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Discovery of novel L-type voltage-gated calcium channel blockers and application for the prevention of inflammation and angiogenesis.
Journal of Neuroinflammation ( IF 9.3 ) Pub Date : 2020-04-25 , DOI: 10.1186/s12974-020-01801-9
Madhu Sudhana Saddala 1 , Anton Lennikov 1 , Anthony Mukwaya 2 , Yan Yang 3 , Michael A Hill 3 , Neil Lagali 2 , Hu Huang 1
Affiliation  

BACKGROUND The ways in which microglia activate and promote neovascularization (NV) are not fully understood. Recent in vivo evidence supports the theory that calcium is required for the transition of microglia from a surveillance state to an active one. The objectives of this study were to discover novel L-type voltage-gated channel (L-VGCC) blockers and investigate their application for the prevention of inflammation and angiogenesis. METHODS Pharmacophore-based computational modeling methods were used to screen for novel calcium channel blockers (CCBs) from the ZINC compound library. The effects of CCBs on calcium blockade, microglial pro-inflammatory activation, and cell toxicity were validated in BV-2 microglial cell and freshly isolated smooth muscle cell (SMC) cultures. Laser-induced choroidal neovascularization (NV) and the suture-induced inflammatory corneal NV models of angiogenesis were used for in vivo validation of the novel CCBs. CX3CR1gfp/+ mice were used to examine the infiltration of GFP-labeled microglial cells. RESULTS We identified three compounds from the ZINC database (Zinc20267861, Zinc18204217, and Zinc33254827) as new blockers of L-type voltage-gated calcium channels (L-VGCC) using a structure-based pharmacophore approach. The effects of the three CCBs on Ca2+ influx into cells were verified in BV-2 microglial cells using Fura-2 fluorescent dye and in freshly isolated SMCs using the voltage-patch clamp. All three CCBs reduced microglial cell migration, activation stimulated by lipopolysaccharide (LPS), and reduced the expression of the inflammatory markers NF-κB (phospho-IκBα) and cyclooxygenase-2 (COX-2) as well as reactive oxygen species. Of the three compounds, we further examined the in vivo activity of Zinc20267861. Topical treatment with Zinc20267861 in a rat model of suture-induced inflammatory cornea neovascularization demonstrated efficacy of the compound in reducing monocyte infiltration and overall corneal NV response. Subconjunctival administration of the compound in the choroidal NV mouse model effectively prevented CNV and microglial infiltration. CONCLUSIONS Our findings suggest that the novel CCBs identified here are effective anti-inflammatory agents that can be further evaluated for treating NV disorders and can be potentially applied in the treatment of ocular inflammatory and pathological angiogenetic disorders.

中文翻译:

新型L型电压门控钙通道阻滞剂的发现及其在预防炎症和血管生成中的应用。

背景技术小胶质细胞激活和促进​​新血管形成(NV)的方式尚不完全清楚。最新的体内证据支持以下理论:小胶质细胞从监视状态向活动状态的转变需要钙。这项研究的目的是发现新型的L型电压门控通道(L-VGCC)阻断剂,并研究其在预防炎症和血管生成中的应用。方法基于药理学的计算模型方法用于从ZINC化合物库中筛选新型钙通道阻滞剂(CCB)。在BV-2小神经胶质细胞和新鲜分离的平滑肌细胞(SMC)培养物中,验证了CCB对钙阻断,小神经胶质促炎激活和细胞毒性的影响。激光诱导的脉络膜新血管形成(NV)和缝合线诱导的血管新生炎症性角膜NV模型用于体内新型CCB的验证。使用CX3CR1gfp / +小鼠检查GFP标记的小胶质细胞的浸润。结果我们使用基于结构的药效团方法从ZINC数据库中确定了三种化合物(Zinc20267861,Zinc18204217和Zinc33254827)作为L型电压门控钙通道(L-VGCC)的新阻滞剂。在使用Fura-2荧光染料的BV​​-2小胶质细胞中以及在使用电压膜片钳的新鲜分离的SMC中,验证了这三种CCB对Ca2 +流入细胞的影响。所有这三个CCB均减少了小胶质细胞迁移,脂多糖(LPS)刺激的激活,并降低了炎症标记物NF-κB(phospho-IκBα)和环氧合酶2(COX-2)的表达以及活性氧的表达。在这三种化合物中,我们进一步检查了Zinc20267861的体内活性。在缝合线诱发的炎症性角膜新血管形成的大鼠模型中用Zinc20267861进行局部治疗证明了该化合物在减少单核细胞浸润和总体角膜NV反应中的功效。在脉络膜NV小鼠模型中结膜下给予该化合物可有效防止CNV和小胶质细胞浸润。结论我们的发现表明,此处鉴定的新型CCB是有效的抗炎药,可以进一步评估其用于治疗NV疾病,并且可以潜在地用于治疗眼部炎症和病理性血管生成疾病。
更新日期:2020-04-25
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