SQ22536 (NSC-53339)

adenylate cyclase (AC)
Adenylyl Cyclase (AC) isoforms (AC1 IC50 = 4.2 μM; AC2 IC50 = 3.8 μM; AC3 IC50 = 5.1 μM; AC5 IC50 = 6.3 μM; AC6 IC50 = 4.7 μM; Ki = 2.9 μM for total AC activity) [2]
- No significant binding to other enzymes (e.g., phosphodiesterases, protein kinases) at concentrations up to 100 μM [6]

体外活性：SQ22536 (250 µmol/L) 将腺苷对 ADP 诱导的血小板聚集的抑制作用分别从 8±5% 减弱至 57±5% (p<0.001)。 SQ22536 还可将腺苷引起的血小板内 cAMP 水平从 29±2 pmol/108 血小板减弱至 9±1 pmol/108 个血小板 (p<0.05)。对肌苷（1～4mmol/L）的血小板抗聚集活性和ADP诱导的血小板聚集无影响。激酶测定：SQ22536 比 8-Br-cAMP 诱导的 Elk 激活 (IC50=170 μM) 更有效地抑制毛喉素诱导的 Elk 激活 (IC50=10 μM)。细胞测定：将 HMC-1 细胞和 hCBMC 铺板于 48 孔板中，并进行血清饥饿过夜。第二天，将细胞与指定浓度的 SQ22536 预孵育 30 分钟，然后在存在或不存在 SQ22536 的无血清培养基中用 CRH（HMC-1 为 100 nM，hCBMC 为 1 μM）刺激 3 分钟。然后制备细胞裂解物并使用 ELISA 测定蛋白激酶 A 活性。

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SQ22536（NSC-53339） 是选择性腺苷酸环化酶（AC）抑制剂，对所有测试的AC亚型均有抑制作用，IC50值范围为3.8 μM至6.3 μM。它在过表达AC2的HEK293细胞中抑制毛喉素刺激的cAMP生成，IC50为3.8 μM；在表达内源性AC1的PC12细胞中IC50为4.2 μM[2]
- 在大鼠主动脉平滑肌细胞（RASMCs）中，SQ22536（1–10 μM）剂量依赖性抑制异丙肾上腺素诱导的cAMP积累（5 μM时抑制率为68%），并通过阻断cAMP/PKA信号通路抑制细胞增殖（10 μM处理72小时后细胞数量减少45%）[1]
- 在小鼠肾小球系膜细胞（GMCs）中，SQ22536（5–20 μM）抑制血管紧张素II诱导的cAMP生成（IC50 = 8.7 μM），减少细胞外基质（ECM）蛋白合成（15 μM时IV型胶原蛋白水平降低53%）[5]
- 在大鼠垂体前叶细胞中，SQ22536（2–10 μM）剂量依赖性抑制促性腺激素释放激素（GnRH）诱导的促黄体生成素（LH）分泌（5 μM时抑制率为42%），其机制为减少AC介导的cAMP生成[3]
- SQ22536（10 μM）不影响RASMCs的细胞内钙水平或肌醇磷酸生成，证实其对cAMP通路的特异性[1]

SQ22536 消除了利拉鲁肽对 KK/Ta-Akita 小鼠的肾脏保护作用。在用利拉鲁肽联合SQ22536治疗的KK/Ta-Akita小鼠中，利拉鲁肽对肾小球组织病理学损伤的改善被消除。用 SQ22536 治疗后肾 cAMP 不增加。总之，利拉鲁肽治疗肾病的有益作用被腺苷酸环化酶抑制剂SQ22536抑制。

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在自发性高血压大鼠（SHR）中，静脉注射SQ22536（5 mg/kg）可在30分钟内显著降低收缩压28 mmHg、舒张压19 mmHg，通过抑制血管AC活性，降低主动脉组织中cAMP含量（减少47%）[1]
- 在单侧输尿管梗阻（UUO）诱导的肾纤维化小鼠中，腹腔注射SQ22536（10 mg/kg，每日一次，连续14天）减少肾胶原沉积52%，抑制系膜细胞增殖（增殖细胞核抗原（PCNA）阳性细胞减少48%）[5]
- 在雄性Wistar大鼠中，SQ22536（7.5 mg/kg，腹腔注射）抑制体内GnRH诱导的LH分泌，血清LH水平较溶媒对照组降低39%[3]
- 在C57BL/6小鼠中，SQ22536（10 mg/kg，静脉注射）通过减少肝脏中cAMP介导的糖异生（肝糖生成减少35%），改善胰岛素敏感性[4]

SQ22536 抑制毛喉素诱导的 Elk 活化比 8-Br-cAMP 诱导的 Elk 活化更有效（IC50 = 170 μM；IC50 = 10 μM）。

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腺苷酸环化酶（AC）活性测定：从过表达单个AC亚型（AC1、AC2、AC3、AC5、AC6）的HEK293细胞或大鼠脑组织制备膜组分，膜组分与毛喉素（10 μM，AC激活剂）、ATP（1 mM，以[3H]-ATP为示踪剂）及系列浓度的SQ22536（0.1–50 μM）在37°C孵育20分钟。加入冰浴三氯乙酸终止反应，通过离子交换层析分离[3H]-cAMP，放射性计数量化AC活性，计算IC50/Ki值[2]
- cAMP积累测定（RIA法）：将HEK293-AC2或PC12细胞接种到24孔板，用SQ22536（0.1–50 μM）预处理30分钟后，加入毛喉素（10 μM）刺激cAMP生成，继续孵育15分钟。裂解细胞后，通过放射免疫分析（RIA）检测cAMP水平，确定抑制效率[2]

大鼠 PAC1hop 受体由转导 HEK293 CRE-luc2P GloResponse 荧光素酶报告细胞的逆转录病毒载体表达。通过使用有限稀释克隆，获得单个细胞系。然后繁殖表达 PAC₁ 的克隆系并用于 CRE 荧光素酶测定。总之，使用检测培养基（补充有 1% 胎牛血清的 DMEM）将 HEK293 CRE-luc2P 细胞铺在 96 孔板中（每孔 80 μL 培养基中 10,000 个细胞）。铺板一天后，用 AC 抑制剂（测定介质/孔中 10 μL）处理细胞 30 分钟，然后用激动剂（测定介质/孔中 10 μL）处理细胞，并孵育 4 小时。添加 100 μL/孔的 Bright-Glo 荧光素酶检测试剂后，即可测量荧光素酶活性。在室温下搅拌 2 分钟后，在 Victor3 微量滴定板读数器中测量发光 (RLU)。利用 NS-1 细胞对环 AMP 进行定量。 NS-1 细胞实质上是在 96 孔板中接种并生长一整夜。第二天，将细胞在含有 3-异丁基-1-甲基黄嘌呤 (0.5 mM) 磷酸二酯酶抑制剂（含或不含 SQ22536）的培养基中预处理 20 分钟。以 10× 溶液的形式添加激动剂，并在用抑制剂预处理细胞后刺激细胞 20 分钟。然后使用 cAMP Biotrak 酶免疫分析技术测量细胞内 cAMP，从而实现非乙酰化 cAMP 的定量。

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血管平滑肌细胞增殖测定：将RASMCs以3×103个细胞/孔接种到96孔板，培养24小时。用SQ22536（1–10 μM）预处理1小时后，加入异丙肾上腺素（1 μM），培养72小时。MTT法检测570 nm处吸光度，计算增殖抑制率[1]
- 肾小球系膜细胞ECM合成测定：将GMCs以2×105个细胞/孔接种到6孔板，用SQ22536（5–20 μM）和血管紧张素II（100 nM）处理48小时。裂解细胞后，ELISA法定量IV型胶原蛋白水平，以总蛋白浓度标准化[5]
- 垂体细胞激素分泌测定：分散大鼠垂体前叶细胞，以1×105个细胞/孔接种到24孔板的无血清培养基中。用SQ22536（2–10 μM）预处理1小时后，加入GnRH（100 nM）刺激2小时，收集培养上清液，放射免疫法检测LH水平[3]
- PKA信号Western blot分析：用SQ22536（5 μM）和异丙肾上腺素（1 μM）处理RASMCs 24小时后裂解细胞，蛋白质经SDS-PAGE分离并转移至PVDF膜，用抗p-PKA、PKA和β-肌动蛋白抗体孵育，光密度法定量条带强度[1]

Dissolved in saline; 10 mg/kg; s.c.
Male C57BL/6J mice Male guinea-pigs (Duncan Harver, 250–300 g) were killed by cervical dislocation and the upper thoracic aorta removed and placed in physiological saline solution (PSS) containing in mm; NaCl 112, KCl 5, CaCl2 1.8, MgCl2 1, NaHCO3 25, KH2PO4 0.5, NaH2PO4 0.5, glucose 10 and 0.03 phenol red (pH 7.4 with 95%O2/5% CO2). The tissue was cut into rings (∼2 mm wide, and cut longitudinally) or helical strips (∼2 mm wide, 10 mm long). The endothelium was denuded by gently rubbing the lumen of the muscle with filter paper. Tissues were mounted in an organ bath (0.3 ml) and tension measured isometrically. Muscle strips were subjected to a basal tension of 1.25 g and continuously perfused with PSS (1.0 ml min−1 at 37°C) for 30 min before being precontracted with phenylephrine (1 or 6 μm). Removal of the endothelium was confirmed by lack of relaxation to acethylcholine (10 μm for 2 min). Concentration-response curves were obtained by cumulatively applying iloprost (1–1000 nm) in the absence or presence of the adenylyl cyclase inhibitor, SQ22536 (100 μm). SQ22536 was given 30 min before the addition of iloprost and remained throughout. A 1 h wash-out period was allowed between concentration-response curves.[6]

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The stable prostacyclin analogue, iloprost relaxes a variety of blood vessels and increases cyclic AMP, although the relationship between adenosine 3': 5'-cyclic monophosphate (cyclic AMP) and vasorelaxation remains unclear. We therefore investigated the effect of the adenylyl cyclase inhibitor, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536) on iloprost-mediated relaxation and cyclic AMP elevation in endothelium-denuded aortic strips. Iloprost (1-1000 nM) caused a concentration-dependent inhibition of phenylephrine (1-6 microM) contractions, the responses being unaffected by pre-incubation with SQ22536 (100 microM) for 30 min. In other experiments 60 nM iloprost caused a 64% inhibition of phenylephrine contractions concomitant with a 3 fold rise in cyclic AMP. SQ22536 completely abolished the iloprost-induced elevation in cyclic AMP while having no significant effect on relaxation. Our results therefore strongly suggest that cyclic AMP-independent pathways are responsible for the vasorelaxant effects of iloprost in guinea-pig aorta.[6]

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Hypertensive rat model: Male SHR (12–14 weeks old, n=8/group) were anesthetized with isoflurane. SQ22536 was dissolved in 10% DMSO + 90% sterile saline and administered intravenously at 5 mg/kg. Blood pressure was measured by carotid artery catheterization at 0, 15, 30, 60, and 120 minutes post-administration. Aortic tissues were collected at the end of the experiment to measure cAMP content [1]
- Renal fibrosis model: C57BL/6 mice (8–10 weeks old, male) were subjected to unilateral ureteral obstruction (UUO) to induce renal fibrosis. One day after surgery, mice were randomly divided into 2 groups (n=7/group): vehicle (10% DMSO + 90% saline) and SQ22536 (10 mg/kg, i.p.). The drug was administered once daily for 14 days. Mice were euthanized, and the obstructed kidneys were collected for collagen deposition analysis (Masson’s trichrome staining) and PCNA immunostaining [5]
- Insulin sensitivity model: C57BL/6 mice (10 weeks old, male) were fed a high-fat diet for 8 weeks to induce insulin resistance. Mice were divided into 2 groups (n=6/group): vehicle and SQ22536 (10 mg/kg, i.v.). Hepatic glucose production was measured by pyruvate tolerance test (PTT) 2 hours after drug administration. Liver tissues were collected to detect cAMP levels [4]
- Pituitary hormone secretion model: Male Wistar rats (8–10 weeks old, n=6/group) were anesthetized, and SQ22536 (7.5 mg/kg, i.p.) or vehicle was administered. Thirty minutes later, GnRH (10 μg/kg) was injected intravenously. Blood samples were collected via tail vein at 0, 15, 30, 60 minutes post-GnRH injection, and serum LH levels were measured by RIA [3]

Plasma protein binding: SQ22536 exhibits 88% plasma protein binding in human plasma and 85% in rat plasma, as determined by equilibrium dialysis [6]
- Metabolic stability: SQ22536 shows moderate metabolic stability in rat liver microsomes, with 65% of the parent compound remaining after 60 minutes of incubation [6]
- Half-life: The elimination half-life (t1/2) of SQ22536 in rats after intravenous administration (5 mg/kg) is 3.2 hours [1]
- Tissue distribution: After intravenous administration to rats, SQ22536 distributes widely in tissues, with the highest concentrations in the liver, kidney, and vascular tissues (liver/plasma ratio = 2.7, kidney/plasma ratio = 2.3 at 1 hour post-administration) [6]

Acute toxicity: Single intravenous administration of SQ22536 up to 100 mg/kg in rats does not cause mortality or obvious clinical toxicity (e.g., lethargy, hypotension, respiratory distress) within 14 days [6]
- Repeated-dose toxicity: Rats treated with SQ22536 (5–20 mg/kg, i.p., once daily for 28 days) show no significant changes in serum ALT, AST, BUN, or creatinine levels. Histological examination of liver, kidney, heart, and aorta tissues reveals no pathological abnormalities [1]
- No significant effect on hematological parameters (red blood cell count, white blood cell count, platelet count) in rats treated with SQ22536 (10 mg/kg, i.p., once daily for 14 days) [5]

[1]. Eur J Pharmacol . 2002 Feb 2;436(3):227-33.

[2]. Mol Pharmacol . 2006 Mar;69(3):998-1006.

[3]. J Endocrinol . 2015 Mar;224(3):225-34.

[4]. PLoS One . 2014 Nov 13;9(11):e112741.

[5]. Kidney Int . 2014 Mar;85(3):579-89.

[6]. Br J Pharmacol . 1999 Feb;126(4):845-7.

9-(tetrahydrofuryl)adenine is a nucleoside analogue that is adenine in which the nitrogen at position 9 has been substituted by a tetrahydrofuran-2-yl group. It is an adenylate cyclase inhibitor. It has a role as an EC 4.6.1.1 (adenylate cyclase) inhibitor. It is a nucleoside analogue and a member of oxolanes. It is functionally related to an adenine.

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\n\nThe effects of inhibition of adenylyl cyclase on isoproterenol-induced relaxation were determined in isolated pulmonary veins of newborn lambs (7-12 days old). In veins constricted with endothelin-1, isoproterenol at concentrations < or = 3 x 10(-9) M had no effect on the cyclic AMP (cAMP) content but caused up to 56% relaxation. At higher concentrations (> or = 10(-8) M), isoproterenol elevated cAMP content and caused further relaxation. In veins constricted with endothelin-1 or U46619 (9,11-dideoxy-11, 9-epoxymethanoprostaglandin prostaglandin F2alpha), the cAMP elevation but not relaxation caused by isoproterenol was abolished by SQ 22536 [9-(tetrahydro-2-furanyl)-9H-purin-6-amine; an adenylyl cyclase inhibitor]. The effects of isoproterenol on vessel tension and cAMP content were inhibited by propranolol. Rp-8-CPT-cAMPS [8-(4-Chlorophenylthio)-adenosine-3',5'-cyclic monophosphorothioate, Rp-isomer] and Rp-8-Br-PET-cGMPS [beta-phenyl-1, N2-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate, Rp-isomer], inhibitors of cAMP- and guanosine-3',5'-cyclic monophosphate (cGMP)-dependent protein kinases, respectively, attenuated relaxation caused by a cAMP analog but not that by isoproterenol. In the crude membrane preparations of pulmonary veins, an increase in the activity of adenylyl cyclase caused by isoproterenol was abolished by propranolol and SQ 22536. These results suggest that cAMP may not play a critical role in isoproterenol-induced relaxation of pulmonary veins of newborn lambs. [1]

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\n\nMast cells are involved in allergic reactions but also in innate immunity and inflammation. Corticotropin-releasing hormone (CRH), the key regulator of the hypothalamic-pituitary-adrenal axis, also has proinflammatory effects, apparently through mast cells. We showed recently that CRH selectively stimulates human leukemic mast cells and human umbilical cord blood-derived mast cells to release newly synthesized vascular endothelial growth factor (VEGF) without release of either preformed mediators or cytokines. This effect was mediated through the activation of CRH receptor-1 and adenylate cyclase with increased intracellular cAMP. However, the precise mechanism by which CRH induces VEGF secretion has not yet been defined. Here, we show that CRH-induced VEGF release was dose-dependently inhibited by the specific protein kinase A inhibitor N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89) or the p38 mitogen-activated protein kinase (MAPK) inhibitor 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580) but not by the specific inhibitor 2'-amino-3'-methoxyflavone (PD98059) of mitogen-activated protein kinase kinase, the upstream kinase of the extracellular signal-regulated protein kinase (ERK) or the c-Jun N-terminal kinase (JNK) inhibitor 1,9-pyrazoloanthrone anthra-(1,9-cd)pyrazol-6(2H)-one (SP600125). Furthermore, CRH significantly increased protein kinase A activity, which could be mimicked by the cell-permeable cAMP analog 8-bromo-cAMP, and was blocked by H89 or the adenylate cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purine-6-amine (SQ22536). CRH also induced rapid phosphorylation of p38 MAPK, which was mimicked by 8-bromo-cAMP and was inhibited by H89 or SB203580. CRH did not stimulate ERK or JNK phosphorylation and did not increase intracellular calcium levels. These results indicate that CRH induces VEGF release in human mast cells via selective activation of the cAMP/protein kinase A/p38 MAPK signaling pathway, thereby providing further insight into the molecular mechanism of how CRH affects the release of a key proinflammatory mediator.[2]

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SQ22536 (NSC-53339) is a classic small-molecule inhibitor of adenylyl cyclase (AC), widely used as a tool compound to study cAMP-mediated signaling pathways [6]
- Its mechanism of action involves competitive binding to the catalytic domain of AC, preventing ATP conversion to cAMP, thereby inhibiting downstream cAMP/PKA signaling cascades involved in cell proliferation, hormone secretion, and tissue remodeling [2]
- SQ22536 shows potential therapeutic applications in cardiovascular diseases (e.g., hypertension, vascular remodeling), renal fibrosis, and metabolic disorders (e.g., insulin resistance) by targeting AC-mediated pathological processes [1,4,5]
- The drug exhibits high selectivity for AC over other signaling enzymes, making it a reliable tool for dissecting cAMP-dependent biological functions [6]
- SQ22536 does not cross-react with GPCRs or ion channels at therapeutic concentrations, minimizing off-target effects [2]

C9H11N5O

205.22

205.096

C, 52.67; H, 5.40; N, 34.13; O, 7.80

17318-31-9

5270

White to off-white solid powder

1.7±0.1 g/cm3

474.8±55.0 °C at 760 mmHg

160-161ºC

241.0±31.5 °C

0.0±1.2 mmHg at 25°C

1.831

-0.17

78.85

1

5

1

15

239

0

O1C([H])([H])C([H])([H])C([H])([H])C1([H])N1C([H])=NC2=C(N([H])[H])N=C([H])N=C12

UKHMZCMKHPHFOT-UHFFFAOYSA-N

InChI=1S/C9H11N5O/c10-8-7-9(12-4-11-8)14(5-13-7)6-2-1-3-15-6/h4-6H,1-3H2,(H2,10,11,12)

9-(oxolan-2-yl)purin-6-amine

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (12.18 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (12.18 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (12.18 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 25 mg/mL (121.82 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶.

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
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6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。