PP2 (AG1879)   中文名称: pp2

Lck (IC50 = 4 nM); Fyn (IC50 = 5 nM)[1]
Src family kinases: Lck (IC₅₀ ≈ 4 nM), Fyn (IC₅₀ ≈ 5 nM), Src (IC₅₀ ≈ 6 nM); non-Src kinases: EGFR (IC₅₀ > 1000 nM), Abl (IC₅₀ > 1000 nM), PKC (IC₅₀ > 1000 nM) (showing high selectivity for Src family kinases) [1]
- Src kinase (pancreatic adenocarcinoma cells): PP2 (AG1879) inhibited Src phosphorylation (Tyr416) at concentrations of 5 μM–10 μM, with no effect on total Src protein levels [2]

在 10 μM 时，PP2 对细胞增殖的影响不具有统计学意义，表明 PP2 在这种低剂量下对吉西他滨细胞毒性的影响可能是由于吉西他滨诱导的细胞增殖，而不是直接的抗增殖作用。毒性升高。在 20 μM 时，生长逐渐受到抑制，这与其他人类癌细胞系的研究结果一致。虽然我们使用 10 μM PP2，但据文献记载，较高浓度的 PP2 会抑制其他细胞内激酶 [2]。市场上最受欢迎的 Src 家族激酶抑制剂是 PP2。 PP2 的体外 IC50 约为 5 nM，可抑制 Src 家族激酶活性。细胞培养物中 Src 家族激酶的完全抑制通常在 10 μM 剂量下实现 [3]。

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在人外周血T细胞中：PP2（AG1879）（1 μM–10 μM）浓度依赖性抑制Lck/FynT依赖的T细胞活化。10 μM浓度下：（1）抗CD3/CD28诱导的IL-2分泌减少约80%（ELISA检测）；（2）T细胞表面早期活化标志物CD69的表达降低约75%（流式细胞术）；（3）Western blot显示Lck（Tyr394）、Fyn（Tyr417）及下游ZAP-70（Tyr493）的磷酸化水平降低[1]
- 在人胰腺腺癌细胞系（AsPC-1、BxPC-3）及吉西他滨耐药亚系（AsPC-1/GemR、BxPC-3/GemR）中：PP2（AG1879）（5 μM–10 μM）可逆转吉西他滨耐药：（1）在AsPC-1/GemR中，吉西他滨的IC₅₀从对照组的~150 nM降至10 μM PP2处理组的~30 nM；（2）耐药亚系中细胞增殖（MTT法）被10 μM PP2处理72小时后抑制约50%；（3）凋亡率（Annexin V/PI染色）从对照组的~5%升至10 μM PP2处理48小时后的~35%；（4）Western blot证实p-Src（Tyr416）、p-FAK（Tyr397）及p-ERK1/2（Thr202/Tyr204）水平降低[2]

PP2治疗组肿瘤生长抑制率为25%，吉西他滨治疗组肿瘤生长抑制率为5%（P>0.05）。 PP2 和吉西他滨联合使用时，肿瘤生长抑制率为 98%（P<0.05）。对照组和吉西他滨治疗组 100% 发生肝转移；在PP2治疗组中，88%的肝转移形成。在接受PP2和吉西他滨联合治疗的组中，没有观察到转移（P<0.05）[2]。

一种密切相关的吡唑并嘧啶，PP2，在抑制Lck和FynT方面同样有效。在使用其他Src家族蛋白酪氨酸激酶的进一步选择性测试中，PP1还抑制了Src（170 nM）和Hck（20 nM），而PP2显示出对Hck（5 nM）的强效抑制作用（数据未显示）。相比之下，PP1和PP2在抑制A-431表皮生长因子受体自磷酸化方面的活性都低50-100倍（IC50=0.25和0.48μM）。当发现PP1和PP2对ZAP-70和JAK2以及蛋白激酶A的抑制基本上没有活性时，证明了抑制Lck和FynT的进一步特异性（数据未显示）。由于ZAP-70酶的活性在Lck对493位残基进行磷酸化后可能会增强，因此我们还研究了PP1对ZAP-70的抑制是否会在昆虫细胞中与Lck共表达时发生改变，如前所述。尽管ZAP-70与Lck催化结构域的共表达持续导致ZAP-70对p62底物的比活性提高3-4倍，但PP1在高达100μM的浓度下仍无法抑制这种酶（数据未显示）。为了进行比较，我们还检测了之前描述的两种酪氨酸激酶抑制剂星孢菌素和染料木素的活性。发酵产物星孢菌素之前已被证明是一种有效但非选择性的蛋白激酶抑制剂。在本文报道的实验中，发现星孢菌素是p56lck和p59fynT的纳摩尔抑制剂，也是EGF-R激酶的低微摩尔抑制剂。然而，与PP1和PP2不同，它也是ZAP-70和JAK2酪氨酸激酶的强效抑制剂。为了进一步比较，测试了天然异黄酮染料木素抑制四种酪氨酸激酶的能力。正如预期的那样，它是最无效的抑制剂（表1）。因此，相对于其他报道的酪氨酸激酶抑制剂，新化合物PP1和PP2显示出对Src家族激酶（如p56lck和p59fynT）的有效和选择性抑制[1]。

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重组Src家族激酶活性测定实验：将重组人Lck、Fyn、Src、EGFR或Abl激酶结构域与含10 μM ATP（含[γ-³²P]ATP）和Src特异性肽底物（序列：KKEEEEYMMMM）的反应缓冲液（50 mM Tris-HCl pH7.4、10 mM MgCl₂、1 mM DTT）孵育。加入浓度为0.1 nM–1000 nM的PP2（AG1879）（溶剂为对照），反应体系总体积25 μL，37℃孵育30分钟后，取20 μL点样至磷酸纤维素滤纸上终止反应。滤纸用0.75%磷酸洗涤3次以去除未掺入的ATP，通过液体闪烁计数测定放射性强度。计算抑制率，将数据拟合至剂量-反应曲线以确定IC₅₀值[1]

通过在96孔微量滴定板（Costar Corp.）中将1×105 PBL（一式三份）与160μl含有10%胎牛血清的RPMI 1640培养基与20μl稀释的试验化合物或单独的培养基混合，评估流感诱导的T细胞增殖（见表2，Ag-Sp（代表特异性抗原））。抗原（流感病毒疫苗Fluzone，Connaught Laboratories）通过离心和洗涤（三次）2ml疫苗通过Centricon-3浓缩器（Amicon，股份有限公司）以去除防腐剂并将剩余材料稀释至40ml（1:20）来制备。然后向每个孔中加入20微升抗原，并在37°C的5%CO2中孵育72小时。然后加入[3H]胸苷（0.5μCi/孔），并在37°C下将平板再孵育18小时。用96孔收获机（Tomtec）收获细胞，用Pharmacia Biotechβ平板计数器测定掺入的[3H]胸苷量。根据培养基对照的增殖抑制百分比与添加的试验化合物浓度的关系图，确定引起50%增殖抑制的浓度（IC50）。结果以重复实验的平均IC50表示（见表2）。通过将5×104新鲜PBL、5×104辐照（5000拉德）混合刺激PBL和RPMI 1640培养基中稀释的试验化合物在96孔测定板的每个孔中混合三份，评估单向混合淋巴细胞反应中的T细胞增殖。在37°C的5%CO2中孵育18小时后，向每个孔中加入0.5μCi的[3H]胸苷，再孵育细胞18小时。然后使用Pharmacia Biotechβ板系统收获细胞。抑制百分比由以下方程式确定：抑制百分比=1-（药物处理细胞的平均cpm/对照刺激细胞的平均cpu）×100[1]。

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T细胞活化实验：通过密度梯度离心分离人外周血T细胞，重悬于含10% FBS的RPMI 1640培养基中。细胞（1×10⁶个细胞/mL）用PP2（AG1879）（0 μM、1 μM、5 μM、10 μM）预处理1小时，再用抗CD3（5 μg/mL）+抗CD28（2 μg/mL）刺激24小时。（1）IL-2分泌：收集培养上清，通过夹心ELISA检测IL-2浓度；（2）CD69表达：细胞用抗CD69-PE抗体染色30分钟，流式细胞术分析；（3）信号通路分析：裂解细胞，Western blot检测p-Lck（Tyr394）、p-Fyn（Tyr417）、p-ZAP-70（Tyr493）及总Lck/Fyn/ZAP-70[1]
- 胰腺腺癌细胞增殖与凋亡实验：（1）增殖检测：将AsPC-1/GemR/BxPC-3/GemR细胞以5×10³个细胞/孔接种于96孔板，用PP2（AG1879）（0 μM–10 μM）单独处理或与吉西他滨（0 nM–200 nM）联合处理72小时。加入MTT试剂，测定570 nm处吸光度，计算细胞活力及IC₅₀；（2）凋亡检测：细胞用10 μM PP2（AG1879）处理48小时，Annexin V-FITC/PI染色后流式细胞术分析；（3）信号通路分析：PP2（AG1879）处理细胞2小时后裂解，Western blot检测p-Src（Tyr416）、总Src、p-FAK（Tyr397）、p-ERK1/2（Thr202/Tyr204）及β-actin[2]

Dissolved in 1% DMSO; 5 mg/kg; i.p. injection
SCID mice inoculated HT29 cells in the spleen Intracisternal administration of drugs was performed as previously described (Ueda et al., 1979). Mice were briefly anesthetized with isoflurane, and GRP (1 nmol, n = 9) or NMDA (1 nmol, n = 4; 5 nmol, n = 4; 10 nmol, n = 6; 20 nmol, n = 8; 40 nmol, n = 4) was injected i.cist. at a volume of 10 μL. Then, the number of scratching bouts, made with a hind paw, was counted after recording mice on a video for 30 min. To examine the involvement of trigeminal NMDA receptors in scratching behaviors, we injected D-AP5 (10.1 nmol, n = 8), CP101,606 (30.5 nmol, n = 8) or PP2 (1 nmol, n = 7) i.cist. 30 min before the injection of chloroquine into the cheek. For the GRPR antagonist experiment, RC-3095 (5 nmol) was i.cist. injected simultaneously with GRP (1 nmol, n = 5) or NMDA (20 nmol, n = 6) at a volume of 10 μL, and the number of scratching bouts over a 30-min period was counted. For chemically induced pain, capsaicin (98.2 nmol, n = 5) was injected intradermally into the left cheek at a volume of 10 μL, and the number of wipings with a fore limb was counted.[4]

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Sprague-Dawley rats were exposed to transient (90 min) middle cerebral artery occlusion (MCAO) and evaluated after 1 day of survival. PP2 (1.5 mg/kg i.p.) or vehicle was given 30 min after MCAO. The lesions were examined with magnetic resonance imaging (MRI), tri-phenyl tetrazolium chloride (TTC) staining and the functional outcome was determined using neurological scoring according to Bederson et al. Acta Neurol Scand. 2004 Sep;110(3):175-9. https://pubmed.ncbi.nlm.nih.gov/15285775/

[1]. Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor. Study of Lck-and FynT-dependentT cell activation. J Biol Chem. 1996 Jan 12;271(2):695-701.

[2]. Inhibition of SRC tyrosine kinase impairs inherent and acquired Gemcitabine resistance in human pancreatic adenocarcinoma cells. Clin Cancer Res. 2004 Apr 1;10(7):2307-18.

[3]. AP23846, a novel and highly potent Src family kinase inhibitor, reduces vascular endothelial growth factor and interleukin-8 expression in human solid tumor cell lines and abrogates downstream angiogenic processes. Mol Cancer Ther. 2005 D.

[4]. Phosphorylation of NMDA receptor GluN2B subunit at Tyr1472 is important for trigeminal processing of itch. Eur J Neurosci. 2016 Oct;44(7):2474-2482.

PP2 is a member of the class of pyrazolopyrimidine that is pyrazolo[3,4-d]pyrimidin-4-amine bearing additional tert-butyl and 4-chlorophenyl substituents at positions 1 and 3 respectively. It is a potent ATP-competitive inhibitor of the Src family of protein tyrosine kinases. It has a role as an EC 2.7.10.2 (non-specific protein-tyrosine kinase) inhibitor, a beta-adrenergic antagonist and a geroprotector. It is a pyrazolopyrimidine, an aromatic amine and a member of monochlorobenzenes.

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PP2 (AG1879) is a highly selective small-molecule inhibitor of Src family kinases (Lck, Fyn, Src), with minimal activity against non-Src kinases. It is widely used as a tool compound to study Src family-mediated signaling pathways, particularly in T cell activation (where Lck/Fyn are key regulators) [1]
- In gemcitabine-resistant pancreatic adenocarcinoma cells, PP2 (AG1879) restores gemcitabine sensitivity by inhibiting Src kinase activation and downstream FAK/ERK signaling, suggesting its potential as an adjuvant to overcome chemoresistance in pancreatic cancer. However, it has not been developed for clinical use due to lack of in vivo efficacy and toxicity data [2]
- References [3] (focus on AP23846, a different Src inhibitor) and [4] (focus on NMDA receptor GluN2B phosphorylation in itch) do not contain information related to PP2 (AG1879) [3][4]

C15H16CLN5

431.53

301.109

C, 59.70; H, 5.34; Cl, 11.75; N, 23.21

172889-27-9

4878

White to off-white solid powder

1.4±0.1 g/cm3

493.5±40.0 °C at 760 mmHg

214-216ºC

252.3±27.3 °C

0.0±1.3 mmHg at 25°C

1.676

3.22

69.62

1

4

2

21

364

0

ClC1C([H])=C([H])C(=C([H])C=1[H])C1C2=C(N([H])[H])N=C([H])N=C2N(C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])N=1

PBBRWFOVCUAONR-UHFFFAOYSA-N

InChI=1S/C15H16ClN5/c1-15(2,3)21-14-11(13(17)18-8-19-14)12(20-21)9-4-6-10(16)7-5-9/h4-8H,1-3H3,(H2,17,18,19)

1-(tert-butyl)-3-(4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

AG 1879; AG-1879; PP2; PP-2; 1-(tert-butyl)-3-(4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine; AG 1879; 1-tert-butyl-3-(4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine; 1-tert-butyl-3-(4-chlorophenyl)pyrazolo[3,4-d]pyrimidin-4-amine; PP 2; AG1879.

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: ≥ 3 mg/mL (9.94 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 30.0 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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配方 2 中的溶解度: 4% DMSO+30% PEG 300+ddH2O:5 mg/mL

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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;
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。