A-804598 (A804598)   中文名称: A 804598

P2X7 Receptor
P2X7 Receptor (purinergic receptor P2X subtype 7) - Ki value: ~1.6 nM (determined by [³H]A-804598 competitive binding assay in rat brain cortex membranes); - IC50 for inhibiting ATP-induced P2X7-mediated responses: ~8.3 nM (inhibition of ATP-induced ethidium bromide (EtBr) uptake, a marker of P2X7-mediated pore formation, in human embryonic kidney (HEK) 293 cells stably expressing human P2X7 (HEK-hP2X7)); - No significant binding to other P2 receptor subtypes (P2X1, P2X2, P2X3, P2X4, P2X5, P2Y1, P2Y2, P2Y4, P2Y6) at concentrations up to 10 μM, indicating high selectivity for P2X7^[1]
[1]

以浓度依赖性方式，与 A-804598（0.1-10 μM；1 小时）预孵育可大大减少 BzATP 诱导的细胞损失。 3 μM A-804598 显示了针对 BzATP 诱导的细胞毒性的最高保护效果[2]。

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为了补充P2X7敲除的结果，我们试图测试P2X7拮抗剂（a-804598）在小鼠小胶质细胞中的作用。在暴露于BzATP之前，将原代小胶质细胞与不同浓度的A-804598一起孵育1小时。通过CCK-8测定法测量细胞活力，并通过共聚焦显微镜检查小胶质细胞形态。如图6a所示，单独使用BzATP会导致约40%的小胶质细胞损失，而与A-804598预孵育会以浓度依赖的方式显著减轻BzATP诱导的细胞损失。三种微摩尔A-804598对BzATP诱导的细胞毒性表现出最大的保护作用（图6b）。此外，我们验证了A-804598对活化小胶质细胞的保护作用。LPS预处理的小胶质细胞首先暴露于不同浓度的A-804598，然后用BzATP处理。正如预期的那样，经预处理的小胶质细胞在BzATP的作用下表现出变形虫形态和显著的细胞损失（图6a）。然而，共聚焦显微镜和CCK-8测定的结果表明，BzATP引起的细胞损失可以通过与A-804598预孵育以浓度依赖的方式抵消（图6c）。综上所述，我们的研究结果揭示了P2X7拮抗剂A-804598对失活和活化小胶质细胞中BzATP诱导的细胞毒性的保护作用，进一步证明了P2X9在ATP诱导的小胶质细胞死亡中的介导作用。[2]

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1. P2X7受体结合亲和力与选择性： - 在大鼠大脑皮层膜制备物中，[³H]A-804598以高亲和力结合P2X7受体：平衡解离常数（Kd）为~2.1 nM，最大结合容量（Bmax）为~11.2 fmol/mg蛋白。未标记的A-804598可竞争性置换[³H]A-804598，Ki值为~1.6 nM。 - 在针对15种其他受体家族（包括肾上腺素能、胆碱能、GABA能、阿片受体）和离子通道的结合实验中，10 μM A-804598对相应放射性配体的置换率<50%，证实无脱靶结合^[1]
2. 抑制P2X7介导的功能反应： - 在HEK-hP2X7细胞中：A-804598呈剂量依赖性抑制ATP（1 mM）诱导的EtBr摄取（小孔形成），IC50为~8.3 nM；100 nM时抑制率超过95%。其还可抑制ATP诱导的细胞内钙（[Ca²⁺]i）升高，IC50为~7.9 nM，与P2X7拮抗作用一致。 - 在大鼠原代小胶质细胞中：100 nM A-804598可完全阻断ATP（5 mM）诱导的EtBr摄取，证实其在天然细胞中对P2X7的抑制作用^[1]
3. P2X7抑制作用的可逆性： - 将HEK-hP2X7细胞中10 nM A-804598洗脱后，ATP诱导的[Ca²⁺]i升高在30分钟内恢复至处理前水平的~90%，表明其与P2X7的结合具有可逆性^[1]
[1]

在疾病末期的腰脊髓中，A-804598慢性治疗（腹腔注射；30 mg/kg；每周五次）可降低LC3B-II和SQSTM1/p62的表达[3]。

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众所周知，在疾病进展过程中，当自噬通量受损时，SQSTM1/p62自噬底物与LC3B-II一起在ALS小鼠的腰椎脊髓中积累（Zhang等人，2011）。因此，我们测量了SOD1-G93A小鼠腰椎脊髓中LC3B-II和SQSTM1/p62蛋白的水平，该小鼠在体内通过用血脑渗透剂a-804598对雌性SOD1-G933A小鼠进行慢性治疗来药理学抑制P2X7受体，证明在啮齿动物中口服或腹腔注射剂量后达到脑浓度（Able等人，2011；Iwata等人，2016），从发病前到疾病末期以30mg/Kg的剂量给药。我们发现，虽然与野生型相比，经赋形剂处理的SOD1-G93A小鼠的LC3B-II（图5A）和SQSTM1/p62（图5B）的蛋白质水平在终末期都有所增加，但在A-804598处理的ALS小鼠中，与赋形剂相比，LC3B-II的蛋白质含量似乎没有变化，而SQSTM1/p62被抑制到基础水平（图5A、B）。如图所示，在雌性SOD1-G93A小鼠中施用A-804598时，行为评分（图5C）、疾病发作（图5D）和存活率（图5E）均不受影响。[3]

ATP敏感的P2X7受体定位在免疫来源的细胞上，包括中枢神经系统中的外周巨噬细胞和神经胶质细胞。P2X7受体的激活导致细胞内钙浓度的快速变化，促炎细胞因子IL-1β的释放，以及在长时间接触激动剂后，质膜中细胞溶解孔的形成。基因敲除研究和最近描述的选择性拮抗剂的数据表明，P2X7受体激活在炎症和疼痛中起作用。虽然存在几种物种选择性P2X7拮抗剂，但A-804598代表了一种结构新颖、具有竞争力和选择性的拮抗剂，对大鼠（IC50=10 nM）、小鼠（IC50=9 nM）和人类（IC50=11 nM）P2X7受体具有同等的高亲和力。A-804598还有效地阻断了激动剂刺激的IL-1β和Yo-Pro从天然表达人P2X7受体的分化THP-1细胞中的摄取释放。A-804598被氚化（[3H]A-804598；8.1Ci/mmol），用于研究1321N1细胞中表达的重组大鼠P2X7受体。[3H]A-804598标记了一类高亲和力结合位点（Kd=2.4 nM，表观Bmax=0.56 pmol/mg）。在未转染的1321N1细胞中没有观察到特异性结合。P2X拮抗剂抑制[3H]A-804598结合的药理学特征与其阻断P2X7受体功能激活的能力相关（r=0.95，P<0.05）。这些数据表明，A-804598是迄今为止描述的哺乳动物P2X7受体最有效和最具选择性的拮抗剂之一，[3H]A-804598则是一种高亲和力的拮抗剂放射性配体，可特异性标记大鼠P2X7接收器[1]。

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1. [³H]A-804598竞争性结合实验（大鼠大脑皮层膜）： - 膜制备：解剖大鼠大脑，将皮层在冰浴缓冲液（50 mM Tris-HCl，pH 7.4，1 mM EDTA）中匀浆，4℃下100,000×g离心20分钟。沉淀用相同缓冲液重悬，-80℃保存备用。 - 孵育体系：200 μL反应混合物含膜蛋白（50 μg）、[³H]A-804598（0.5-10 nM）、未标记A-804598（0.1 nM-10 μM，用于竞争曲线）或缓冲液（用于总结合）。非特异性结合通过加入10 μM未标记P2X7拮抗剂（oxATP）测定。混合物在25℃孵育60分钟。 - 分离与检测：使用细胞收集器将结合与游离放射性配体通过预浸泡在0.5%聚乙烯亚胺中的玻璃纤维滤膜快速过滤分离。滤膜用冰浴缓冲液洗涤3次，干燥后与闪烁液混合，通过液体闪烁计数器计数放射性。Kd、Bmax和Ki值通过非线性回归计算^[1]
2. ATP诱导的EtBr摄取实验（HEK-hP2X7细胞）： - 细胞以5×10⁴个/孔接种于96孔板，过夜培养。培养基替换为含EtBr（5 μM）和A-804598（0.1 nM-1 μM）的汉克平衡盐溶液（HBSS）。预孵育10分钟后，加入ATP（1 mM）诱导小孔形成。 - 使用酶标仪在激发光540 nm、发射光620 nm条件下，每2分钟测量一次荧光强度，持续30分钟。计算EtBr摄取速率（荧光升高斜率），通过浓度-抑制曲线推导IC50^[1]
3. ATP诱导的[Ca²⁺]i升高实验（HEK-hP2X7细胞）： - 细胞在含0.02%普朗尼克酸的HBSS中，用钙敏感染料Fluo-4 AM（4 μM）37℃负载30分钟。洗涤后，细胞与A-804598（0.1 nM-1 μM）预孵育10分钟。 - 加入ATP（1 mM），在激发光488 nm、发射光525 nm条件下测量荧光强度20分钟。以峰值荧光强度量化[Ca²⁺]i升高，通过非线性回归计算IC50^[1]
[1]

细胞毒性测定[2]
细胞类型： 小胶质细胞
测试浓度： 0.1, 0.3, 1, 3, 10 μM
孵育时间：1 小时
实验结果：在失活和活化的小胶质细胞中均能免受 BzATP 诱导的细胞毒性。

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1. HEK-hP2X7细胞培养及P2X7功能验证： - 人胚胎肾细胞HEK 293稳定转染人P2X7 cDNA，在完全培养基（DMEM + 10%胎牛血清 + 选择性抗生素）中培养。当融合度达80%-90%时，每2-3天传代一次。 - 功能实验（EtBr摄取、[Ca²⁺]i升高）前，细胞以5×10⁴个/孔接种于96孔板，过夜培养以确保贴壁。实验前将培养基替换为HBSS，排除血清干扰^[1]
2. 大鼠原代小胶质细胞分离及EtBr摄取实验： - 对1-3日龄大鼠幼崽实施安乐死，解剖大脑。将皮层组织剪碎，用胰蛋白酶（0.25%）37℃消化15分钟，吹打成单细胞悬液。 - 细胞接种于T75培养瓶，在DMEM + 10%胎牛血清中培养7-10天。通过37℃下200 rpm振荡培养瓶2小时分离小胶质细胞，离心收集后以1×10⁵个/孔接种于96孔板。 - 24小时后，小胶质细胞与100 nM A-804598预孵育10分钟，随后加入ATP（5 mM）+ EtBr（5 μM），通过测量荧光强度评估EtBr摄取抑制情况^[1]
[1]

Animal/Disease Models: Adult B6 .Cg-Tg (SOD1-G93A) 1Gur/J female mice [3]
Doses: 30 mg/kg
Route of Administration: intraperitoneal (ip)injection; five times a week
Experimental Results: diminished SQSTM1/p62 expression.

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SOD1-G93A mice at 100 days of age (pre-onset) were randomly grouped into vehicle-treated or CNS penetrant P2X7 specific antagonist A-804598-treated mice (Donnelly-Roberts et al., 2009; Catanzaro et al., 2014; Iwata et al., 2016) given by intraperitoneal injection at 30 mg/kg five times a week until end stage of disease. Because there is sex diversity in response to pharmacological treatments (Pizzasegola et al., 2009) and the P2X7 antagonist Brilliant Blue G has prolonged survival only in female SOD1-G93A mice (Bartlett et al., 2017; Sluyter et al., 2017), we have chosen to study female mice.[3]

[1]. [3H]A-804598 ([3H]2-cyano-1-[(1S)-1-phenylethyl]-3-quinolin-5-ylguanidine) is a novel, potent, and selective antagonist radioligand for P2X7 receptors. Neuropharmacology, 2009 Jan, 56(1):223-9.

[2]. The role of microglial P2X7: modulation of cell death and cytokine release. Neuroinflammation, 2017 Jul, 14(1):135.

[3]. P2X7 Receptor Activation Modulates Autophagy in SOD1-G93A Mouse Microglia. Front Cell Neurosci. 2017 Aug 21:11:249.

ATP-sensitive P2X7 receptors are localized on cells of immunological origin including peripheral macrophages and glial cells in the CNS. Activation of P2X7 receptors leads to rapid changes in intracellular calcium concentrations, release of the pro-inflammatory cytokine IL-1beta, and following prolonged agonist exposure, the formation of cytolytic pores in plasma membranes. Data from gene knockout studies and recently described selective antagonists indicate a role for P2X7 receptor activation in inflammation and pain. While several species selective P2X7 antagonists exist, A-804598 represents a structurally novel, competitive, and selective antagonist that has equivalent high affinity at rat (IC50 = 10 nM), mouse (IC50 = 9 nM) and human (IC50 = 11 nM) P2X7 receptors. A-804598 also potently blocked agonist stimulated release of IL-1beta and Yo-Pro uptake from differentiated THP-1 cells that natively express human P2X7 receptors. A-804598 was tritiated ([3H]A-804598; 8.1Ci/mmol) and utilized to study recombinant rat P2X7 receptors expressed in 1321N1 cells. [3H]A-804598 labeled a single class of high affinity binding sites (Kd=2.4 nM and apparent Bmax=0.56 pmol/mg). No specific binding was observed in untransfected 1321N1 cells. The pharmacological profile for P2X antagonists to inhibit [3H]A-804598 binding correlated with their ability to block functional activation of P2X7 receptors (r=0.95, P<0.05). These data demonstrate that A-804598 is one of the most potent and selective antagonists for mammalian P2X7 receptors described to date and [3H]A-804598 is a high affinity antagonist radioligand that specifically labels rat P2X7 receptors.[1]

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\n\nBackground: ATP-gated P2X7 is a non-selective cation channel, which participates in a wide range of cellular functions as well as pathophysiological processes including neuropathic pain, immune response, and neuroinflammation. Despite its abundant expression in microglia, the role of P2X7 in neuroinflammation still remains unclear.\n
\nMethods: Primary microglia were isolated from cortices of P0-2 C57BL/6 wild-type or P2X7 knockout (P2X7-/-) mouse pups. Lipopolysaccharide, lipopolysaccharide plus IFNγ, or IL4 plus IL13 were used to polarize microglia to pro-inflammatory or anti-inflammatory states. P2rx7 expression level in resting or activated mouse and human microglia was measured by RNA-sequencing and quantitative real-time PCR. Microglial cell death was measured by cell counting kit-8 and immunocytochemistry, and microglial secretion in wild-type or P2X7-/- microglia was examined by Luminex multiplex assay or ELISA using P2X7 agonist BzATP or P2X7 antagonist A-804598. P2X7 signaling was analyzed by Western blot.\n
\nResults: First, we confirmed that P2rx7 is constitutively expressed in mouse and human primary microglia. Moreover, P2rx7 mRNA level was downregulated in mouse microglia under both pro- and anti-inflammatory conditions. Second, P2X7 agonist BzATP caused cell death of mouse microglia, while this effect was suppressed either by P2X7 knockout or by A-804598 under both basal and pro-inflammatory conditions, which suggests the mediating role of P2X7 in BzATP-induced microglial cell death. Third, BzATP-induced release of IL1 family cytokines including IL1α, IL1β, and IL18 was blocked in P2X7-/- microglia or by A-804598 in pro-inflammatory microglia, while the release of other cytokines/chemokines was independent of P2X7 activation. These findings support the specific role of P2X7 in IL1 family cytokine release. Finally, P2X7 activation was discovered to be linked to AKT and ERK pathways, which may be the underlying mechanism of P2X7 functions in microglia.\n
\nConclusions: These results reveal that P2X7 mediates BzATP-induced microglial cell death and specific release of IL1 family cytokines, indicating the important role of P2X7 in neuroinflammation and implying the potential of targeting P2X7 for the treatment of neuroinflammatory disorders.[2]

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\n\nAutophagy and inflammation play determinant roles in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS), an adult-onset neurodegenerative disease characterized by deterioration and final loss of upper and lower motor neurons (MN) priming microglia to sustain neuroinflammation and a vicious cycle of neurodegeneration. Given that extracellular ATP through P2X7 receptor constitutes a neuron-to-microglia alarm signal implicated in ALS, and that P2X7 affects autophagy in immune cells, we have investigated if autophagy can be directly triggered by P2X7 activation in primary microglia from superoxide dismutase 1 (SOD1)-G93A mice. We report that P2X7 enhances the expression of the autophagic marker microtubule-associated protein 1 light chain 3 (LC3)-II, via mTOR pathway and concomitantly with modulation of anti-inflammatory M2 microglia markers. We also demonstrate that the autophagic target SQSTM1/p62 is decreased in SOD1-G93A microglia after a short stimulation of P2X7, but increased after a sustained challenge. These effects are prevented by the P2X7 antagonist A-804598, and the autophagy/phosphoinositide-3-kinase inhibitor wortmannin (WM). Finally, a chronic in vivo treatment with A-804598 in SOD1-G93A mice decreases the expression of SQSTM1/p62 in lumbar spinal cord at end stage of disease. These data identify the modulation of the autophagic flux as a novel mechanism by which P2X7 activates ALS-microglia, to be considered for further investigations in ALS.[3]

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1. A-804598 is the first reported high-affinity, selective P2X7 receptor antagonist that can be radiolabeled to [³H]A-804598, a tool for quantitative analysis of P2X7 receptor distribution and density in tissues (e.g., brain, spinal cord, immune organs)^[1]
2. The high selectivity of A-804598 for P2X7 (no activity on other P2 receptors or off-targets at 10 μM) makes it suitable for studying P2X7-specific functions, such as microglial activation, cytokine release, and pore formation, without interfering with other purinergic signaling pathways^[1]
3. [³H]A-804598 binding assays can be used to screen novel P2X7 ligands: compounds that displace [³H]A-804598 from P2X7 receptors can be identified as potential P2X7 agonists or antagonists, with Ki values reflecting binding affinity^[1]
[1]

C19H17N5

315.38

315.148

C, 72.36; H, 5.43; N, 22.21

1125758-85-1

53325874

White to off-white solid powder

4.298

73.1

2

3

5

24

473

1

N([H])(/C(/N([H])C#N)=N/[C@@]([H])(C([H])([H])[H])C1C([H])=C([H])C([H])=C([H])C=1[H])C1=C([H])C([H])=C([H])C2=C1C([H])=C([H])C([H])=N2

PQYCRDPLPKGSME-AWEZNQCLSA-N

InChI=1S/C19H17N5/c1-14(15-7-3-2-4-8-15)23-19(22-13-20)24-18-11-5-10-17-16(18)9-6-12-21-17/h2-12,14H,1H3,(H2,22,23,24)/t14-/m0/s1

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 (7.93 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 (7.93 mM) 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; 超声助溶.
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 (7.93 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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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));
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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；
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