The calcium–calmodulin‐dependent protein kinase kinase inhibitor, STO‐609, inhibits nicotine‐induced currents and intracellular calcium increase in insect neurosecretory cells

Article

作者: Taillebois, Emiliane ; Thany, Steeve H. ; Taha, Maria ; Houchat, Jean‐Noël

AbstractInsect neuronal nicotinic acetylcholine receptors (nAChRs) are transmembrane receptors that play a key role in the development and synaptic plasticity of both vertebrates and invertebrates and are considered to be major targets of neonicotinoid insecticides. We used dorsal unpaired median (DUM) neurons, which are insect neurosecretory cells, in order to explore the intracellular mechanisms leading to the regulation of insect neuronal nAChRs in more detail. Using whole‐cell patch‐clamp and fura‐2AM calcium imaging techniques, we found that a novel CaMKK/AMPK pathway could be involved in the intracellular regulation of DUM neuron nAChRs. The CaMKK selective inhibitor, STO, reduced nicotinic current amplitudes, and strongly when co‐applied with α‐Bgt. Interestingly, intracellular application of the AMPK activator, A‐76, prevented the reduction in nicotine‐induced currents observed in the presence of the AMPK inhibitor, dorsomorphin. STO prevented the increase in intracellular calcium induced by nicotine, which was not dependent on α‐Bgt. Currents induced by 1 mM LMA, a selective activator of nAChR2, were reduced under bath application of STO, and mecamylamine, which blocked nAChR2 subtype, inhibited the increase in intracellular calcium induced by LMA. These findings provide insight into potential complex mechanisms linked to the modulation of the DUM neuron nAChRs and CaMKK pathway.

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2024-01-01·Cell Biology International

Pachymic acid (PA) inhibits ferroptosis of cardiomyocytes via activation of the AMPK in mice with ischemia/reperfusion‐induced myocardial injury

Article

作者: Liu, Dongmin ; Li, Zhenzhen ; Ding, Jiru ; Lu, Youquan

AbstractPachymic acid (PA) is a lanostane‐type triterpenoid with various pharmacological effects. However, little is known about the effect of PA on myocardial infarction (MI) induced by ischemia/reperfusion (I/R). In this study, we aimed to investigate the protective effect of PA and its underlying mechanism. A cellular MI model was established by oxygen‐glucose deprivation and reperfusion (OGD/R) treatment in HL‐1 cardiomyocytes, and we found that OGD/R treatment decreased cell viability and glutathione peroxide (GSH‐Px) activity, increased Fe2+ concentration and lactate dehydrogenase (LDH) activity, promoted malondialdehyde (MDA) and reactive oxygen species (ROS) production, and inhibited the expression of ferroptosis marker proteins SLC7A11 and GPX4 in a time‐dependent manner. OGD/R‐induced HL‐1 cells were pretreated with different concentrations of PA (0, 20, 40, 60 μg/mL) for 24 h, and toxicological experiments showed that 150 μg/mL PA decreased cell viability, while low concentrations of PA had no toxic effect on cells. 20 μg/mL PA reversed the inhibitory effect of OGD/R on cell viability, reduced MDA and ROS production, and Fe2+ accumulation, increased GSH‐Px activity and the expression of SLC7A11 and GPX4, and decreased LDH activity, especially at 60 μg/mL PA. Meanwhile, PA promoted the phosphorylation of IRS‐1, AKT, and AMPK proteins in a dose‐dependent manner. AICAR, an AMPK activator, inhibited ferroptosis, while STO‐609, an AMPK inhibitor, largely abolished the effect of PA on OGD/R‐induced ferroptosis of HL‐1 cells. In addition, PA inhibited ferroptosis and myocardial I/R injury in wild‐type mice and AMPK knockout (AMPK−/−) mice. Collectively, PA inhibited ferroptosis of cardiomyocytes through activating of the AMPK pathway, thereby alleviating myocardial I/R injury in mice.

2023-09-01·Molecular metabolism

CaMKK2 is not involved in contraction-stimulated AMPK activation and glucose uptake in skeletal muscle.

Article

作者: Hellberg, Kristina ; Hafen, Paul S ; Agerholm, Marianne ; Hossain, Mohammad A ; Sakamoto, Kei ; Witczak, Carol A ; Sankar, Uma ; Ling, Naomi X Y ; Brault, Jeffrey J ; Sermersheim, Tyler J ; Oakhill, Jonathan S ; Ochala, Julien ; Addinsall, Alex B ; Bringas, Conchita Fraguas ; Negoita, Florentina ; Drewry, David H ; Larsen, Jeppe K ; Lewis, Christopher T A ; Deshmukh, Atul S ; Ahwazi, Danial ; Scott, John W

OBJECTIVEThe AMP-activated protein kinase (AMPK) gets activated in response to energetic stress such as contractions and plays a vital role in regulating various metabolic processes such as insulin-independent glucose uptake in skeletal muscle. The main upstream kinase that activates AMPK through phosphorylation of α-AMPK Thr172 in skeletal muscle is LKB1, however some studies have suggested that Ca²⁺/calmodulin-dependent protein kinase kinase 2 (CaMKK2) acts as an alternative kinase to activate AMPK. We aimed to establish whether CaMKK2 is involved in activation of AMPK and promotion of glucose uptake following contractions in skeletal muscle.METHODSA recently developed CaMKK2 inhibitor (SGC-CAMKK2-1) alongside a structurally related but inactive compound (SGC-CAMKK2-1N), as well as CaMKK2 knock-out (KO) mice were used. In vitro kinase inhibition selectivity and efficacy assays, as well as cellular inhibition efficacy analyses of CaMKK inhibitors (STO-609 and SGC-CAMKK2-1) were performed. Phosphorylation and activity of AMPK following contractions (ex vivo) in mouse skeletal muscles treated with/without CaMKK inhibitors or isolated from wild-type (WT)/CaMKK2 KO mice were assessed. Camkk2 mRNA in mouse tissues was measured by qPCR. CaMKK2 protein expression was assessed by immunoblotting with or without prior enrichment of calmodulin-binding proteins from skeletal muscle extracts, as well as by mass spectrometry-based proteomics of mouse skeletal muscle and C2C12 myotubes.RESULTSSTO-609 and SGC-CAMKK2-1 were equally potent and effective in inhibiting CaMKK2 in cell-free and cell-based assays, but SGC-CAMKK2-1 was much more selective. Contraction-stimulated phosphorylation and activation of AMPK were not affected with CaMKK inhibitors or in CaMKK2 null muscles. Contraction-stimulated glucose uptake was comparable between WT and CaMKK2 KO muscle. Both CaMKK inhibitors (STO-609 and SGC-CAMKK2-1) and the inactive compound (SGC-CAMKK2-1N) significantly inhibited contraction-stimulated glucose uptake. SGC-CAMKK2-1 also inhibited glucose uptake induced by a pharmacological AMPK activator or insulin. Relatively low levels of Camkk2 mRNA were detected in mouse skeletal muscle, but neither CaMKK2 protein nor its derived peptides were detectable in mouse skeletal muscle tissue.CONCLUSIONSWe demonstrate that pharmacological inhibition or genetic loss of CaMKK2 does not affect contraction-stimulated AMPK phosphorylation and activation, as well as glucose uptake in skeletal muscle. Previously observed inhibitory effect of STO-609 on AMPK activity and glucose uptake is likely due to off-target effects. CaMKK2 protein is either absent from adult murine skeletal muscle or below the detection limit of currently available methods.

项与 STO-609 相关的新闻（医药）

2022-12-14

·生物谷

Redox Biology: 巨噬细胞球蛋白4缺乏减轻脓毒症所致小鼠急性肺损伤

脓毒症是一种毁灭性的临床疾病，在重症监护病房的患者中经常导致多器官衰竭。肺部是脓毒症期间最脆弱的器官之一，尽管机械支持通气和对症治疗取得了进展，但脓毒症合并急性肺损伤(ALI)的患者也表现出更高的死亡脓毒症是一种毁灭性的临床疾病，在重症监护病房的患者中经常导致多器官衰竭。肺部是脓毒症期间最脆弱的器官之一，尽管机械支持通气和对症治疗取得了进展，但脓毒症合并急性肺损伤(ALI)的患者也表现出更高的死亡率和更差的预后。众所周知，炎症和氧化应激在脓毒症所致的ALI中起关键作用，抑制过度产生的炎性细胞因子和活性氧物种(ROS)显著改善肺功能和生存状态。AMPK主要作为细胞内能量平衡的中央调节器，将消耗ATP的合成代谢转变为产生ATP的分解代谢；然而，新的研究表明，AMPK远远超出了其能量调节功能，还参与调节炎症和氧化应激。图片来源: https://doi.org/10.1016/j.redox.2022.102513 近日，来自武汉大学人民医院的研究者们在Redox Biology杂志上发表了题为“Macrophage Sprouty4 deficiency diminishes sepsis-induced acute lung injury in mice”的文章，本研究确定巨噬细胞SPRY4是脓毒症所致ALI的一个有前景的预测和治疗靶点。炎症和氧化应激在脓毒症所致的急性肺损伤(ALI)中起重要作用。Sprout4(SPRY4)参与调节炎症和组织损伤，但其在脓毒症所致ALI中的作用和机制尚不清楚。研究者建立巨噬细胞特异性SPRY4基因敲除(Spry4MKO)、转基因(Spry4MTG)小鼠和配对对照小鼠，应用盲肠结扎和穿孔(CLP)手术建立细菌性败血症诱导的ALI模型。从Spry4MKO或Spry4MTG小鼠分离骨髓来源的巨噬细胞(BMDM)，并在脂多糖(LPS)刺激下进一步验证SPRY4的体外作用。另外，研究者采用SPRY4和AMPK双基因敲除小鼠和化合物C进行体内和体外实验，以验证AMPK的必要性。用STO-609抑制钙/钙调素依赖的蛋白激酶2(CAMKK2)。研究者发现巨噬细胞SPRY4在CLP小鼠体内表达增加，并与脓毒症所致的ALI呈正相关。巨噬细胞SPRY4缺乏预防，而巨噬细胞SPRY4过表达加剧了脓毒症诱导的炎症、氧化应激和小鼠和骨髓基质细胞的ALI。机制研究表明，巨噬细胞SPRY4缺乏通过激活CAMKK2/AMPK通路减轻脓毒症所致的ALI。巨噬细胞SPRY4缺陷通过激活AMPK通路减轻脓毒症所致ALI 图片来源: https://doi.org/10.1016/j.redox.2022.102513 综上所述，本研究揭示巨噬细胞SPRY4缺乏以CAMKK2/AMPK依赖的方式预防脓毒症所致的炎症、氧化应激和ALI，并将其定义为脓毒症所致ALI的一个有前景的预测和治疗靶点。（生物谷 Bioon.com）参考文献 Rong Chen et al. Macrophage Sprouty4 deficiency diminishes sepsis-induced acute lung injury in mice. Redox Biol. 2022 Dec; 58:102513. doi: 10.1016/j.redox.2022.102513.

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适应症	最高研发状态	国家/地区	公司	日期
移植物抗宿主病	临床前	美国	Duke University School of Medicine	-
移植物抗宿主病	临床前	美国	Eyedesis Biosciences LLC	-
湿性年龄相关性黄斑变性	临床前	美国	Duke University School of Medicine	-
湿性年龄相关性黄斑变性	临床前	美国	Eyedesis Biosciences LLC	-