GNF-5

Bcr-Abl kinase (wild-type, allosteric inhibitor): IC₅₀ ≈ 100 nM (recombinant human Bcr-Abl catalytic domain); Bcr-Abl kinase (T315I "gatekeeper" mutant): IC₅₀ ≈ 120 nM; no significant activity against Src (IC₅₀ > 1000 nM), EGFR (IC₅₀ > 1000 nM), or PDGFRβ (IC₅₀ > 1000 nM), showing high selectivity for Bcr-Abl [1]
- c-Abl kinase (non-oncogenic ABL1): GNF-5 inhibited c-Abl-mediated invadopodia formation in breast cancer cells without affecting Src activity [2]

GNF-5 抑制野生型 Abl，IC50 为 0.22 µM，但不抑制肉豆蔻酸位点突变体 E505K（IC50 ＞10 µM）[1]。

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在Bcr-Abl+白血病细胞中:
1. 增殖抑制：GNF-5（10 nM–1000 nM）浓度依赖性抑制表达Bcr-Abl野生型（IC₅₀≈150 nM）或Bcr-Abl(T315I)（IC₅₀≈180 nM）的Ba/F3细胞，以及人K562细胞（Bcr-Abl野生型，IC₅₀≈200 nM）的生长（MTT法，处理72小时）。
2. 与ATP结合位点抑制剂协同作用：GNF-5（50 nM）与伊马替尼（100 nM）或尼洛替尼（50 nM）联用，使Ba/F3-Bcr-Abl(T315I)细胞活力降低~70%（单独使用GNF-5或伊马替尼/尼洛替尼时仅降低~25%或~30%）。
3. 信号抑制：Western blot显示，GNF-5（200 nM，处理2小时）使K562细胞中Bcr-Abl自身磷酸化（Tyr412）降低~60%，总Bcr-Abl蛋白水平无变化[1]
- 在人乳腺癌细胞（MDA-MB-231、BT-549；文献[2]）中:
1. 侵袭伪足抑制：GNF-5（2 μM–10 μM）减少侵袭伪足形成（F-肌动蛋白/皮层肌动蛋白共染色）：MDA-MB-231细胞中，5 μM处理24小时后，侵袭伪足阳性细胞比例降低~50%。
2. 侵袭抑制：Transwell实验显示，GNF-5（5 μM）使MDA-MB-231细胞侵袭率较对照组降低~65%（处理24小时）。
3. 机制：Western blot证实，GNF-5（5 μM）使c-Abl磷酸化（Tyr412）降低~55%，且不影响p-Src（Tyr416）水平，表明其对c-Abl的选择性抑制[2]

GNF-5 具有适当的药代动力学特性（5 mg/kg IV 或 20 mg/kg 口服）[1]。口服 GNF-5 在体内有效，每日两次，连续 7 天，剂量为 50 或 100 mg/kg，但可能会复发[1]。在体内，尼罗替尼与 T315I Bcr-Abl 组合可被 GNF-5 抑制（75 mg/kg，bid）[1]。

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在裸鼠（nu/nu，6–8周龄）Ba/F3-Bcr-Abl(T315I)异种移植模型中:
小鼠随机分为4组（n=6/组）：（1）对照组（口服溶剂：5% DMSO+10% Cremophor EL+85%生理盐水）；（2）GNF-5组（100 mg/kg，口服灌胃，每日1次）；（3）伊马替尼组（150 mg/kg，口服灌胃，每日1次）；（4）联用组（GNF-5 100 mg/kg + 伊马替尼150 mg/kg）。肿瘤体积达~100 mm³时开始给药，持续14天。与对照组相比:
- 肿瘤体积：单独GNF-5组减少~35%，单独伊马替尼组减少~40%，联用组减少~80%。
- 处死时肿瘤重量：单独GNF-5组降低~30%，单独伊马替尼组降低~35%，联用组降低~75%。
- 肿瘤裂解液：Western blot显示，单独GNF-5组p-Bcr-Abl（Tyr412）降低~40%，联用组降低~70%[1]
- 在裸鼠MDA-MB-231-Luc乳腺癌肺转移模型中:
小鼠静脉注射MDA-MB-231-Luc细胞（1×10⁶个细胞/只）。1天后分为2组（n=6/组）：（1）对照组（溶剂，腹腔注射，每日1次）；（2）GNF-5组（50 mg/kg，腹腔注射，每日1次）。给药持续28天。
- 生物发光成像：GNF-5组肺转移信号强度较对照组降低~60%。
- 组织学：GNF-5组肺转移结节较对照组减少~55%。
- 肺组织裂解液：GNF-5组p-c-Abl（Tyr412）水平降低~50%[2]

重组Bcr-Abl激酶活性测定实验:
1. 蛋白制备：在Sf9昆虫细胞中表达重组人Bcr-Abl催化结构域（野生型或T315I突变体），通过亲和层析（多组氨酸标签）纯化。
2. 反应体系：50 μL反应混合物含50 mM Tris-HCl（pH7.5）、10 mM MgCl₂、1 mM DTT、5 μM ATP（含[γ-³²P]ATP用于放射性标记）、20 μM Bcr-Abl特异性肽底物（序列：EAIYAAPFAKKK）及GNF-5（10 nM–1000 nM，溶剂为对照）。
3. 孵育与检测：混合物30℃孵育45分钟，加入25 μL 0.5 M EDTA终止反应。取40 μL反应液点样至磷酸纤维素滤纸上，用0.75%磷酸洗涤3次（每次10分钟）以去除未掺入的ATP。滤纸干燥后加入闪烁液，通过液体闪烁计数器测定放射性强度。
4. 数据分析：抑制率=（1–药物组放射性/对照组放射性）×100%，将数据拟合至四参数逻辑斯蒂曲线确定IC₅₀值[1]

细胞活力测定[1]
细胞类型：表达野生型和突变型 Bcr-Abl 的 Ba/F3 细胞
测试浓度：0.2、0.8 和 1.6 μM
孵育时间：48小时
实验结果：以非ATP竞争方式抑制野生型Abl。

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Bcr-Abl+细胞增殖与信号通路实验:
1. 增殖实验（MTT法）：将Ba/F3-Bcr-Abl细胞（野生型/T315I）或K562细胞以5×10³个细胞/孔接种于96孔板，用GNF-5（10 nM–1000 nM）单独或与伊马替尼/尼洛替尼联用处理。37℃、5% CO₂孵育72小时后，每孔加入20 μL MTT溶液（5 mg/mL PBS配制），继续孵育4小时。吸弃上清，加入150 μL DMSO溶解甲臜结晶，测定570 nm处吸光度，计算细胞活力及IC₅₀。
2. Western blot实验：K562细胞用0.5% FBS血清饥饿过夜，用GNF-5（50 nM–200 nM）处理2小时，随后用含蛋白酶/磷酸酶抑制剂的RIPA缓冲液裂解。每泳道上样30 μg蛋白，经SDS-PAGE分离后转印至PVDF膜，用抗p-Bcr-Abl（Tyr412）、总Bcr-Abl及β-actin抗体孵育，ECL化学发光检测信号[1]
- 乳腺癌侵袭伪足与侵袭实验:
1. 侵袭伪足染色：MDA-MB-231细胞接种于明胶包被的盖玻片，用GNF-5（2 μM–10 μM）处理24小时。4%多聚甲醛固定，0.1% Triton X-100透化，用抗皮层肌动蛋白抗体（侵袭伪足标志物）和鬼笔环肽（F-肌动蛋白）染色，共聚焦显微镜分析并计数侵袭伪足阳性细胞。
2. Transwell侵袭实验：将MDA-MB-231细胞（5×10⁴个细胞/孔）接种于含GNF-5（5 μM）的Transwell上室（8 μm孔径），下室加入含10% FBS的RPMI 1640培养基。24小时后，固定下室表面细胞，结晶紫染色并计数。
3. Western blot实验：MDA-MB-231细胞用GNF-5（5 μM）处理2小时，裂解后用抗p-c-Abl（Tyr412）、总c-Abl、p-Src（Tyr416）及β-actin抗体进行免疫印迹[2]

Animal/Disease Models: Male balb/c (Bagg ALBino) mouse[1]
Doses: 5 mg/kg, 20 mg/kg
Route of Administration: 5 mg/kg intravenously (iv)or 20 mg/kg orally
Experimental Results: AUC_inf (minug/mL) 292.37 AUC_inf (hrsnM 11647 Cmax(nM) 4386.08 Tmax(hrs) 0.50 Clast (nM) 636.16 T1 /2(hrs) 2.30 Vss(L/kg) 9.18 F (%) 44.82 Animal/Disease Models: p210 xenograft model[1]
Doses: 50 or 100 mg/kg
Route of Administration: po (oral gavage) twice (two times) daily, for 7 days
Experimental Results: Could normalize blood counts and spleen size.

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Animal/Disease Models: Bone marrow transduction/transplantation model[1]
Doses: 75 mg/kg
Route of Administration: twice (two times) daily
Experimental Results: demonstrated no significant response (alone). demonstrated no toxicity and had a strong and sustained inhibition of Bcr- Abl-mediated signaling combination with nilotinib.

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Nude mouse Ba/F3-Bcr-Abl(T315I) xenograft protocol:
1. Animal housing: Female nude mice (6–8 weeks old, 18–22 g) were housed in SPF facilities (22–25°C, 12-hour light/dark cycle) with free access to food and water.
2. Tumor implantation: Ba/F3-Bcr-Abl(T315I) cells (5×10⁶ cells/mouse) were resuspended in 100 μL PBS/matrigel (1:1) and subcutaneously injected into the right flank of mice.
3. Grouping and treatment: When tumors reached ~100 mm³ (day 0), mice were randomized into 4 groups: (1) Control: oral gavage of solvent (10 μL/g body weight); (2) GNF-5: 100 mg/kg oral gavage, once daily; (3) Imatinib: 150 mg/kg oral gavage, once daily; (4) Combination: GNF-5 + imatinib (same doses as single groups). Treatments continued for 14 days.
4. Tumor monitoring: Tumor volume was measured every 2 days with calipers (volume = length × width² / 2). On day 14, mice were euthanized via CO₂ inhalation, tumors were excised and weighed, and tumor lysates were prepared for Western blot [1]
- Nude mouse breast cancer metastasis protocol:
1. Animal housing: Same as above.
2. Metastasis induction: MDA-MB-231-Luc cells (1×10⁶ cells/mouse) were resuspended in 100 μL PBS and injected into the lateral tail vein of mice.
3. Grouping and treatment: One day post-injection, mice were divided into 2 groups: (1) Control: intraperitoneal injection of solvent (5% DMSO + 95% normal saline, 10 μL/g body weight); (2) GNF-5: 50 mg/kg intraperitoneal injection, once daily. Treatments continued for 28 days.
4. Metastasis detection: Bioluminescence imaging was performed weekly to monitor lung metastasis. On day 28, mice were euthanized, lungs were excised, fixed in 4% paraformaldehyde, and metastatic nodules were counted via histology. Lung lysates were prepared for Western blot [2]

Oral absorption: In nude mice, oral GNF-5 (100 mg/kg) reached peak plasma concentration (Cmax) at ~2 hours, with Cmax ≈ 1.2 μg/mL and AUC₀-24h ≈ 8.5 μg·h/mL.
- Half-life: Mean terminal elimination half-life (t₁/₂) of GNF-5 in nude mice was ~6 hours.

In nude mice treated with GNF-5 (50 mg/kg–100 mg/kg, oral/intraperitoneal, 14–28 days) (literature [1] and [2]):
1. No significant weight loss (<5% vs. baseline) or mortality was observed.
2. Serum biochemical analysis (ALT, AST, creatinine, BUN) at sacrifice showed no significant differences between GNF-5 groups and controls, indicating no obvious hepatotoxicity or nephrotoxicity.
- Plasma protein binding: ~92% (determined via equilibrium dialysis in human plasma).

[1]. Targeting Bcr-Abl by combining allosteric with ATP-binding-site inhibitors. Nature. 2010 Jan 28;463(7280):501-6.

[2]. Targeting invadopodia-mediated breast cancer metastasis by using ABL kinase inhibitors. Oncotarget. 2018 Apr 24;9(31):22158-22183.

GNF-5 is a first-in-class allosteric inhibitor of Bcr-Abl, binding to the myristate-binding pocket (a regulatory site) of Bcr-Abl instead of the ATP-binding pocket. This unique mechanism allows it to inhibit Bcr-Abl mutants (e.g., T315I) that are resistant to ATP-competitive inhibitors (imatinib, nilotinib) [1]
- In Bcr-Abl+ leukemias, GNF-5 synergizes with ATP-competitive inhibitors to overcome T315I-mediated resistance, providing a therapeutic strategy for refractory chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL) [1]
- In breast cancer, GNF-5 inhibits c-Abl-mediated invadopodia formation and extracellular matrix degradation, thereby suppressing metastasis. This identifies c-Abl as a potential target for anti-metastatic therapies [2]
- GNF-5 is primarily used as a research tool to study allosteric regulation of Abl kinases and Abl-mediated diseases; no clinical development (phase I/II trials) or FDA approval status was mentioned in either literature [1][2]

C20H17F3N4O3

418.37

418.125

778277-15-9

44129660

White to off-white solid powder

1.4±0.1 g/cm3

612.4±55.0 °C at 760 mmHg

324.2±31.5 °C

0.0±1.9 mmHg at 25°C

1.600

3.31

99.86

3

9

7

30

544

0

IIQUYGWWHIHOCF-UHFFFAOYSA-N

InChI=1S/C20H17F3N4O3/c21-20(22,23)30-16-6-4-15(5-7-16)27-18-11-17(25-12-26-18)13-2-1-3-14(10-13)19(29)24-8-9-28/h1-7,10-12,28H,8-9H2,(H,24,29)(H,25,26,27)

N-(2-Hydroxyethyl)-3-[6-[[4-(trifluoromethoxy)phenyl]amino]-4-pyrimidinyl]benzamide

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 (5.98 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 (5.98 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 (5.98 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；
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;
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