Ca2+/Calmodulin-dependent protein kinase kinase(CaM-KK); CaM-KKα (Ki = 80 ng/mL); CaM-KKβ (Ki = 15 ng/mL)

STO-609 suppresses both the autophosphorylation and activity of recombinant CaM-KKα and CaM-KKβ isoforms, with Ki values of 80 and 15 ng/mL, respectively. STO-609 has an IC50 value of 10 μg/mL against CaM-KII and is highly selective for CaM-KK with no discernible effect on the downstream CaM kinases (CaM-KI and -IV). Both the wild-type enzyme and constitutively active CaM-KKα are inhibited by STO-609. STO-609 suppresses the Ca2+-induced activation of CaM-KIV in transfected HeLa cells in a dose-dependent manner. At a concentration of 1μg/mL (80% inhibitory rate), STO-609 significantly lowers the endogenous activity of CaM-KK in SH-SY5Y neuroblastoma cells[1].

In vivo administration of STO-609 results in increased osteoblasts and diminished osteoclasts, conferring significant protection from ovariectomy (OVX)-induced osteoporosis in adult mice. ICV administration of STO-609 in vivo did not affect the counterregulatory responses to neuroglucopenia and AMPK activation induced by glucopenia.

In Vitro Assay for CaM-KK Activity [1]
Purified recombinant CaM-KKs (CaM-KKα, 0.28 μg/ml; CaM-KKβ, 0.52 μg/ml; constitutively active CaM-KK, 0.3 μg/ml) were incubated with 10 μg of GST-CaM-KI-(1–293)-K49E at 30 °C for 5 min in a solution (25 μl) containing 50 mm HEPES (pH 7.5), 10 mm Mg(Ac)2, 1 mm DTT, various concentrations (50–400 μm) of [γ-32P]ATP (650–6500 cpm/pmol) with various concentrations of STO-609 (0–10 μg/ml in Me2SO at a final concentration of 4%) in the presence of either 1 mm EGTA (for constitutively active CaM-KK) or 1 mm CaCl2, 2 μm CaM. The reaction was initiated by the addition of [γ-32P]ATP and terminated by spotting aliquots (15 μl) onto phosphocellulose paper followed by several washes with 75 mm phosphoric acid. Phosphate incorporation into GST-CaM-KI-(1–293)-K49E was determined by liquid scintillation counting of the filters. A 5-min reaction was chosen to determine CaM-KK activity based on the time course experiment described recently. Specific activities of CaM-KKα, CaM-KKβ, and constitutively active CaM-KK in the absence of STO-609 were calculated to be 723 ± 7 μmol/min/mg, 338 ± 18 μmol/min/mg, and 927 ± 40 μmol/min/mg, respectively.

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Autophosphorylation of CaM-KKα and -β [1]
Purified recombinant CaM-KKα and -β (0.8 μg) were assayed at 30 °C for 5 min in a solution (25 μl) containing 50 mm HEPES (pH 7.5), 10 mm Mg(Ac)2, 1 mm DTT, 50 μm [γ-32P]ATP (6500 cpm/pmol) with various concentrations of STO-609 (0–10 μg/ml in Me2SO at a final concentration of 4%) in the presence of either 1 mm EGTA (for CaM-KKα and CaM-KKβ) or 1 mmCaCl2, 2 μm CaM (for CaM-KKα). The reaction was initiated by the addition of [γ-32P]ATP and terminated by the addition of SDS-PAGE sample buffer. The samples were subjected to SDS-10% PAGE followed by autoradiography. 32P incorporation into CaM-KK was estimated by densitometric scanning of the x-ray film.

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In Vitro Assay for CaM-KI, -II, and -IV and MLCK Activities [1]
CaM-KI (2.5 μg/ml), CaM-KII (0.75 μg/ml), CaM-KIV (9 μg/ml), and MLCK (0.6 μg/ml) were incubated with 40 μm syntide-2 or 50 μm MLC peptide (for MLCK) at 30 °C for 5 min in a solution (25 μl) containing 50 mm HEPES (pH 7.5), 10 mm Mg(Ac)2, 1 mm DTT, 50 μm [γ-32P]ATP (4500 cpm/pmol) with various concentrations of STO-609 (0–10 μg/ml in Me2SO at a final concentration of 4%) in the presence of 1 mm CaCl2, 2 μm CaM. Protein kinase activity was measured by the phosphocellulose filter method as described above. Specific activities of CaM-KI, CaM-KII, CaM-KIV, and MLCK in the absence of STO-609 were calculated to be 24 ± 1 μmol/min/mg, 122 ± 3 μmol/min/mg, 48 ± 1 μmol/min/mg, and 178 ± 6 μmol/min/mg, respectively.

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In Vitro Assay for PKA, PKC, and p42 MAP Kinase Activities [1]
PKA (8 μg/ml), PKC (25 ng/ml), and p42 MAP kinase (2 μg/ml) were incubated with 100 μm kemptamide (for PKA), 100 μm neurogranin peptide (for PKC, Promega), or 0.4 mg/ml myelin basic protein (for p42 MAP kinase) at 30 °C for 5 min in a solution (25 μl) containing 50 mm HEPES (pH 7.5), 10 mm Mg(Ac)2, 1 mm DTT, 50 μm [γ-32P]ATP (4500 cpm/pmol) with various concentrations of STO-609 (0–10 μg/ml in Me2SO at a final concentration of 4%) in the absence (for PKA and p42 MAP kinase) or presence of 1 mm CaCl2, 0.4 mg/ml phosphatidylserine, and 0.1 mg/ml bovine serum albumin. Protein kinase activity was measured by the phosphocellulose filter method as described above. Specific activities of PKA, PKC and p42 MAP kinase in the absence of STO-609 were calculated to be 22 ± 1 μmol/min/mg, 7.522 ± 0.062 mmol/min/mg, and 181 ± 3 μmol/min/mg, respectively. Protein kinase activity was measured under linear conditions based on the results obtained from titration experiments for each enzyme.

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Kinase Assays [1]
The AMPK peptide, including the sequence surrounding the phosphorylation site of AMPK (167GEFLRTSCGSP177), was synthesized at the Support Unit for Bio-material Analysis in the RIKEN Brain Science Institute (BSI) Research Resources Center (RRC). Appropriate quantities of the purified CaMKKβ KD and full-length CaMKKβ were each incubated in the presence or absence of 500 μm AMPK peptide at 30 °C, in a reaction solution (20 μl) containing 50 mm HEPES (pH 7.5), 300 mm NaCl, 1 mm DTT, 10 mm MgCl2, 400 μm ATP, and 10% glycerol, with or without 0.5 μm STO-609. For the full-length CaMKKβ, 5 μm calmodulin and 1 mm CaCl2 were added to the reaction solution. ATP consumption was determined by using a Kinase-GloTM Max luminescent kinase assay (Promega) kit, which quantifies the amount of ATP in the reaction solution. Glow-type luminescence was recorded after 10 min, using a FusionTM universal microplate analyzer

Transient Expression and Immunoprecipitation of HA-CaM-KIV [1]
HeLa cells were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. Cells were subcultured in 6-cm dishes 12 h before transfection. The cells were then transferred to serum-free medium and treated with a mixture of either 3 μg of pME18s plasmid DNA or 3 μg of HA (hemagglutinin-tagged)-CaM-KIV and 20 μg of LipofectAMINE Reagent in 2.5 ml of medium. After 20 h of incubation, the cells were further cultured in serum-free medium for 6 h in either the absence or presence of various concentrations of STO-609 (0.01–10 μg/ml in Me2SO at a final concentration of 0.5%) and then treated with or without 1 μm ionomycin for 5 min. Stimulation was terminated by the addition of 1 ml of lysis buffer (150 mm NaCl, 20 mm Tris-HCl (pH 7.5), 2 mm EDTA, 2 mm EGTA, 1% Nonidet P-40, 10% glycerol, 0.2 mm phenylmethylsulfonyl fluoride, 10 mg/liter leupeptin, 10 mg/liter trypsin inhibitor, and 1 μm microcystin LR), and the cells were lysed for 30 min on ice. The cell extract was collected and centrifuged at 15,000 × g for 15 min, the supernatant was precleared with 40 μl of Protein G-Sepharose (50% slurry) for 2 h at 4 °C, and the supernatant was mixed with 4 μg of anti-HA antibody for 3 h. 40 μl of Protein G-Sepharose was then applied to the extract and incubated overnight. The immunoprecipitated resin was washed three times with 1 ml of the lysis buffer as described above and then washed with 1 ml of kinase buffer (50 mm HEPES (pH 7.5), 10 mmMg(Ac)2, 1 mm DTT, 1 mm EGTA, and 1 μm microcystin LR). Protein G-Sepharose with immunoprecipitated HA-CaM-KIV was subjected to the protein kinase assay (50 μl reaction volume) in the presence of 1 mm EGTA using syntide-2 as a substrate as described above. To estimate the amount of immunoprecipitated HA-CaM-KIV, SDS-PAGE sample buffer (50 μl) was added to immunoprecipitated samples and then heated at 95 °C for 10 min. After centrifugation, 10 μl of the sample was subjected to SDS-10% PAGE followed by Western blotting using anti-CaM-KIV antibody (1:2000).

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Expression of Ca2+/CaM-independent CaM-KIV in SH-SY5Y Neuroblastoma Cells by Infection with Recombinant Adenovirus [1]
Recombinant adenoviruses carrying cDNAs encoding Ca2+/CaM-independent CaM-KIV (305HMDT-DEDD), a kinase-deficient mutant (305HMDT-DEDD, K71E), or a constitutively active CaM-KK-(1–434) (3) were constructed as follows. Briefly, CaM-KIV mutants and constitutively active CaM-KK cDNAs in pME18s plasmid were digested, blunt-ended, and then ligated into pShuttle (CLONTECH). Recombinant viruses were obtained from HEK293 cells using the Adeno-X Expression System (CLONTECH) according to the manufacturer’s protocol. For virus infection, confluent SY5Y cells in 6-well culture plates were infected with viruses at a multiplicity of infection of 10 plaque-forming units/cell at 37 °C for 1 h. After infection, virus was aspirated, and the cells were further cultured in RPMI medium containing 10% fetal bovine serum for 12 h. The cells were then serum-starved for 6 h in either the absence or presence of various concentrations of STO-609 (0.01–10 μg/ml in Me2SO at a final concentration of 0.5%). The cells were stimulated with 1 μm ionomycin for 10 min (or not subjected to ionomycin treatment) in the absence or presence of various concentrations of STO-609 and then lysed, and the extract was subjected to SDS-7.5% PAGE followed by Western blotting using anti-CaM-KIV antibody. The intensity of the immunoreactive band was measured by densitometric scanning of the x-ray film.

[1]. STO-609, a specific inhibitor of the Ca(2+)/calmodulin-dependent protein kinase kinase. J Biol Chem. 2002 May 3;277(18):15813-8.

[2]. Crystal structure of the Ca2+/calmodulin-dependent protein kinase kinase in complex with the inhibitor STO-609. J Biol Chem. 2011 Jun 24;286(25):22570-9.

LSM-3164 is a naphthoic acid. STO-609 is a selective Ca(2+)/calmodulin-dependent protein kinase inhibitor (CaM-KK), which has been synthesized and its in vitro and in vivo inhibitory properties have been investigated. STO-609 inhibits the activity of recombinant CaM-KK α and CaM-KK β isoforms with K(i) values of 80 and 15 ng/ml, respectively, and also inhibits their autophosphorylation activity. Comparison of the inhibitory efficacy of this compound against various protein kinases revealed that STO-609 exhibits high selectivity for CaM-KK, with no significant effect on downstream CaM kinases (CaM-KI and -IV), and its IC(50) value for CaM-KII is approximately 10 μg/ml. STO-609 inhibits both constitutively active CaM-KKα (glutathione S-transferase (GST)-CaM-KK-(84-434)) and wild-type CaM-KKα. Kinetic analysis indicates that this compound is a competitive inhibitor of ATP. In transfected HeLa cells, STO-609 inhibits Ca²⁺-induced CaM-KIV activation in a dose-dependent manner. Consistent with this observation, the inhibitor significantly reduced the endogenous activity of CaM-KK in SH-SY5Y neuroblastoma cells at a concentration of 1 μg/ml (inhibition rate of approximately 80%). In summary, these results indicate that STO-609 is a selective and highly cell-penetrating CaM-KK inhibitor, which can serve as an effective tool for assessing the physiological significance of the CaM-KK-mediated pathway in vivo and in vitro. [1]
In summary, we recently developed a highly efficient and relatively selective CaM-KK inhibitor, STO-609, which is cellularly penetrable and a competitive inhibitor of ATP. Recent studies have shown that CaM-KK is a regulatory protein kinase of CaM-KI and CaM-KIV in vitro and in transfected cells; however, this property has not been directly confirmed in vivo. This report shows that STO-609 can inhibit the activity of CaM-KK in intact cells, thereby inhibiting the activity of downstream CaM-KIV, although it cannot inhibit the activity of downstream CaM kinases in vitro. Therefore, STO-609 can serve as an effective tool for assessing the regulatory role of CaM-KK in various physiological functions of the CaM kinase cascade, such as gene expression regulation mediated by the CaM-KIV pathway. In addition, STO-609 can be used to distinguish the functions of the two CaM-KK isoforms, as CaM-KKβ is about 5 times more sensitive to this compound than the α isoform. The mechanism of the difference in sensitivity of CaM-KK isoforms to STO-609 is unclear, but it is unlikely to be due to their different affinity for ATP, since the apparent Km values of CaM-KKα and CaM-KKβ to ATP are indistinguishable (approximately 33 μM, Fig. 3B). In addition, the physiological functions regulated by the CaM-KK/CaM-KI cascade compared to CaM-KIV have not been well studied, and STO-609 may be able to solve this problem. [1]

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Ca(2+)/calmodulin (CaM) dependent protein kinase (CaMK) kinase (CaMKK) is a member of the CaMK cascade and mediates the response to increased intracellular Ca(2+) concentration. CaMKK phosphorylates and activates CaMKI and CaMKIV, the latter of which directly activates transcription factors. In this study, we resolved the 2.4 Å crystal structure of the catalytic kinase domain of the human CaMKKβ isoform and its selective inhibitor STO-609 complex. The structure reveals that CaMKKβ lacks an αD helix, and its corresponding region exhibits a hydrophobic molecular surface, which may reflect its unique substrate recognition and self-inhibition mechanism. Although CaMKKβ lacks an activation ring phosphorylation site, its activation ring folds into an active conformation, which is stabilized by multiple interactions between conserved amino acid residues across CaMKK isoforms. In vitro kinase activity analysis confirmed the intrinsic activity of the CaMKKβ kinase domain. Structural and sequence analysis of the STO-609 binding site revealed amino acid substitutions that may affect inhibitor binding. Indeed, mutagenesis experiments showed that the Pro(274) residue of CaMKKβ (which replaces a conserved acidic residue in other protein kinases) is a crucial determinant of selective STO-609 inhibition. Therefore, the structure presented in this study provides a molecular basis for elucidating the known biochemical properties of CaMKKβ and designing novel inhibitors targeting CaMKKβ and its related protein kinases. [2] In summary, this study revealed the unique structural characteristics of CaMKKβ KD, providing a molecular basis for understanding the known biochemical properties of CaMKKβ and the differences in sensitivity of CaMKKα and CaMKKβ isoforms to STO-609. The structure of the CaMKKβ·STO-609 complex that we resolved also provides a structural basis for designing novel inhibitors to specifically block CaMKKβ and related protein kinases. [2]

C21H14N2O5

374.35

374.09

C, 67.38; H, 3.77; N, 7.48; O, 21.37

1173022-21-3

STO-609;52029-86-4

16760660

Typically exists as solid at room temperature

3.381

2

6

1

28

596

0

C1=CC=C2C(=C1)N=C3C4=CC=C(C5=C4C(=CC=C5)C(=O)N23)C(=O)O.CC(=O)O

WNRSTFUVBWNELX-UHFFFAOYSA-N

InChI=1S/C19H10N2O3.C2H4O2/c22-18-13-5-3-4-10-11(19(23)24)8-9-12(16(10)13)17-20-14-6-1-2-7-15(14)21(17)18;1-2(3)4/h1-9H,(H,23,24);1H3,(H,3,4)

acetic acid;11-oxo-3,10-diazapentacyclo[10.7.1.02,10.04,9.016,20]icosa-1(20),2,4,6,8,12,14,16,18-nonaene-17-carboxylic acid

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)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
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Oral Formulation 6: Mixing with food powders

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