KN-62 is a selective and reversible inhibitor of calmodulin-dependent protein kinase II (CaMK-II) with a K_i of 0.9 μM for rat brain CaMK-II. KN-62 directly binds to the calmodulin binding site of CaMK-II. KN-62 displays noncompetitive antagonism at P2X₇ receptors in HEK293 cells, with an IC₅₀ value of approximately 15 nM.

KN-62 potently antagonizes ATP-stimulated Ba²⁺ influx into fura-2 loaded human lymphocytes with an IC₅₀ of 12.7 nM and complete inhibition of the flux at a concentration of 500 nM^[1].
KN-62 does not inhibit the activity of autophosphorylated Ca2+/CaM kinase II. KN-62 inhibits the Ca2+/calmodulin-dependent autophosphorylation of both alpha (50 kDa) and beta (60 kDa) subunits of Ca2+/CaM kinase II dose dependently in the presence or absence of exogenous substrate^[2].
In human leukemic B lymphocytes, KN-62 reduces the rate of permeability increase to larger permeant cations, like ethidium, induced by Bz-ATP with an IC₅₀ of 13.1 nM^[4].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

KN62 (5 mg/kg/day; ip; three times a week for 6 weeks) significantly reduces the liver metastatic tumor burden in five weeks old BALB/c athymic nude mice inoculated with TAMR-MCF-7 cells^[3].
KN-62 (1 μg/site, i.c.v.) prevents the antidepressant-like behavior and antidepressant-like behaviors of ZnCl₂ (10 mg/kg, p.o.)^[5].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

721.84

C₃₈H₃₅N₅O₆S₂

127191-97-3

Room temperature in continental US; may vary elsewhere.

Powder	-20°C	3 years
	4°C	2 years
In solvent	-80°C	6 months
	-20°C	1 month

In Vitro: 

DMSO : ≥ 100 mg/mL (138.53 mM)

* “≥” means soluble, but saturation unknown.

*

请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。
储备液的保存方式和期限：-80°C, 6 months; -20°C, 1 month。-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。

In Vivo:

请根据您的实验动物和给药方式选择适当的溶解方案。以下溶解方案都请先按照 In Vitro 方式配制澄清的储备液，再依次添加助溶剂：

——为保证实验结果的可靠性，澄清的储备液可以根据储存条件，适当保存；体内实验的工作液，建议您现用现配，当天使用； 以下溶剂前显示的百
分比是指该溶剂在您配制终溶液中的体积占比；如在配制过程中出现沉淀、析出现象，可以通过加热和/或超声的方式助溶

• 1.

  请依序添加每种溶剂： 10% DMSO    40% PEG300    5% Tween-80    45% saline

  Solubility: ≥ 2.5 mg/mL (3.46 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (3.46 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀；向上述体系中加入50 μL Tween-80，混合均匀；然后继续加入 450 μL生理盐水定容至 1 mL。

  将 0.9 g 氯化钠，完全溶解于 100 mL ddH₂O 中，得到澄清透明的生理盐水溶液

• 2.

  请依序添加每种溶剂： 10% DMSO    90% corn oil

  Solubility: ≥ 2.5 mg/mL (3.46 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (3.46 mM，饱和度未知) 的澄清溶液，此方案不适用于实验周期在半个月以上的实验。

  以 1 mL 工作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 900 μL玉米油中，混合均匀。

• [1]. Gargett CE, et al. The isoquinoline derivative KN-62 a potent antagonist of the P2Z-receptor of human lymphocytes. Br J Pharmacol. 1997 Apr;120(8):1483-90.

  [2]. H Hidaka, et al. Pharmacology of protein kinase inhibitors. Annu Rev Pharmacol Toxicol. 1992;32:377-97.

  [3]. Miso Park, et al. Involvement of the P2X7 receptor in the migration and metastasis of tamoxifen-resistant breast cancer: effects on small extracellular vesicles production. Sci Rep. 2019 Aug 12;9(1):11587.

  [4]. Ravi RG, et al. Potent P2X7 Receptor Antagonists: Tyrosyl Derivatives Synthesized Using a Sequential Parallel Synthetic Approach. Drug Dev Res. 2001 Oct;54(2):75-87.

  [5]. Manosso LM, et al. Antidepressant-like effect of zinc is dependent on signaling pathways implicated in BDNF modulation. Prog Neuropsychopharmacol Biol Psychiatry. 2015 Jun 3;59:59-67.

Lymphocytes (1×10⁷/mL) are cultured with [³H]-oleic acid (2-5 μCi/mL, specific activity 10 Ci/mmol) for 20-24 h in RPMI-1640 medium supplemented with Gentamicin (40 μg/mL), 10% heat inactivated foetal calf serum (FCS) at 37°C to label membrane phospholipids. Labelled cells are washed twice in HEPES buffered saline followed by a final wash in either HEPES buffered saline or 150 mM KCl medium containing HEPES 10 mM, pH 7.4, bovine serum albumin (BSA) 1 g/L and D-glucose 5 mM and CaCl₂ 1 mM. Three mL aliquots containing 1.1×10< sup>7/mL lymphocytes are warmed to 37°C and incubated with or without KN-62 or KN-04 (1 nM-500 nM) for 5 min, then 900 mL aliquots are added to 100 uL butanol (final concentration 30 mM) for a further 5 min, and stimulated with 1 mM ATP for 15 min with gentle mixing in the continued presence of inhibitor or diluent. The phospholipase D reaction is terminated by addition of 1 mL of 20 mM MgCl₂ followed by centrifugation and addition of 1 mL ice cold methanol. Membrane lipids are extracted into chloroform/HCl at 4°C under N₂, and separated by silica gel thin layer chromatography (t.l.c.) with the solvent system, ethyl acetate/iso-octane/acetic acid/water (13:2:3:10, v/v) under saturating conditions. Sample spots are located by autoradiography and [³H]-phosphatidylbutanol ([³H]-PBut) spots identified by an authentic standard. [³H]-PBut and [³H]-phospholipid spots are scraped into scintillant fluid (PPO in toluene, 4 g/L) and counted in a liquid scintillation counter. The quantity of [³H]-PBut is presented as a percentage of total ³H labelled-cellular phospholipids. Phospholipase D assays are performed in triplicate^[1].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

All experiments are performed using adherent HEK293 cells stably transfected with cDNA encoding the human P2X₇ receptor. Adherent cells on 12-well polylysine-coated plates are incubated at 37°C in 1 mL physiological salt solution (125 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1.5 mM CaCl₂, 25 mM NaHEPES (pH 7.5), 10 mM D-glucose, 1 mg/mL BSA). Antagonists(e.g., KN-62) are added from 1,000× stock solutions dissolved in DMSO. Cells are preincubated with antagonists (e.g., KN-62) for 15 min prior to stimulation for 10 min with 3 mM ATP (final concentration). Reactions are terminated by rapid aspiration of the extracellular medium in each well. The adherent cells in each well are then extracted overnight with 1 mL 10% HNO₃. K⁺ content in these nitric acid extracts is assayed by atomic absorbance spectrophotometry. Duplicate or triplicate wells are run for all test conditions in each separate experiment^[4].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[3]
Female Swiss mice (45-55 days old, weighing 30-45 g) are used. The following drugs are used: ZnCl₂ (1 or 10 mg/kg), H-89 (1 μg/site, PKA inhibitor), KN-62 (1 μg/site, CAMKII inhibitor), chelerythrine (1 μg/site, PKC inhibitor), PD98059 (5 μg/site, MAPKK/MEK 1/2 inhibitor), U0126 (5 μg/site, MEK1/2 inhibitor), LY294002 (10 nmol/site, PI3K inhibitor), AR-A014418 (0.001 μg/site, selective GSK-3β inhibitor). ZnCl₂ is dissolved in distilled water and administered orally (p.o.). H-89, KN-62, chelerythrine, PD98059, U0126, LY294002, AR-A014418 are dissolved in saline (0.9% NaCl) at a final concentration of 1% dimethyl sulfoxide (DMSO) and administered by intracerebroventricular (i.c.v.) route. The drugs are freshly prepared before treatment and administered in a volume of 10 mL/kg body weight (p.o. route) or 5 μL/site (i.c.v. route). Control animals receive the appropriate vehicle.

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Gargett CE, et al. The isoquinoline derivative KN-62 a potent antagonist of the P2Z-receptor of human lymphocytes. Br J Pharmacol. 1997 Apr;120(8):1483-90.

  [2]. H Hidaka, et al. Pharmacology of protein kinase inhibitors. Annu Rev Pharmacol Toxicol. 1992;32:377-97.

  [3]. Miso Park, et al. Involvement of the P2X7 receptor in the migration and metastasis of tamoxifen-resistant breast cancer: effects on small extracellular vesicles production. Sci Rep. 2019 Aug 12;9(1):11587.

  [4]. Ravi RG, et al. Potent P2X7 Receptor Antagonists: Tyrosyl Derivatives Synthesized Using a Sequential Parallel Synthetic Approach. Drug Dev Res. 2001 Oct;54(2):75-87.

  [5]. Manosso LM, et al. Antidepressant-like effect of zinc is dependent on signaling pathways implicated in BDNF modulation. Prog Neuropsychopharmacol Biol Psychiatry. 2015 Jun 3;59:59-67.