Orai1 is a plasma membrane Ca²⁺ channel involved in store operated calcium entry (SOCE). SOCE can regulate cell growth, exocytosis, gene expression and inflammation. We previously found that short palate lung and nasal epithelial clone 1's (SPLUNC1) sixth α-helix (α6) bound Orai1 to inhibit SOCE. SPLUNC1 was not proteolytically stable, so we developed ELD607, an 11 amino acid peptide based on SPLUNC1's α6 region which was more stable and more potent than SPLUNC1/α6. Here, we studied ELD607's mechanism of action. We overexpressed either Orai1-HA or Orai1-YFP in HEK293T cells to probe ELD607-Orai1 interactions by confocal microscopy. We also measured changes in Fluo-4 fluorescence in a multiplate reader as a marker of cytoplasmic Ca²⁺ levels. ELD607 internalized Orai1 independently of STIM1. Both 15 min and 3 h exposure to ELD607 similarly depleted Orai1 in the plasma membrane. However, 3 h exposure to ELD607 yielded greater inhibition of SOCE. ELD607 continued to colocalize with Orai1 after internalization and this process was dependent on the presence of the ubiquitin ligase NEDD4.2. Similarly, ELD607 increased the colocalization between Orai1 and ubiquitin. ELD607 also increased the colocalization between Orai1 and Rab5 and 7, but not Rab11, suggesting that Orai1 trafficked through early and late but not recycling endosomes. Finally, ELD607 caused Orai1, but not Orai2, Orai3, or STIM1 to traffic to lysosomes. We conclude that ELD607 rapidly binds to Orai1 and works in an identical fashion as full length SPLUNC1 by internalizing Orai1 and sending it to lysosomes, leading to a decrease in SOCE.

2024-03-15·American journal of respiratory and critical care medicine

Specific Inhibition of Orai1-mediated Calcium Signalling Resolves Inflammation and Clears Bacteria in an Acute Respiratory Distress Syndrome Model

Article

作者: Ahmad, Saira ; Greenbaum, Alon ; Wrennall, Joe A. ; Tarran, Robert ; Sekhri, Malika ; Moran, Timothy P. ; Mahida, Rahul Y. ; Abdelwahab, Sabri H. ; Goriounova, Alexandra S. ; Iskarpatyoti, Jason A. ; Krajewski, Krzysztof ; Ghosh, Arunava ; Ignar, Diane M. ; Rai, Mani ; Thickett, David R. ; Sassano, M. Flori ; Voeller, Alexis ; Tilley, Stephen L.

Rationale: Acute respiratory distress syndrome (ARDS) has an unacceptably high mortality rate (35%) and is without effective therapy. Orai1 is a Ca²⁺ channel involved in store-operated Ca²⁺ entry (SOCE), a process that exquisitely regulates inflammation. Orai1 is considered a druggable target, but no Orai1-specific inhibitors exist to date. Objectives: To evaluate whether ELD607, a first-in-class Orai1 antagonist, can treat ARDS caused by bacterial pneumonia in preclinical models. Methods: ELD607 pharmacology was evaluated in HEK293T cells and freshly isolated immune cells from patients with ARDS. A murine acute lung injury model caused by bacterial pneumonia was then used: mice were infected with Pseudomonas aeruginosa, Staphylococcus aureus, methicillin-resistant S. aureus, or multidrug-resistant P. aeruginosa and then treated with ELD607 intranasally. Measurements and Main Results: ELD607 specifically inhibited SOCE in HEK293T cells with a half-maximal inhibitory concentration of 9 nM. ELD607 was stable in ARDS airway secretions and inhibited SOCE in ARDS immune cells. In vivo, inhaled ELD607 significantly reduced neutrophilia and improved survival. Surprisingly, Orai1 inhibition by ELD607 caused a significant reduction in lung bacteria, including methicillin-resistant S. aureus. ELD607 worked as an immunomodulator that reduced cytokine levels, reduced neutrophilia, and promoted macrophage-mediated resolution of inflammation and clearance of bacteria. Indeed, when alveolar macrophages were depleted with inhaled clodronate, ELD607 was no longer able to resolve inflammation or clear bacteria. Conclusions: These data indicate that specific Orai1 inhibition by ELD607 may be a novel approach to reduce multiorgan inflammation and treat antibiotic-resistant bacteria.

2015-07-15·American journal of physiology. Lung cellular and molecular physiology2区 · 医学

Automated acquisition and analysis of airway surface liquid height by confocal microscopy

2区 · 医学

Article

作者: Kim, Christine Seul Ki ; Tarran, Robert ; Choi, Hyun-Chul

The airway surface liquid (ASL) is a thin-liquid layer that lines the luminal side of airway epithelia. ASL contains many molecules that are involved in primary innate defense in the lung. Measurement of ASL height on primary airway cultures by confocal microscopy is a powerful tool that has enabled researchers to study ASL physiology and pharmacology. Previously, ASL image acquisition and analysis were performed manually. However, this process is time and labor intensive. To increase the throughput, we have developed an automatic ASL measurement technique that combines a fully automated confocal microscope with novel automatic image analysis software that was written with image processing techniques derived from the computer science field. We were able to acquire XZ ASL images at the rate of ∼1 image/s in a reproducible fashion. Our automatic analysis software was able to analyze images at the rate of ∼32 ms/image. As proofs of concept, we generated a time course for ASL absorption and a dose response in the presence of SPLUNC1, a known epithelial sodium channel inhibitor, on human bronchial epithelial cultures. Using this approach, we determined the IC50 for SPLUNC1 to be 6.53 μM. Furthermore, our technique successfully detected a difference in ASL height between normal and cystic fibrosis (CF) human bronchial epithelial cultures and detected changes in ATP-stimulated Cl−/ASL secretion. We conclude that our automatic ASL measurement technique can be applied for repeated ASL height measurements with high accuracy and consistency and increased throughput.

项与 ELD-607 相关的新闻（医药）

2023-12-23

·今日头条

新思路！中南湘雅学者发现鼻咽癌最新免疫治疗策略

本文为转化医学网原创，转载请注明出处作者：Rainbow 导读：将巨噬细胞重编程为M1表型是诱导抗癌免疫的一种新策略。 12月21日，中南大学湘雅医院研究团队在期刊《Cell Death & Disease》上在线发表题为“USP7 inhibits the progression of nasopharyngeal carcinoma via promoting SPLUNC1-mediated M1 macrophage polarization through TRIM24”的研究论文，研究表明， USP7通过去泛素化和稳定TRIM24来重编程巨噬细胞为M1表型，从而增强SPLUNC1的表达，最终抑制了鼻咽癌的生长和转移。研究表明，针对USP7/TRIM24/SPLUNC1级联可能抑制免疫抑制作用，代表了一种有效的鼻咽癌免疫治疗策略。研究发现极大地贡献了对USP7在调控鼻咽癌进展机制和功能的理解。因此，研究结果为鼻咽癌的治疗提供了一种新颖的治疗思路，有望极大改善患者的预后。 https://www.nature.com/articles/s41419-023-06368-w 研究背景 01 鼻咽癌（NPC）是发生在鼻咽部的常见恶性肿瘤，占所有头颈肿瘤的约78％。目前，NPC的主要治疗方法是手术切除后联合放疗和化疗，然而，对于出现转移的NPC患者预后并不理想。因此，迫切需要阐明NPC细胞转移的机制并找到新的干预措施。巨噬细胞，在癌症中也被称为肿瘤相关巨噬细胞，通常表现为M2表型，并促进肿瘤的发展。而M1表型巨噬细胞则产生促炎症Th1免疫反应并具有杀伤肿瘤的活性。肿瘤相关巨噬细胞存在于NPC的肿瘤微环境中，并已报道与NPC患者的预后有关。然而，在NPC进展过程中，巨噬细胞极化的调节机制尚未得到阐明。短腭、肺和鼻上皮克隆1（SPLUNC1）是在鼻咽部表达的PLUNC家族成员，具有宿主防御性质。已经证明SPLUNC1能够保护免受革兰氏阴性细菌引起的呼吸道炎症。到目前为止，SPLUNC1是否能通过调节巨噬细胞极化来延缓NPC进展仍不清楚。泛素特异性加工酶7（USP7）是一个关键的去泛素化酶。已经有文献证实，USP7通过调节特定蛋白质的稳定性和功能，在肿瘤发展中发挥着重要作用。通过生物信息学分析，研究人员发现USP7具有去泛素化和稳定TRIM24的潜力。然而，USP7是否可以通过去泛素化和稳定TRIM24来影响NPC的进展，并随后调节SPLUNC1介导的M1型巨噬细胞极化，仍然不清楚。 SPLUNC1诱导的M1型巨噬细胞极化抑制了鼻咽癌细胞的生长和迁移 02 为了确定SPLUNC1介导的M1型巨噬细胞极化是否发挥了抗肿瘤作用，将鼻咽癌细胞（NPC/HK-1和CNE3）与THP-1源Mφs的培养基混合。与对照组相比，使用该培养基处理抑制了NPC细胞的生长，而在THP-1源Mφs中敲除SPLUNC1则破坏了这一结果。培养基的添加抑制了NPC细胞的迁移，而SPLUNC1沉默则对此产生了相反的效应。相反，使用SPLUNC1过表达的THP-1源Mφs的培养基进一步加强了THP-1源Mφs培养基对NPC细胞增殖和迁移的抑制作用。这些发现表明，在体外SPLUNC1诱导的M1型巨噬细胞极化抑制了NPC细胞的生长和转移。研究结果 03 综上所述，研究表明， USP7通过去泛素化和稳定TRIM24来重编程巨噬细胞为M1表型，从而增强SPLUNC1的表达，最终抑制了鼻咽癌的生长和转移。研究表明，针对USP7/TRIM24/SPLUNC1级联可能抑制免疫抑制作用，代表了一种有效的鼻咽癌免疫治疗策略。研究发现极大地贡献了对USP7在调控鼻咽癌进展机制和功能的理解。因此，研究结果为鼻咽癌的治疗提供了一种新颖的治疗思路，有望极大改善患者的预后。参考资料： https://www.nature.com/articles/s41419-023-06368-w 注：本文旨在介绍医学研究进展，不能作为治疗方案参考。如需获得健康指导，请至正规医院就诊。热门·直播/活动 🕓 北京｜12月19日-20日 ▶第四届单细胞测序技术应用研讨会暨单细胞&空间组学研讨会（日程稍后公布）点击对应文字查看详情

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适应症	最高研发状态	国家/地区	公司	日期
肠杆菌感染	临床前	美国	Eldec Pharmaceuticals, Inc.	-
粪肠球菌感染	临床前	美国	Eldec Pharmaceuticals, Inc.	-
碳青霉烯类耐药鲍曼不动杆菌感染	临床前	美国	Eldec Pharmaceuticals, Inc.	-
细菌性肺炎	临床前	美国	Eldec Pharmaceuticals, Inc.	-
假单胞菌感染	临床前	美国	Eldec Pharmaceuticals, Inc.	-
葡萄球菌感染	临床前	美国	Eldec Pharmaceuticals, Inc.	-