20, oral administration at 40 mg/kg resulted in the highest exposure (area under the curve, AUC) compared with IV injection at 2 mg/kg [1]

[1]. A small-molecule inhibitor of PIM kinases as a potential treatment for urothelial carcinomas. Neoplasia. 2014 May;16(5):403-12.

Nuvisertib is an orally available, second-generation and selective ATP-competitive inhibitor of proviral integration site for Moloney murine leukemia virus (PIM) kinases, with potential antineoplastic activity. Upon oral administration, nuvisertib selectively binds to and prevents the activation of the PIM kinases. This prevents the activation of PIM-mediated signaling pathways and inhibits proliferation in cells that overexpress PIM. PIMs, constitutively active proto-oncogenic serine/threonine kinases, are upregulated in various types of cancers and play key roles in tumor cell proliferation and survival.

---

1. TP-3654 is a second-generation small-molecule inhibitor of PIM kinases (PIM-1, PIM-2, PIM-3), which are serine/threonine kinases involved in cancer cell signaling pathways (inhibitors of apoptosis, positive regulators of G1-S phase cell cycle progression) [1]
2. PIM kinases are upregulated in multiple cancers (lymphoma, leukemia, multiple myeloma, prostate, gastric, head and neck cancers, urothelial carcinoma), and overexpression correlates with poor prognosis [1]
3. High levels of PIM-1, PIM-2, and PIM-3 were observed in both noninvasive and invasive urothelial carcinomas (137 cases of surgical biopsy/resection specimens) [1]
4. TP-3654 is a potential therapeutic agent for human urothelial carcinomas, as PIM kinase inhibition reduces tumor growth in bladder cancer xenograft models [1]
5. TP-3654 has advantages over first-generation PIM inhibitor SGI-1776 in hERG/cytochrome P450 inhibition and oral bioavailability [1]

C22H25F3N4O

418.4553

418.198

1361951-15-6

1418143-09-5 (HCl);1361951-15-6;

66598080

White to light yellow solid powder

4.6

62.4

2

7

4

30

579

0

FC(C1=C([H])C([H])=C([H])C(=C1[H])C1=C([H])N=C2C([H])=C([H])C(=NN21)N([H])C1([H])C([H])([H])C([H])([H])C([H])(C(C([H])([H])[H])(C([H])([H])[H])O[H])C([H])([H])C1([H])[H])(F)F

XRNVABDYQLHODA-UHFFFAOYSA-N

InChI=1S/C22H25F3N4O/c1-21(2,30)15-6-8-17(9-7-15)27-19-10-11-20-26-13-18(29(20)28-19)14-4-3-5-16(12-14)22(23,24)25/h3-5,10-13,15,17,30H,6-9H2,1-2H3,(H,27,28)

2-[4-[[3-[3-(trifluoromethyl)phenyl]imidazo[1,2-b]pyridazin-6-yl]amino]cyclohexyl]propan-2-ol

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: ~84 mg/mL ( 200.73 mM）
Water: <6 mg/mL Ethanol: Insoluble

配方 1 中的溶解度: 2.5 mg/mL (5.97 mM) 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), 悬浮液；超声助溶。
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。

---

配方 2 中的溶解度: ≥ 2.08 mg/mL (4.97 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 20.8 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入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。

---

View More

配方 3 中的溶解度: ≥ 2.08 mg/mL (4.97 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 20.8 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

---

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。