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

Over a concentration range of 0.1 to 10 μM, XMU-MP-1 phosphorylated less endogenous MOB1, LATS1/2, and YAP in HepG2 cells in a dose-dependent manner. In several cell lines, such as mouse macrophage-like cells, human osteosarcoma, and human colorectal adenocarcinoma cells, XMU-MP-1 therapy suppresses hydrogen peroxide-stimulated MOB1 phosphorylation and MST1/2 autophosphorylation. By inhibiting MST1/2 kinase activity, XMU-MP-1 stimulates the downstream effector Yes-related protein and encourages cell division. In cells, XMU-MP-1 can more potently and reversibly suppress the activity of kinase MST1/2 and improve its subsequent YAP activation [1].

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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[3]. To determine whether the Hippo signaling pathway mediated the biological functions of MST1 in BCa, the Hippo signaling pathway inhibitor, XMU-MP-1 (an inhibitor of MST1/2), was used to inhibit the function of the Hippo signaling pathway in BCa cells overexpressing MST1. RT-qPCR analysis demonstrated that the treatment of MST1-overexpressing cells with the inhibitor downregulated the expression levels of MST1 (Fig. 5A). The results of the CCK-8 and EdU incorporation assays revealed that the inhibited proliferative ability in the LV-MST1 group was partially restored in the LV-MST1 + XMU-MP-1 group (Fig. 5B and C). BCa cell migration was analyzed using wound healing assays; the results revealed that cell migration was also significantly increased in the MST1 + XMU-MP-1 cell group compared with the LV-MST1 group (Fig. 5D). Finally, western blotting analysis was performed to analyze the expression levels of key proteins in the Hippo signaling pathway. The expression levels of LATS1 and Bax were significantly downregulated, while the expression levels of YAP, Bcl-2 and Ki-67 were significantly upregulated in the LV-MST1 + XMU-MP-1 group compared with the LV-MST1 group in both cell lines (Fig. 6A and B).

For intraperitoneal administration, XMU-MP-1 shows outstanding in vivo pharmacokinetics in mice with acute and chronic liver injury at dosages ranging from 1 mg/kg to 3 mg/kg. In the Fah-deficient mice model, XMUMP-1 treatment demonstrated a significantly higher rate of human hepatocyte repopulation than vehicle-treated controls, suggesting that XMU-MP-1 treatment may encourage human liver regeneration [1].

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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 [2].

In vitro and in vivo kinase inhibition assays [1]
For the in vivo inhibition assays, human embryonic kidney (HEK) 293T cells were transfected with 0.5 μg of empty plasmid or pCMV plasmids expressing various forms of Flag-tagged full-length MST1 or MST2 kinase in 12-well plates each. Twenty-four hours after transfection, cells were treated with the indicated doses of XMU-MP-1 for 3 hours. Cell lysates were analyzed via immunoblotting with the indicated antibodies. For the in vitro kinase inhibition assays, recombinant GST-tagged MOB1a and various forms of recombinant His-tagged full-length MST1 or MST2 kinase were expressed and purified from Escherichia coli. The enzyme, ATP, and GST-MOB1 consumption were kept consistent with the previously optimized conditions. The assays were performed with the indicated doses of XMU-MP-1 in the kinase assay buffer for 30 min at 30°C followed by SDS–polyacrylamide gel electrophoresis and immunoblot analyses.

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KINOMEscan profiling of XMU-MP-1 [1]
XMU-MP-1 was profiled against a panel of 468 kinases using KINOMEscan technology, an active-site–dependent competition-binding assay at 1 μM. The KINOMEscan selectivity score is a quantitative measure of a compound’s selectivity. It is calculated by dividing the number of kinases that bind to the compound by the total number of kinases tested. The results are reported as “control%” (ctrl%) in which lower numbers represent higher-affinity binding; ctrl% = (test compound signal − positive control signal)/(negative control signal − positive control signal) × 100, where the negative control = DMSO (ctrl% = 100%) and the positive control = control compound (ctrl% = 0%); S(10) = (number of kinases with ctrl% ≤10%)/(number of kinases tested). ctrl% <10% means very strong inhibition, and ctrl% >70% means very weak inhibition. The kinase group or individual kinase names in the current study includes TK (tyrosine kinase), TKL (TK-like), STE (homologs of yeast Sterile 7, Sterile 11, and Sterile 20 kinases), AGC [protein kinase A, G, and C families], CAMK (calcium/calmodulin-dependent protein kinase), CK1 (casein kinase 1), and CMGC [cyclin-dependent kinase (CDK), mitogen-activated protein kinase (MAPK), glycogen synthase kinase 3 (GSK3), and CDC2-like kinase (CLK) families].

The human BCa cell lines, MCF-7, MDA-MB-231 and SKBR3, and the normal mammary epithelial cell line, MCF-10A, were obtained from the American Type Culture Collection. All cell lines were cultured in DMEM supplemented with 10% FBS, and maintained in a humidified incubator at 37°C with 5% CO2. XMU-MP-1, which is an inhibitor of MST1, was dissolved in DMSO and added to the medium at a final concentration of 0.1%. Cell lines were cultured in the medium for 1 h at 37°C [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 by single intravenous or oral administration. XMU-MP-1 showed good pharmacokinetic characteristics 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 studies, the inhibition of phosphorylation of MOB1 and YAP reached its maximum between 1.5 and 6 hours after intraperitoneal injection of XMU-MP-1 (1 mg/kg) (Figure 4A). Dose escalation studies of XMU-MP-1 showed that the phosphorylation of MOB1 in liver tissue could be blocked at the minimum dose (1 mg/kg, intraperitoneal injection) (Figure 4B). [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 aims to address the critical medical need to replace damaged tissue with functional tissue. While most regenerative medicine strategies focus on the delivery of biomaterials and cells, drug-induced regeneration, with its good specificity and safety profile, has not yet been fully explored. The Hippo signaling pathway plays a crucial regulatory role in organ size and regeneration by inhibiting cell proliferation and promoting apoptosis. MST1 and MST2 (MST1/2) are homologous proteins of the mammalian Hippo signaling pathway and are core components of this pathway, making them ideal targets for drug-induced tissue regeneration. We discovered a reversible and selective MST1/2 inhibitor—4-((5,10-dimethyl-6-oxo-6,10-dihydro-5H-pyrimidino[5,4-b]thieno[3,2-e][1,4]diazaphen-2-yl)amino)benzenesulfonamide (XMU-MP-1)—using a high-throughput biochemical method based on enzyme-linked immunosorbent assay (ELISA). Co-crystal structure and structure-activity relationship studies confirmed that XMU-MP-1 targets MST1/2. XMU-MP-1 can block the activity of MST1/2 kinases, thereby activating downstream effector molecules Yes-related proteins and promoting cell growth. XMU-MP-1 has excellent in vivo pharmacokinetic properties. Intraperitoneal injection of 1-3 mg/kg doses can enhance intestinal repair and liver repair and regeneration in mouse models of acute and chronic liver injury. In the Fah-deficient mouse model, the regeneration rate of human hepatocytes in the XMU-MP-1 treatment group was significantly higher than that in the vector control group, indicating that XMU-MP-1 treatment may promote human liver regeneration. Therefore, the pharmacological regulation of MST1/2 kinase activity provides a new approach to enhance tissue repair and regeneration, and XMU-MP-1 is the first lead compound for developing targeted regenerative therapy. [1]

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Breast cancer (BCa) is the most common malignant tumor in women worldwide, and its incidence has increased significantly in the past 10 years. Mammalian STE20-like protein kinase 1 (MST1) is involved in the development and progression of various malignant tumors. This study aimed to investigate the role of MST1 in breast cancer and its potential association with poor prognosis in breast cancer patients. We used reverse transcription quantitative PCR and immunohistochemistry to analyze the expression level of MST1 in bladder cancer (BCa), and further explored the relationship between MST1 and clinicopathological features and prognosis of BCa patients through statistical analysis. Results showed that MST1 was highly expressed in BCa cell lines (MCF-7, MDA-MB-231, and SKBR3). Cell proliferation and apoptosis were detected using CCK-8 assay, 5-ethynyl-2′-deoxyuridine (EDU), and flow cytometry, respectively, and cell migration was assessed using a scratch assay. The results indicated that downregulation of MST1 expression in BCa was closely associated with poor prognosis, and MST1 may be an independent risk factor for BCa. MST1 overexpression significantly inhibited BCa cell proliferation and migration and promoted apoptosis. Furthermore, MST1 overexpression significantly activated the Hippo signaling pathway. XMU-MP-1 treatment downregulated the expression level of MST1 and partially reversed the inhibitory effect of MST1 on proliferation, migration and apoptosis-related proteins, while inhibiting the Hippo signaling pathway. In summary, the results of this study indicate that the expression level of MST1 may be downregulated in bladder cancer (BCa) and is closely related to tumor size, clinical stage and poor patient prognosis. In addition, MST1 may inhibit the progression of bladder cancer by targeting the Hippo signaling pathway. [3]

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Background: Ascending aortic aneurysm and abdominal aortic aneurysm (AA) are asymptomatic permanent dilatations of the aorta, and the only current treatment is surgery. The Hippo-Yap signaling pathway plays a key role in stem cell self-renewal, tissue regeneration and organ size control. This study used XMU-MP-1 (a pharmacological inhibitor of MST1/2, a key component of the Hippo-Yap signaling pathway) to investigate the functional role of Hippo-Yap in the development of abdominal aortic aneurysms (AAs) in hypercholesterolemia mice perfused with angiotensin II (AngII). Methods and Results: Immunohistochemistry and Western blotting were used to detect the expression of MST, p-MST, p-YAP, p-MOB, and TAZ proteins in the ascending aorta and abdominal aorta of AngII-perfused mice. To investigate the effect of MST1/2 inhibition on aneurysms (AAs), low-density lipoprotein (LDL) receptor knockout (LDL-R^-/-) mice fed a high-fat diet were perfused with AngII and then administered either a vector or XMU-MP-1 for 5 weeks. Results showed that the expression of Hippo-Yap signaling pathway proteins was significantly increased in the ascending and abdominal aorta of AngII-perfused mice. XMU-MP-1 administration alleviated AngII-induced ascending aortic aneurysms without affecting abdominal aortic aneurysms or aortic atherosclerosis. Inhibition of the Hippo-YAP signaling pathway also suppressed AngII-induced matrix metalloproteinase 2 (MMP2) activity, macrophage aggregation, aortic media hypertrophy, and ascending aortic elastin rupture. Conclusion: This study shows that the Hippo-YAP signaling pathway plays a key role in the development of AngII-induced ascending aortic aneurysms. [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

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)

Solubility in Formulation 1: ≥ 0.83 mg/mL (1.99 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 8.3 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 0.83 mg/mL (1.99 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 8.3 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 0.83 mg/mL (1.99 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 8.3 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: ≥ 0.4 mg/mL (0.96 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)