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.

摘要 蛋白激酶协调大多数正常细胞功能所必需的信号转导通路。 重要是，蛋白激酶信号的失调是几种人类疾病的原因和结果 疾病，尤其是癌症。由于它们的可药性，70多种小分子疗法 阻断激酶的ATP结合位点已被批准，主要用于肿瘤疾病。因为ATP- 结合部位相对保守，大多数靶向治疗药物可以同时抑制ON-以及OFF- 靶向激酶，影响疗效和毒性。值得注意的是，化学蛋白质组学现在已经 研究发现，与靶外蛋白激酶一样，蛋白激酶抑制剂也可以结合和抑制非蛋白激酶 蛋白质。尤其是线粒体定位的血红素生物合成酶，铁络合酶(FECH) 成为一个常见的靶点，可以被10%以上的激酶抑制剂抑制。FECH如何 抑制会影响药物的反应，特别是毒性，但仍不清楚。鉴于他们的 广泛的临床应用，还有一个明显的未得到满足的需要，以了解激酶抑制剂的机制基础 毒性，特别是与抑制非激酶蛋白有关的毒性。在这方面，我们最近的研究提供了 BRAF-激酶抑制剂维莫拉非尼非靶向抑制FECH对肾脏影响的首次证据 肾小管上皮细胞(RTEC)体外死亡和体内肾毒性。然而，在以下方面存在着知识差距 我们对激酶抑制剂如何转运到正常细胞的理解，以及随后的FECH是如何 抑制导致线粒体功能障碍，以及这些通路在男性和男性中是如何差异调节的 女性。为了解决这些问题，我们进行了基于全基因组RNAi和CRISPR的筛选， 它们提供了两个关键见解：(1)我们确定了维莫拉非尼的推定机制 摄取、FECH抑制、线粒体功能障碍和RTEC细胞死亡。(Ii)我们发现了一种独特的 不同性别的毒性差异，其中维莫拉非尼治疗导致女性特异性FECH RTECs表达上调使其对肾毒性具有抵抗力。在当前应用程序中，我们建议使用 一套体外和体内化学遗传和基因敲除方法进一步阐明了 控制与维莫拉非尼诱导的FECH抑制相关的毒性的机制。使用这种方法， 在目标1中，我们将使用RTEC特异性条件性基因敲除小鼠、原代细胞和基于CRISPR的基因敲除 检测Cubilin依赖的内吞作用在维莫拉非尼摄取、FECH抑制和 线粒体功能障碍。在目标2中，我们将利用条件基因敲除小鼠和原代细胞来检查 RUNX1在女性特异性FECH上调和抵抗维莫拉非尼肾毒性中的作用这些 研究有望为正常人群中激酶抑制剂的药理作用提供更广泛的见解。 组织包括细胞摄取、非激酶靶向抑制、线粒体功能障碍和转录 毒性的性别差异的潜在机制。