Identification of a Selective Pharmacologic IRE1/XBP1s Activator with Enhanced Tissue Exposure

Activation of the IRE1/XBP1s signaling arm of the unfolded protein response (UPR) has emerged as a promising strategy to mitigate etiologically-diverse diseases. Despite this promise, few compounds are available to selectively activate IRE1/XBP1s signaling to probe the biologic and therapeutic implications of this pathway in human disease. Recently, we identified the compound IXA4 as a highly-selective activator of protective IRE1/XBP1s signaling. While IXA4 has proven useful for increasing IRE1/XBP1s signaling in cultured cells and mouse liver, the utility of this compound is restricted by its limited activity in other tissues. To broaden our ability to pharmacologically interrogate the impact of IRE1/XBP1s signaling in vivo, we sought to identify IRE1/XBP1s activators with greater tissue activity than IXA4. We re-analyzed 'hits' from the high throughput screen used to identify IXA4, selecting compounds from structural classes not previously pursued. We then performed global RNAseq to confirm that these compounds showed transcriptome-wide selectivity for IRE1/XBP1s activation. Functional profiling revealed compound IXA62 as a selective IRE1/XBP1s activator that reduced A? secretion from CHO7PA2 cells and enhanced glucose-stimulated insulin secretion from rat insulinoma cells, mimicking the effects of IXA4 in these assays. IXA62 robustly and selectively activated IRE1/XBP1s signaling in the liver of mice dosed compound intraperitoneally or orally. In treated mice, IXA62 showed broader tissue activity, relative to IXA4, inducing expression of IRE1/XBP1s target genes in additional tissues such as kidney and lung. Collectively, our results designate IXA62 as a selective IRE1/XBP1s signaling activating compound with enhanced tissue activity, which increases our ability to pharmacologically probe the biologic significance and potential therapeutic utility of enhancing adaptive IRE1/XBP1s signaling in vivo. Overall design: RNA-seq profilling from liver or kidney of mice IP administered vehicle or IXA62 (50 mpk) daily for 7 days. RNA-seq profilling from HEK293 treated for 6 h with the indicated IRE1/XBP1s activating compound (10 µM) or thapsigargin (Tg; 0.5 µM).

未折叠蛋白反应（unfolded protein response, UPR）的IRE1/XBP1s信号通路分支的激活，已成为缓解多种病因异质性疾病的极具潜力的策略。尽管具备此应用潜力，但目前可用于选择性激活IRE1/XBP1s信号通路、以探究该通路在人类疾病中的生物学与治疗意义的化合物寥寥无几。近期，我们鉴定出化合物IXA4作为保护性IRE1/XBP1s信号通路的高选择性激活剂。尽管IXA4已被证实可在体外培养细胞与小鼠肝脏中增强IRE1/XBP1s信号通路的活性，但该化合物的应用受到其在其他组织中活性有限的限制。为拓展我们通过药理学手段在体内探究IRE1/XBP1s信号通路影响的能力，我们旨在筛选出相较于IXA4具有更强组织活性的IRE1/XBP1s激活剂。我们重新分析了用于鉴定IXA4的高通量筛选所得的“命中化合物”，从此前未被探索的结构类别中筛选候选化合物。随后我们开展了全局RNA测序（RNA-seq）实验，以证实这些化合物在转录组层面对IRE1/XBP1s激活具有选择性。功能表征实验显示，化合物IXA62是一种选择性IRE1/XBP1s激活剂，可减少CHO7PA2细胞的β淀粉样蛋白（Aβ）分泌，并增强大鼠胰岛素瘤细胞的葡萄糖刺激胰岛素分泌，在上述实验中重现了IXA4的作用效果。IXA62在经腹腔注射或口服给药的小鼠肝脏中可稳健且选择性地激活IRE1/XBP1s信号通路。与IXA4相比，经IXA62处理的小鼠展现出更广泛的组织活性，可在肾脏、肺脏等额外组织中诱导IRE1/XBP1s靶基因的表达。综上，我们的研究结果将IXA62定为一种具备增强组织活性的选择性IRE1/XBP1s信号通路激活化合物，这提升了我们通过药理学手段在体内探究增强适应性IRE1/XBP1s信号通路的生物学意义与潜在治疗价值的能力。整体实验设计：对每日腹腔注射赋形剂或IXA62（50 mg/kg）持续7天的小鼠的肝脏与肾脏开展RNA测序分析；对经指定IRE1/XBP1s激活化合物（10 μM）或毒胡萝卜素（thapsigargin, Tg；0.5 μM）处理6小时的HEK293细胞进行RNA测序分析。