PF-431396

FAK/focal adhesion kinase (IC50 = 2 nM); PYK2/proline-rich tyrosine kinase 2 (IC50 = 11 nM); BRD4 (Kd = 445 nM)

体外活性：在 A20 细胞中，PF-431396 阻断抗 Ig 和聚集 LFA-1 诱导的 Pyk2 和 FAK 酪氨酸磷酸化，并进一步阻断 B 细胞扩散。 PF-431396 始终如一地抑制因不添加钙而诱导的蛋白质酪氨酸磷酸化 (PY) 的增加，以及在有钙存在的情况下由 W-7 诱导的蛋白质酪氨酸磷酸化 (PY) 的增加。激酶测定：PF-431396 是粘着斑激酶 (FAK) 和富含脯氨酸酪氨酸激酶 2 (PYK2) 的双重抑制剂（IC50 值分别为 2 和 11 nM），PF-431396 对 BRD4 的 Kd 值为 445 nM。 IC50值：2nM（FAK）； 11nM（PYK2）； 445 nM（BRD4 的 KD）[1] [2] 目标：FAK； PYK2； BRD4 体外：PF-431396 是 Pyk2 和 FAK 的有效且高选择性的基于嘧啶的抑制剂，与 Pyk2 和 FAK 的酪氨酸磷酸化涉及初始自磷酸化或转磷酸化步骤的观点一致，用 PF-431396 处理 A20 细胞当细胞在悬浮液中受到 ECM 刺激时，可阻断抗 Ig 诱导的 Pyk2 和 FAK 酪氨酸磷酸化。还测定了 PF-431396 (Kd = 445 ± 42 nM) 和 PIM 抑制剂 (Kd = 565 ± 63 nM) 的纳摩尔亲和力。

PYK2抑制剂可增加OVX大鼠的骨形成并防止骨丢失。[3]
接下来，我们使用PF-431396 (PF-46)（PF-46）测试了PYK2活性的药理学调节是否会影响OVX大鼠的骨量，这是一种已建立的绝经后骨质疏松症临床前疾病模型，PF-431396 (PF-46)是一种基于嘧啶的强效PYK2抑制剂，对重组PYK2酶的IC50为31 nM（图5A和SI方法）。4个月大的OVX大鼠每天用赋形剂PF-431396（PF-46）（10和30mg/kg）或EE（一种抗骨吸收剂）治疗28天。药代动力学研究表明，PF-431396的游离血浆浓度在高剂量下覆盖PYK2 IC50至少8小时（数据未显示）。如股骨远端中期的μCT图像所示（图5B），载体治疗的OVX大鼠的骨小梁质量比假对照组少。EE和PF-431396都抵消了OVX诱导的骨丢失，完全保留了总骨含量和总骨密度（图5C和D）。与之前的研究一致，与假对照组相比，载体治疗的OVX大鼠表现出高骨转换率，其特征是骨形成（每骨表面矿化表面和每骨表面骨形成率）和骨吸收（破骨细胞表面和血清CTX）增加（表1和图5E和F）。EE对OVX大鼠的治疗抑制了高骨转换，与赋形剂治疗相比，骨吸收和形成减少。与EE相比，两种剂量的PF-431396均显著提高了骨形成率，同时矿化表面和矿物附着率也升高（表1和图5E），表明PF-431396-促进了成骨细胞的募集和活性。与此一致，PF-431396增加了7天hMSC培养物中的碱性磷酸酶活性（P.C.B.和L.B.，数据未显示）。尽管高剂量PF-431396降低了胫骨近端的破骨细胞表面（骨吸收的参考参数），但在治疗2周（数据未显示）和4周（图5F）后，两种剂量都没有改变血清CTX（骨吸收系统生物标志物）。这些结果表明，PF-431396是一种强效的PYK2抑制剂，主要通过刺激骨形成来预防雌激素缺乏引起的大鼠骨丢失，为PYK2在调节骨形成中的作用提供了独立的药理学证实。

PYK2抑制剂的IC50测定。[3]
将含有150 pM PYK2结构域酶、30 mM肽底物和10 mM DTT的反应混合物在测定缓冲液[50 mM Hepes（pH 7.0）、1 mM MgCl2和0.1%BSA]中分配到384孔测定板的所有孔中。将稀释在二甲亚砜（DMSO）中的化合物分三次分配到测定板中。每项试验中都包括仅DMSO（阳性）对照和强效PYK2抑制剂（阴性）对照，分别用于设定最大和最小反应。将在测定缓冲液中稀释的ATP分配到整个板上，使其终浓度为50 mM（ATP的浓度为»kM）。然后将测定板在30°C下孵育2小时。向板的每个孔中加入含有45 mM EDTA、10´PTK绿色示踪剂和10´抗磷酸酪氨酸抗体的停止/检测混合物。将检测反应在室温下在黑暗中孵育1小时，然后使用荧光偏振协议（485 nm激发和530 nm发射，使用505 nm二向色滤光片）在Analyst GT上读取荧光偏振。通过使用GraphPad Prism 4或类似的专有软件并拟合到S形剂量反应曲线，从剂量反应曲线计算IC50测定值。

细胞扩散[2]
组织培养板在4°C下用大鼠抗小鼠LFA-1单克隆抗体或纤维连接蛋白包被过夜，然后用含有2%牛血清白蛋白的磷酸盐缓冲盐水封闭1小时。A20细胞（10~5个细胞在0.5ml RPMI 1640培养基中，含有2%胎牛血清和50μm 2-巯基乙醇）用DMSO或PF-431396预处理45分钟，加入包被的孔中，在37°C下孵育。标记为铺展的细胞呈深色，形状细长或不规则，有明显的膜突起。

Sprague–Dawley female rats were used for the PYK2 pharmacology studies. Rats were sham-operated or ovariectomized at 4.5–5 months of age. Beginning the day after surgery, animals were dosed every day by oral gavage with either vehicle (20% β-cyclodextrin in water) or the PYK2 inhibitor (PF-431396 (PF-46)) at 10 or 30 mg/kg in vehicle for 28 consecutive days.[3]

[1]. Structural characterization of proline-rich tyrosine kinase 2 (PYK2) reveals a unique (DFG-out) conformation and enables inhibitor design. J Biol Chem. 2009 May 8;284(19):13193-201.
[2]. B cell receptor-induced phosphorylation of Pyk2 and focal adhesion kinase involves integrins and the Rap GTPases and is required for B cell spreading. J Biol Chem. 2009 Aug 21;284(34):22865-77.
[3]. Proline-rich tyrosine kinase 2 regulates osteoprogenitor cells and bone formation, and offers an anabolic treatment approach for osteoporosis. Proc Natl Acad Sci U S A. 2007 Jun 19;104(25):10619-24.

N-methyl-N-[2-[[[2-[(2-oxo-1,3-dihydroindol-5-yl)amino]-5-(trifluoromethyl)-4-pyrimidinyl]amino]methyl]phenyl]methanesulfonamide is a sulfonamide.

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Proline-rich tyrosine kinase 2 (PYK2) is a cytoplasmic, non-receptor tyrosine kinase implicated in multiple signaling pathways. It is a negative regulator of osteogenesis and considered a viable drug target for osteoporosis treatment. The high-resolution structures of the human PYK2 kinase domain with different inhibitor complexes establish the conventional bilobal kinase architecture and show the conformational variability of the DFG loop. The basis for the lack of selectivity for the classical kinase inhibitor, PF-431396, within the FAK family is explained by our structural analyses. Importantly, the novel DFG-out conformation with two diarylurea inhibitors (BIRB796, PF-4618433) reveals a distinct subclass of non-receptor tyrosine kinases identifiable by the gatekeeper Met-502 and the unique hinge loop conformation of Leu-504. This is the first example of a leucine residue in the hinge loop that blocks the ATP binding site in the DFG-out conformation. Our structural, biophysical, and pharmacological studies suggest that the unique features of the DFG motif, including Leu-504 hinge-loop variability, can be exploited for the development of selective protein kinase inhibitors.[1]

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Signaling by the B cell receptor (BCR) promotes integrin-mediated adhesion and cytoskeletal reorganization. This results in B cell spreading, which enhances the ability of B cells to bind antigens and become activated. Proline-rich tyrosine kinase (Pyk2) and focal adhesion kinase (FAK) are related cytoplasmic tyrosine kinases that regulate cell adhesion, cell morphology, and cell migration. In this report we show that BCR signaling and integrin signaling collaborate to induce the phosphorylation of Pyk2 and FAK on key tyrosine residues, a modification that increases the kinase activity of Pyk2 and FAK. Activation of the Rap GTPases is critical for BCR-induced integrin activation as well as for BCR- and integrin-induced reorganization of the actin cytoskeleton. We now show that Rap activation is essential for BCR-induced phosphorylation of Pyk2 and for integrin-induced phosphorylation of Pyk2 and FAK. Moreover Rap-dependent phosphorylation of Pyk2 and FAK required an intact actin cytoskeleton as well as actin dynamics, suggesting that Rap regulates Pyk2 and FAK via its effects on the actin cytoskeleton. Importantly B cell spreading induced by BCR/integrin co-stimulation or by integrin engagement was inhibited by short hairpin RNA-mediated knockdown of either Pyk2 or FAK expression and by treatment with PF-431396, a chemical inhibitor that blocks the kinase activities of both Pyk2 and FAK. Thus Pyk2 and FAK are downstream targets of the Rap GTPases that play a key role in regulating B cell morphology.[2]

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Bone is accrued and maintained primarily through the coupled actions of bone-forming osteoblasts and bone-resorbing osteoclasts. Cumulative in vitro studies indicated that proline-rich tyrosine kinase 2 (PYK2) is a positive mediator of osteoclast function and activity. However, our investigation of PYK2-/- mice did not reveal evidence supporting an essential function for PYK2 in osteoclasts either in vivo or in culture. We find that PYK2-/- mice have high bone mass resulting from an unexpected increase in bone formation. Consistent with the in vivo findings, mouse bone marrow cultures show that PYK2 deficiency enhances differentiation and activity of osteoprogenitor cells, as does expressing a PYK2-specific short hairpin RNA or dominantly interfering proteins in human mesenchymal stem cells. Furthermore, the daily administration of a small-molecule PYK2 inhibitor increases bone formation and protects against bone loss in ovariectomized rats, an established preclinical model of postmenopausal osteoporosis. In summary, we find that PYK2 regulates the differentiation of early osteoprogenitor cells across species and that inhibitors of the PYK2 have potential as a bone anabolic approach for the treatment of osteoporosis.[3]

C22H21F3N6O3S

506.5

506.134

C, 52.17; H, 4.18; F, 11.25; N, 16.59; O, 9.48; S, 6.33

717906-29-1

11598628

Beige solid powder

1.5±0.1 g/cm3

1.643

1.36

127.93

3

11

7

35

854

0

S(C([H])([H])[H])(N(C([H])([H])[H])C1=C([H])C([H])=C([H])C([H])=C1C([H])([H])N([H])C1C(C(F)(F)F)=C([H])N=C(N=1)N([H])C1C([H])=C([H])C2=C(C=1[H])C([H])([H])C(N2[H])=O)(=O)=O

POJZIZBONPAWIV-UHFFFAOYSA-N

InChI=1S/C22H21F3N6O3S/c1-31(35(2,33)34)18-6-4-3-5-13(18)11-26-20-16(22(23,24)25)12-27-21(30-20)28-15-7-8-17-14(9-15)10-19(32)29-17/h3-9,12H,10-11H2,1-2H3,(H,29,32)(H2,26,27,28,30)

N-Methyl-N-[2-[[[2-[(2,3-dihydro-2-oxo-1H-indol-5-yl)amino]-5-(trifluoromethyl)-4-pyrimidinyl]amino]methyl]phenyl]methanesulfonamide

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)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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;
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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;
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