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 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

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.)