XMU-MP-1

Mammalian STE20-like protein kinase/MST1 (IC50 = 71.1 nM); MST2 (IC50 = 38.1 nM)

在 0.1 至 10 μM 的浓度范围内，XMU-MP-1 以剂量依赖性方式磷酸化 HepG2 细胞中内源性较低的 MOB1、LATS1/2 和 YAP。在小鼠巨噬细胞样细胞、人骨肉瘤和人结直肠腺癌细胞等多种细胞系中，XMU-MP-1 疗法可抑制过氧化氢刺激的 MOB1 磷酸化和 MST1/2 自磷酸化。通过抑制 MST1/2 激酶活性，XMU-MP-1 刺激下游效应器 Yes 相关蛋白并促进细胞分裂。在细胞中，XMU-MP-1 可以更有效、可逆地抑制激酶 MST1/2 的活性，并改善其随后的 YAP 激活 [1]。

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用XMU-MP-1治疗可下调MST1的表达水平，部分逆转MST1对增殖、迁移和凋亡相关蛋白的抑制作用，并抑制Hippo信号通路[3]。 为了确定Hippo信号通路是否介导了MST1在BCa中的生物学功能，使用Hippo信息通路抑制剂XMU-MP-1（MST1/2的抑制剂）抑制过表达MST1的BCa细胞中Hippo的信号通路功能。RT-qPCR分析表明，用抑制剂处理MST1过表达细胞会下调MST1的表达水平（图5A）。CCK-8和EdU掺入试验的结果显示，LV-MST1+XMU-MP-1组中LV-MST1组的抑制增殖能力部分恢复（图5B和C）。使用伤口愈合试验分析BCa细胞迁移；结果显示，与LV-MST1组相比，MST1+XMU-MP-1细胞组的细胞迁移也显著增加（图5D）。最后，进行蛋白质印迹分析，分析Hippo信号通路中关键蛋白的表达水平。与LV-MST1组相比，LV-MST1+XMU-MP-1组中LATS1和Bax的表达水平显著下调，而YAP、Bcl-2和Ki-67的表达水平在两种细胞系中显著上调（图6A和B）。

对于腹腔给药，XMU-MP-1在1 mg/kg至3 mg/kg的剂量范围内，在急性和慢性肝损伤小鼠中表现出出色的体内药代动力学。在 Fah 缺陷小鼠模型中，XMUMP-1 治疗表现出比媒介物治疗的对照组显着更高的人肝细胞再生率，表明 XMU-MP-1 治疗可能促进人肝再生 [1]。

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通过免疫组织化学和蛋白质印迹分析评估AngII输注的升主动脉和腹主动脉中的MST、p-MST、p-YAP、p-MOB和TAZ蛋白。为了研究MST1/2抑制对AA的影响，用输注AngII的西方饮食喂养的低密度脂蛋白（LDL）受体-/-小鼠服用赋形剂或XMU-MP-15周。在输注AngII的升主动脉和腹主动脉中，Hippo YAP信号蛋白显著升高XMU-MP-1给药导致AngII诱导的上升AA减弱，而不影响腹部AA和主动脉粥样硬化。抑制Hippo-YAP信号传导也导致AngII诱导的基质金属蛋白酶2（MMP2）活性、巨噬细胞积聚、主动脉内侧肥大和升主动脉弹性蛋白断裂的抑制[2]。

体外和体内激酶抑制试验[1]
对于体内抑制试验，用0.5μg空质粒或pCMV质粒转染人胚胎肾（HEK）293T细胞，每个质粒在12孔板中表达各种形式的Flag标记的全长MST1或MST2激酶。转染后24小时，用指定剂量的XMU-MP-1处理细胞3小时。用指定的抗体通过免疫印迹分析细胞裂解物。对于体外激酶抑制试验，从大肠杆菌中表达并纯化了重组GST标记的MOB1a和各种形式的重组His标记的全长MST1或MST2激酶。酶、ATP和GST-MOB1的消耗量与之前优化的条件保持一致。在30°C下，在激酶测定缓冲液中用指定剂量的XMU-MP-1进行30分钟的测定，然后进行SDS-聚丙烯酰胺凝胶电泳和免疫印迹分析。

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XMU-MP-1的KINOMEscan分析[1]
XMU-MP-1使用KINOMEscan技术对468种激酶进行了分析，这是一种1μM的活性位点依赖性竞争结合分析。KINOMEscan选择性得分是化合物选择性的定量度量。它是通过将与化合物结合的激酶数量除以所测试的激酶总数来计算的。结果被报告为“对照%”（ctrl%），其中较低的数字表示较高的亲和力结合；ctrl%=（试验化合物信号-阳性对照信号）/（阴性对照信号-阳性控制信号）×100，其中阴性对照=DMSO（ctrl%=100%），阳性对照=对照化合物（ctrl%=0%）；S（10）=（ctrl%≤10%的激酶数量）/（测试的激酶的数量）。ctrl%<10%表示非常强的抑制作用，ctrl%>70%表示非常弱的抑制作用。当前研究中的激酶组或单个激酶名称包括TK（酪氨酸激酶）、TKL（TK样）、STE（酵母Sterile 7、Sterile 11和Sterile 20激酶的同源物）、AGC[蛋白激酶A、G和C家族]、CAMK（钙/钙调素依赖性蛋白激酶）、CK1（酪蛋白激酶1）和CMGC[细胞周期蛋白依赖性激酶（CDK）、丝裂原活化蛋白激酶（MAPK）、糖原合酶激酶3（GSK3）和CDC2样激酶（CLK）家族]。

人BCa细胞系MCF-7、MDA-MB-231和SKBR3以及正常乳腺上皮细胞系MCF-10A购自美国典型培养物保藏中心。所有细胞系均在添加了10%FBS的DMEM中培养，并在37°C、5%CO2的加湿培养箱中保持。 将MST1抑制剂XMU-MP-1溶解在DMSO中，并以0.1%的终浓度加入培养基中。细胞系在37°C的培养基中培养1小时[3]。

Both LDL receptor −/− and C57BL/6J mice were used. LDL receptor −/− mice were backcrossed 10-fold into a C57BL/6 background. Age-matched male littermates (8–10 weeks old) were used for the present study. Mice were maintained in a barrier facility and fed normal mouse laboratory diet. To induce hypercholesterolemia, mice were fed a diet supplemented with saturated fat (21% wt/wt milk fat and 0.15% cholesterol) for 5 weeks. XMU-MP-1 was dissolved in dimethyl sulfoxide at a concentration of 30 mg/mL and administered daily by gavage at a dose of 3 mg/kg/day for different intervals of time ranging from 7 to 35 days.[2]

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1 mg/kg; i.p.

FRG mice

The pharmacokinetic properties of XMU-MP-1 were first evaluated in Sprague-Dawley rats administered a single intravenous or oral dose. XMU-MP-1 exhibited favorable pharmacokinetics with a half-life of 1.2 hours, an area under the curve of 1035 (h·ng)/ml, and a bioavailability of 39.5% (table S3). In pharmacodynamic experiments, the maximal phosphorylation inhibition of MOB1 and YAP was achieved between 1.5 and 6 hours after intraperitoneal dosing with XMU-MP-1 (1 mg/kg) (Fig. 4AOpens in image viewer). A dose escalation study of XMU-MP-1 revealed that the phosphorylation of MOB1 in liver tissue was blocked at a minimal dose (1 mg/kg, intraperitoneally) (Fig. 4BOpens in image viewer).[1]

[1]. Pharmacological targeting of kinases MST1 and MST2 augments tissue repair and regeneration. Sci Transl Med. 2016 Aug 17;8(352):352ra108.

[2]. Mst1/2 Kinases Inhibitor, XMU-MP-1, Attenuates Angiotensin II-Induced Ascending Aortic Expansion in Hypercholesterolemic Mice. Circ Rep. 2021 Apr 20;3(5):259–266.

[3]. MST1 inhibits the progression of breast cancer by regulating the Hippo signaling pathway and may serve as a prognostic biomarker. Mol Med Rep. 2021 Mar 18;23(5):383.

Tissue repair and regenerative medicine address the important medical needs to replace damaged tissue with functional tissue. Most regenerative medicine strategies have focused on delivering biomaterials and cells, yet there is the untapped potential for drug-induced regeneration with good specificity and safety profiles. The Hippo pathway is a key regulator of organ size and regeneration by inhibiting cell proliferation and promoting apoptosis. Kinases MST1 and MST2 (MST1/2), the mammalian Hippo orthologs, are central components of this pathway and are, therefore, strong target candidates for pharmacologically induced tissue regeneration. We report the discovery of a reversible and selective MST1/2 inhibitor, 4-((5,10-dimethyl-6-oxo-6,10-dihydro-5H-pyrimido[5,4-b]thieno[3,2-e][1,4]diazepin-2-yl)amino)benzenesulfonamide (XMU-MP-1), using an enzyme-linked immunosorbent assay–based high-throughput biochemical assay. The cocrystal structure and the structure-activity relationship confirmed that XMU-MP-1 is on-target to MST1/2. XMU-MP-1 blocked MST1/2 kinase activities, thereby activating the downstream effector Yes-associated protein and promoting cell growth. XMU-MP-1 displayed excellent in vivo pharmacokinetics and was able to augment mouse intestinal repair, as well as liver repair and regeneration, in both acute and chronic liver injury mouse models at a dose of 1 to 3 mg/kg via intraperitoneal injection. XMU-MP-1 treatment exhibited substantially greater repopulation rate of human hepatocytes in the Fah-deficient mouse model than in the vehicle-treated control, indicating that XMU-MP-1 treatment might facilitate human liver regeneration. Thus, the pharmacological modulation of MST1/2 kinase activities provides a novel approach to potentiate tissue repair and regeneration, with XMU-MP-1 as the first lead for the development of targeted regenerative therapeutics. [1]

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Breast cancer (BCa) is the most common malignancy threatening the health of women worldwide, and the incidence rate has significantly increased in the last 10 years. Mammalian STE20-like protein kinase 1 (MST1) is involved in the development of various types of malignant tumor. The present study aimed to investigate the role of MST1 in BCa and its potential involvement in the poor prognosis of patients with BCa. Reverse transcription-quantitative PCR and immunohistochemistry were used to analyze the expression levels of MST1 in BCa, and the clinicopathological characteristics and prognosis of patients with BCa were further analyzed by statistical analysis. MST1 was overexpressed in BCa cell lines (MCF-7, MDA-MB-231 and SKBR3). Cell Counting Kit-8, 5-ethynyl-2′-deoxyuridine and flow cytometry assays were used to analyze cell proliferation and apoptosis, respectively, and a wound healing assay was used to analyze cell migration. The results of the present study revealed that the downregulated expression levels of MST1 in BCa were closely associated with the poor prognosis of patients, and MST1 may be an independent risk factor for BCa. The overexpression of MST1 significantly inhibited the proliferation and migration, and promoted the apoptosis of BCa cells. In addition, the overexpression of MST1 significantly activated the Hippo signaling pathway. Treatment with XMU-MP-1 downregulated the expression levels of MST1 and partially reversed the inhibitory effects of MST1 on proliferation, migration and apoptosis-related proteins, and inhibited the Hippo signaling pathway. In conclusion, the results of the present study suggested that MST1 expression levels may be downregulated in BCa and closely associated with tumor size and clinical stage, as well as the poor prognosis of affected patients. Furthermore, MST1 may inhibit the progression of BCa by targeting the Hippo signaling pathway.[3]

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Background: Ascending and abdominal aortic aneurysms (AAs) are asymptomatic, permanent dilations of the aorta with surgical intervention as the currently available therapy. Hippo-Yap signaling cascade plays a critical role in stem cell self-renewal, tissue regeneration and organ size control. By using XMU-MP-1, a pharmacological inhibitor of the key component of Hippo-Yap signaling, MST1/2, we examined the functional contribution of Hippo-Yap in the development of AAs in Angiotensin II (AngII)-infused hypercholesterolemic mice. Methods and Results: MST, p-MST, p-YAP, p-MOB and TAZ proteins in AngII-infused ascending and abdominal aortas were assessed by immunohistochemical and western blot analyses. To examine the effect of MST1/2 inhibition on AAs, western diet-fed low density lipoprotein (LDL) receptor −/− mice infused with AngII were administered with either vehicle or XMU-MP-1 for 5 weeks. Hippo-YAP signaling proteins were significantly elevated in AngII infused ascending and abdominal aortas. XMU-MP-1 administration resulted in the attenuation of AngII-induced ascending AAs without influencing abdominal AAs and aortic atherosclerosis. Inhibition of Hippo-YAP signaling also resulted in the suppression of AngII-induced matrix metalloproteinase 2 (MMP2) activity, macrophage accumulation, aortic medial hypertrophy and elastin breaks in the ascending aorta. Conclusions: The present study demonstrates a pivotal role for the Hippo-YAP signaling pathway in AngII-induced ascending AA development.[3]

C17H16N6O3S2

416.4773

416.072

C, 49.03; H, 3.87; N, 20.18; O, 11.52; S, 15.40

2061980-01-4

121499143

Light yellow to yellow solid powder

1.8

158

2

9

3

28

694

0

S1C([H])=C([H])C2=C1C(N(C([H])([H])[H])C1=C([H])N=C(N([H])C3C([H])=C([H])C(=C([H])C=3[H])S(N([H])[H])(=O)=O)N=C1N2C([H])([H])[H])=O

YRDHKIFCGOZTGD-UHFFFAOYSA-N

InChI=1S/C17H16N6O3S2/c1-22-12-7-8-27-14(12)16(24)23(2)13-9-19-17(21-15(13)22)20-10-3-5-11(6-4-10)28(18,25)26/h3-9H,1-2H3,(H2,18,25,26)(H,19,20,21)

4-[(2,9-dimethyl-8-oxo-6-thia-2,9,12,14-tetrazatricyclo[8.4.0.03,7]tetradeca-1(14),3(7),4,10,12-pentaen-13-yl)amino]benzenesulfonamide

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: ≥ 0.83 mg/mL (1.99 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 8.3 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: ≥ 0.83 mg/mL (1.99 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 8.3 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: ≥ 0.83 mg/mL (1.99 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 8.3 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 4 中的溶解度: ≥ 0.4 mg/mL (0.96 mM) (饱和度未知) in 5% DMSO + 95% (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。
*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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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。