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STO-609 Sale

目录号 : GC37694

A CaMKK inhibitor

STO-609 Chemical Structure

Cas No.:52029-86-4

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10mM (in 1mL DMSO)
¥762.00
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5mg
¥693.00
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10mg
¥1,215.00
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25mg
¥2,655.00
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50mg
¥4,770.00
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100mg
¥8,550.00
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产品描述

STO-609 is a calcium/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor (IC50s = 120 and 40 ng/ml for CaMKKα and CaMKKβ, respectively).1 It is selective for CaMKKs over CaMKI, CaMKII, CaMKIV, MLCK, PKC, PKA, and p42 MAPK (IC50s = ≥10,000 ng/ml for all). STO-609 inhibits phosphorylation of CaMKI and AMP-activated protein kinase (AMPK) in chicken sperm in the presence of extracellular calcium, as well as reduces chicken sperm motility, in a concentration-dependent manner.2 It decreases hypothalamic neuropeptide Y (Npy) and agouti-related protein (Agrp) expression and cumulative food intake in mice when administered intracerebroventricularly as a 20 μM solution at a continuous rate of 0.5 μL/hour .3 STO-609 (3 μg/animal, i.c.v.) increases cortical, striatal, and total infarct volume in a mouse model of focal transient cerebral ischemia induced by middle cerebral artery occlusion (MCAO).4

1.Tokumitsu, H., Inuzuka, H., Ishikawa, Y., et al.STO-609, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase kinaseJ. Biol. Chem.278(13)10908-10913(2013) 2.Nguyen, T.M.D., Combarnous, Y., Praud, C., et al.Ca2+/calmodulin-dependent protein kinase kinases (CaMKKs) effects on AMP-activated protein kinase (AMPK) regulation of chicken sperm functionsPLoS One11(1)e0147559(2016) 3.Anderson, K.A., Ribar, T.J., Lin, F., et al.Hypothalamic CaMKK2 contributes to the regulation of energy balanceCell Metab.7(5)377-388(2008) 4.McCullough, L.D., Tarabishy, S., Liu, L., et al.Inhibition of calcium/calmodulin-dependent protein kinase kinase β and calcium/calmodulin-dependent protein kinase IV is detrimental in cerebral ischemiaStroke44(9)2559-2566(2013)

Chemical Properties

Cas No. 52029-86-4 SDF
Canonical SMILES O=C(C1=C2C3=C(C4=NC5=CC=CC=C5N4C(C3=CC=C2)=O)C=C1)O
分子式 C19H10N2O3 分子量 314.29
溶解度 DMSO: 5.6 mg/mL (17.82 mM and warming); Water: < 0.1 mg/mL (insoluble) 储存条件 Store at -20°C
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1 mM 3.1818 mL 15.9089 mL 31.8177 mL
5 mM 0.6364 mL 3.1818 mL 6.3635 mL
10 mM 0.3182 mL 1.5909 mL 3.1818 mL
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Research Update

STO-609, a specific inhibitor of the Ca(2+)/calmodulin-dependent protein kinase kinase

J Biol Chem 2002 May 3;277(18):15813-8.PMID:11867640DOI:10.1074/jbc.M201075200.

STO-609, a selective inhibitor of Ca(2+)/calmodulin-dependent protein kinase kinase (CaM-KK) was synthesized, and its inhibitory properties were investigated both in vitro and in vivo. STO-609 inhibits the activities of recombinant CaM-KK alpha and CaM-KK beta isoforms, with K(i) values of 80 and 15 ng/ml, respectively, and also inhibits their autophosphorylation activities. Comparison of the inhibitory potency of the compound against various protein kinases revealed that STO-609 is highly selective for CaM-KK without any significant effect on the downstream CaM kinases (CaM-KI and -IV), and the IC(50) value of the compound against CaM-KII is approximately 10 microg/ml. STO-609 inhibits constitutively active CaM-KK alpha (glutathione S-transferase (GST)-CaM-KK-(84-434)) as well as the wild-type enzyme. Kinetic analysis indicates that the compound is a competitive inhibitor of ATP. In transfected HeLa cells, STO-609 suppresses the Ca(2+)-induced activation of CaM-KIV in a dose-dependent manner. In agreement with this observation, the inhibitor significantly reduces the endogenous activity of CaM-KK in SH-SY5Y neuroblastoma cells at a concentration of 1 microg/ml (approximately 80% inhibitory rate). Taken together, these results indicate that STO-609 is a selective and cell-permeable inhibitor of CaM-KK and that it may be a useful tool for evaluating the physiological significance of the CaM-KK-mediated pathway in vivo as well as in vitro.

Using the fluorescent properties of STO-609 as a tool to assist structure-function analyses of recombinant CaMKK2

Biochem Biophys Res Commun 2016 Jul 22;476(2):102-7.PMID:27178209DOI:10.1016/j.bbrc.2016.05.045.

Calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) has been implicated in the regulation of metabolic activity in cancer and immune cells, and affects whole-body metabolism by regulating ghrelin-signalling in the hypothalamus. This has led to efforts to develop specific CaMKK2 inhibitors, and STO-609 is the standardly used CaMKK2 inhibitor to date. We have developed a novel fluorescence-based assay by exploiting the intrinsic fluorescence properties of STO-609. Here, we report an in vitro binding constant of KD ∼17 nM between STO-609 and purified CaMKK2 or CaMKK2:Calmodulin complex. Whereas high concentrations of ATP were able to displace STO-609 from the kinase, GTP was unable to achieve this confirming the specificity of this association. Recent structural studies on the kinase domain of CaMKK2 had implicated a number of amino acids involved in the binding of STO-609. Our fluorescent assay enabled us to confirm that Phe(267) is critically important for this association since mutation of this residue to a glycine abolished the binding of STO-609. An ATP replacement assay, as well as the mutation of the 'gatekeeper' amino acid Phe(267)Gly, confirmed the specificity of the assay and once more confirmed the strong binding of STO-609 to the kinase. In further characterising the purified kinase and kinase-calmodulin complex we identified a number of phosphorylation sites some of which corroborated previously reported CaMKK2 phosphorylation and some of which, particularly in the activation segment, were novel phosphorylation events. In conclusion, the intrinsic fluorescent properties of STO-609 provide a great opportunity to utilise this drug to label the ATP-binding pocket and probe the impact of mutations and other regulatory modifications and interactions on the pocket. It is however clear that the number of phosphorylation sites on CaMKK2 will pose a challenge in studying the impact of phosphorylation on the pocket unless the field can develop approaches to control the spectrum of modifications that occur during recombinant protein expression in Escherichia coli.

Analysis of Distinct Roles of CaMKK Isoforms Using STO-609-Resistant Mutants in Living Cells

Biochemistry 2015 Jun 30;54(25):3969-77.PMID:26050738DOI:10.1021/acs.biochem.5b00149.

To assess the isoform specificity of the Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK)-mediated signaling pathway using a CaMKK inhibitor (STO-609) in living cells, we have established A549 cell lines expressing STO-609-resistant mutants of CaMKK isoforms. Following serial mutagenesis studies, we have succeeded in obtaining an STO-609-resistant CaMKKα mutant (Ala292Thr/Leu233Phe) and a CaMKKβ mutant (Ala328Thr/Val269Phe), which showed sensitivity to STO-609 that was 2-3 orders of magnitude lower without an appreciable effect on kinase activity or CaM requirement. These results are consistent with the results obtained for CaMKK activities in the extracts of A549 cells stably expressing the mutants of CaMKK isoforms. Ionomycin-induced 5'-AMP-activated protein kinase (AMPK) phosphorylation at Thr172 in A549 cells expressing either the wild-type or the STO-609-resistant mutant of CaMKKα was completely suppressed by STO-609 treatment but resistant to the inhibitor in the presence of the CaMKKβ mutant (Ala328Thr/Val269Phe). This result strongly suggested that CaMKKβ is responsible for ionomycin-induced AMPK activation, which supported previous reports. In contrast, ionomycin-induced CaMKIV phosphorylation at Thr196 was resistant to STO-609 treatment in A549 cells expressing STO-609-resistant mutants of both CaMKK isoforms, indicating that both CaMKK isoforms are capable of phosphorylating and activating CaMKIV in living cells. Considering these results together, STO-609-resistant CaMKK mutants developed in this study may be useful for distinguishing CaMKK isoform-mediated signaling pathways in combination with the use of an inhibitor compound.

Pharmacological inhibition of CaMKK2 with the selective antagonist STO-609 regresses NAFLD

Sci Rep 2017 Sep 18;7(1):11793.PMID:28924233DOI:10.1038/s41598-017-12139-3.

Binding of calcium to its intracellular receptor calmodulin (CaM) activates a family of Ca2+/CaM-dependent protein kinases. CaMKK2 (Ca2+/CaM-dependent protein kinase kinase 2) is a central member of this kinase family as it controls the actions of a CaMK cascade involving CaMKI, CaMKIV or AMPK. CaMKK2 controls insulin signaling, metabolic homeostasis, inflammation and cancer cell growth highlighting its potential as a therapeutic target for a variety of diseases. STO-609 is a selective, small molecule inhibitor of CaMKK2. Although STO-609 has been used extensively in vitro and in cells to characterize and define new mechanistic functions of CaMKK2, only a few studies have reported the in vivo use of STO-609. We synthesized functional STO-609 and assessed its pharmacological properties through in vitro (kinase assay), ex vivo (human liver microsomes) and in vivo (mouse) model systems. We describe the metabolic processing of STO-609, its toxicity, pharmacokinetics and bioavailability in a variety of mouse tissues. Utilizing these data, we show STO-609 treatment to inhibit CaMKK2 function confers protection against non-alcoholic fatty liver disease. These data provide a valuable resource by establishing criteria for use of STO-609 to inhibit the in vivo functions of CaMKK2 and demonstrate its utility for treating metabolically-related hepatic disease.

Macrophage Sprouty4 deficiency diminishes sepsis-induced acute lung injury in mice

Redox Biol 2022 Dec;58:102513.PMID:36334381DOI:10.1016/j.redox.2022.102513.

Objective: Inflammation and oxidative stress play critical roles in sepsis-induced acute lung injury (ALI). Sprout4 (Spry4) is involved in regulating inflammation and tissue injury; however, its role and mechanism in sepsis-induced ALI remain elusive. Methods: Macrophage-specific Spry4 knockout (Spry4MKO), transgenic (Spry4MTG) mice and matched control littermates were generated and exposed to cecum ligation and puncture (CLP) surgery to establish bacterial sepsis-induced ALI. Bone marrow-derived macrophages (BMDMs) from Spry4MKO or Spry4MTG mice were isolated and subjected to lipopolysaccharide (LPS) stimulation to further validate the role of Spry4 in vitro. To verify the necessity of AMP-activated protein kinase (AMPK), Spry4 and AMPK double knockout mice and compound C were used in vivo and in vitro. BMDMs were treated with STO-609 to inhibit calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2). Results: We found that macrophage Spry4 was increased in CLP mice and positively correlated with sepsis-induced ALI. Macrophage Spry4 deficiency prevented, while macrophage Spry4 overexpression exacerbated sepsis-induced inflammation, oxidative stress and ALI in mice and BMDMs. Mechanistic studies revealed that macrophage Spry4 deficiency alleviated sepsis-induced ALI through activating CaMKK2/AMPK pathway. Conclusion: Our study identify macrophage Spry4 as a promising predictive and therapeutic target of sepsis-induced ALI.