Off target mechanisms of kinase inhibitor toxicities

激酶抑制剂毒性的脱靶机制

• 批准号: 10584567
• 负责人: Amandeep Bajwa
• 金额: $ 50.44万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2026-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10584567
• 关键词: Acute Renal Failure with Renal Papillary Necrosis; Address; Albumins; Binding; Binding Sites; Biological Assay; Biology; CRISPR screen; Cell Death; Cell Line; Cell physiology; Cells; Chemicals; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Data; Development; Disease; Drug toxicity; Endocytosis; Enzymes; Epithelial Cells; FDA approved; Female; Gender; Genes; Genetic; Genetic Transcription; Heme; Human; In Vitro; Inflammatory; Injury to Kidney; Kidney; Knock-in; Knock-out; Knockout Mice; Knowledge; Link; Malignant Neoplasms; Mediating; Mitochondria; Mitochondrial Proteins; Molecular; Mus; Mutation; Normal Cell; Normal tissue morphology; Oncology; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacologic Actions; Phosphotransferases; Prevalence; Protein Kinase; Proteins; Proteomics; Proto-Oncogene Proteins B-raf; RNA Interference; RNA interference screen; RUNX1 gene; Resistance; Role; Signal Transduction; Signal Transduction Pathway; Testing; Therapeutic; Therapeutic Intervention; Tissues; Toxic effect; Tubular formation; Up-Regulation; Work; cancer therapy; chemical genetics; conditional knockout; enzyme biosynthesis; ferrochelatase; functional genomics; gender difference; genome-wide; heme biosynthesis; human disease; in vivo; insight; interdisciplinary approach; intrinsic factor-cobalamin receptor; kinase inhibitor; knock-down; knockout gene; male; mitochondrial dysfunction; mouse model; nephrotoxicity; protein kinase inhibitor; response; screening; small molecule; small molecule therapeutics; targeted treatment; transcription factor; transport inhibitor; tumorigenesis; uptake

ABSTRACT Protein kinases orchestrate signal transduction pathways that are essential for most normal cellular functions. Importantly, dysregulation of protein kinase signaling is both a cause and consequence of several human diseases, especially cancer. Due to their druggability, more than seventy small-molecule therapeutics that block the ATP-binding site of kinases have been approved, mostly for oncological diseases. Since the ATP- binding site is relatively conserved, most targeted therapeutics can simultaneously inhibit on- as well as off- target kinases, impacting efficacy as well as toxicity. Remarkably, chemical proteomic profiling has now revealed that along with off-target kinases, protein kinase inhibitors can also bind and inhibit non-kinase proteins. Particularly, the mitochondria-localized heme biosynthesis enzyme, ferrochelatase (FECH) has emerged as a common target which can be inhibited by more than 10% of kinase inhibitors. How FECH inhibition influences drug responses, especially toxicities, nevertheless remains unknown. Given their widespread clinical use, there is a clear unmet need to understand the mechanistic basis of kinase inhibitor toxicities, especially related to inhibition of non-kinase proteins. In this regard, our recent study has provided the first evidence that off-target FECH inhibition by BRAF-kinase inhibitor vemurafenib contributes to renal tubular epithelial cell (RTEC) death in vitro and nephrotoxicity in vivo. However, there is a knowledge gap in our understanding of how kinase inhibitors are transported into normal cells, how the subsequent FECH inhibition drives mitochondrial dysfunction, and how these pathways are differentially regulated in males versus females. To address these questions, we have performed genome-wide RNAi and CRISPR based screens, which have provided two key insights: (i) we have identified putative mechanisms responsible for vemurafenib uptake, FECH inhibition, mitochondrial dysfunction, and RTEC cell death. (ii) We have uncovered a unique gender-specific difference in toxicity, wherein vemurafenib treatment induces a female specific FECH upregulation in RTECs imparting resistance to nephrotoxicity. In the current application we propose to utilize a suite of in vitro and in vivo chemical genetic and gene knockout approaches to further illuminate the regulatory mechanisms that govern toxicities associated with vemurafenib-induced FECH inhibition. Using this approach, in Aim 1 we will employ RTEC-specific conditional knockout mice, primary cells, and CRISPR-based knockout cell lines to examine the role of cubilin-dependent endocytosis in vemurafenib uptake, FECH inhibition, and mitochondrial dysfunction. In Aim 2 we will utilize conditional knockout mice and primary cells to examine the role of RUNX1 in female specific FECH upregulation and resistance to vemurafenib nephrotoxicity. These studies are expected to provide broad insights into the pharmacological action of kinase inhibitors in normal tissues including cellular uptake, non-kinase target inhibition, mitochondrial dysfunction, and transcriptional mechanisms underlying gender differences in toxicities.

抽象的 蛋白激酶还编排对大多数正常细胞功能必不可少的信号转导途径。 重要的是，蛋白激酶信号传导的失调既是几个人的原因和结果 疾病，尤其是癌症。由于它们的可毒性，超过70种小分子治疗剂 阻止激酶的ATP结合部位已被批准，主要用于肿瘤疾病。由于ATP- 结合位点相对保守，大多数靶向疗法可以同时抑制和外 靶向激酶，影响功效和毒性。值得注意的是，化学蛋白质组学分析现已 揭示了蛋白激酶抑制剂与脱靶激酶一起，也可以结合并抑制非激酶 蛋白质。特别是，线粒体 - 定位的血红素生物合成酶，铁螯合酶（FECH）具有 出现是一个共同的靶标，可以被超过10％的激酶抑制剂抑制。多么狂热 抑制会影响药物反应，尤其是毒性，但仍未知。鉴于他们 广泛的临床用途，明显的未满足需要了解激酶抑制剂的机理基础 毒性，特别是与非激酶蛋白抑制有关的毒性。在这方面，我们最近的研究提供了 BRAF-激酶抑制剂vemurafenib抑制脱靶FECH抑制的第一个证据有助于肾脏 体内的肾小管上皮细胞（RTEC）死亡。但是，有一个知识差距 我们对激酶抑制剂如何转运到正常细胞的理解，随后的FECH 抑制作用驱动线粒体功能障碍，以及这些途径在男性与男性的差异调节 女性。为了解决这些问题，我们进行了全基因组RNAi和基于CRISPR的屏幕， 提供了两个关键见解：（i）我们已经确定了负责Vemurafenib的假定机制 摄取，FECH抑制，线粒体功能障碍和RTEC细胞死亡。 （ii）我们发现了一个独特的 性别特定的毒性差异，其中vemurafenib治疗诱导了女性特异性FECH RTEC的上调，对肾毒性具有抗性。在当前的应用程序中，我们建议使用 体外和体内化学遗传和基因敲除方法进一步阐明调节的方法 控制与维美富尼诱导的FECH抑制相关的毒性的机制。使用这种方法， 在AIM 1中，我们将采用RTEC特异性的条件敲除小鼠，原代细胞和基于CRISPR的淘汰 细胞系检查Cubilin依赖性内吞作用在Vemurafenib摄取，FECH抑制和 线粒体功能障碍。在AIM 2中，我们将利用条件敲除小鼠和原代细胞检查 runx1在女性特异性fech上调和对维美富尼肾毒性的抗性中的作用。这些 预计研究将为激酶抑制剂在正常情况下的药理作用提供广泛的见解 包括细胞摄取，非激酶靶标抑制，线粒体功能障碍和转录的组织 毒性性别差异的基础机制。