Beta-catenin inhibition as a novel therapeutic strategy for porphyria

β-连环蛋白抑制作为卟啉症的新型治疗策略

基本信息

批准号：
10264151
负责人：
Kari N Nejak-Bowen
金额：
$ 43.52万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-15 至 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10264151
关键词：
Acute Acute Intermittent Porphyria Address Affect Aminolevulinic Acid Appearance Autophagocytosis Cell physiology ChIP-seq Chemicals Clinical Defect Deferoxamine Deposition Diet Disease Disease Progression Enzymes Erythropoietic Protoporphyria Event Extrahepatic FRAP1 gene Gene Expression Genes Genetic Genetic Models Glutamate-Ammonia Ligase Glutamine Heme Hemin Hepatic Porphyrias Hepatobiliary Hepatocyte Human Hydroxymethylbilane Synthase Immunohistochemistry In Vitro Injury Knock-out Knockout Mice Ligase Liver Measures Medical Metabolic Diseases Modeling Mus Mutant Strains Mice Oxygenases Palliative Care Pathology Pathway interactions Patients Phenobarbital Phenotype Phototoxicity Porphobilinogen Synthase Porphyrias Porphyrins Production Protoporphyrins Rare Diseases Regulation Reporter Role Severities Signal Pathway Signal Transduction Site-Directed Mutagenesis Source Testing Tetrapyrroles Therapeutic Therapeutic Intervention Time Treatment Efficacy Validation WNT Signaling Pathway Work base beta catenin chromatin immunoprecipitation clinically relevant drug metabolism efficacy evaluation enzyme biosynthesis enzyme pathway ferrochelatase heme biosynthesis improved improved functioning in silico in vitro Assay inhibitor/antagonist liquid chromatography mass spectrometry liver injury mouse model novel novel therapeutic intervention patient subsets prevent promoter protein aggregation standard of care therapeutic development

项目摘要

The porphyrias are a group of metabolic disorders that are caused by defects in heme biosynthesis pathway enzymes, and liver is commonly either a source or target of excess porphyrins. Treatment for most of the porphyrias is limited, and often focused on symptomatic relief and palliative care. Thus, mechanistic-based studies that emphasize therapeutic development are desperately needed. Previous work has identified the Wnt/b-catenin signaling pathway as a modulatable target in the 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet-induced mouse model of porphyria. Inhibition of b-catenin in mice subjected to DDC results in a significant reduction in liver injury due to decreased porphyrin accumulation. We identified a network of key heme biosynthesis enzymes such as δ-aminolevulinic acid (ALA) synthetase and ALA-dehydratase that are suppressed in the absence of b-catenin, resulting in decreased production of porphyrin intermediates and DDC-associated protein aggregation. Autophagy is also increased in mice lacking b-catenin, which may further contribute to protection from injury. Thus, the overarching hypothesis of the proposal is that inhibiting Wnt/b-catenin signaling in clinically-relevant models of porphyria will alleviate injury and progression of disease through decreased production of porphyrin intermediates and/or increased autophagy. In aim 1, we will determine the most proximal step in the pathway that is affected by b-catenin inhibition by treating hepatocytes with ALA and measuring the appearance of porphyrin intermediates; investigate the mechanism by which b-catenin regulates heme enzymes through site-directed mutagenesis, in silico studies, and chromatin immunoprecipitation; and demonstrate therapeutic relevance of targeting β-catenin in patients by utilizing immunohistochemistry to correlate the extent of b-catenin expression with expression of heme enzymes. In aim 2, we will characterize the role and regulation of autophagy in porphyria after Wnt/b-catenin inhibition. We will use RFP-EGFP-LC3 mice, a pH-dependent fluorescent reporter strain, and a genetic knockout of glutamine synthetase, a component of the b-catenin/mTOR pathway, as well as in vitro assays, to comprehensively address the contribution of this cellular process to the observed protected phenotype. In aim 3, we will determine whether inhibiting b-catenin in genetic mouse models of porphyria decreases porphyrin accumulation and improves liver pathology. Two well-characterized porphyria models: the ferrochelatase (Fechm1Pas) mutant mice, which mimics human erythropoietic protoporphyria with significant liver involvement; and the T1/T2 mouse, which is compound heterozygous for hydroxymethylbilane synthase and mimics acute intermittent porphyria upon stimulation with phenobarbitol, will be utilized to determine whether therapeutic intervention with a b-catenin inhibitor can prevent progression or provide protection during acute attacks. Thus, the proposed studies will further our understanding of the potential for inhibiting b-catenin to treat porphyria, & will provide highly significant information for therapeutic and translational use.

卟啉症是由血红素生物合成途径缺陷引起的一组代谢性疾病酶，肝脏通常是多余的卟啉的来源或靶标。大多数卟啉症是有限的，通常专注于症状缓解和姑息治疗。那是基于机械的迫切需要强调治疗发展的研究。以前的工作已经确定了 Wnt/B-catenin信号通路作为3,5-二氧基碳酸1,4-二氢核苷的模块化靶标（DDC）饮食诱导的卟啉症小鼠模型。在受到DDC的小鼠中抑制B-catenin导致A 由于卟啉积累降低而导致的肝损伤显着减少。我们确定了一个密钥网络血红素生物合成酶，例如δ-氨基乙酸（ALA）合成酶和ALA-脱水酶是在不存在B-catenin的情况下被抑制，导致卟啉中间体的产生降低和 DDC相关的蛋白质聚集。缺乏B-catenin的小鼠也增加了自噬，这可能会进一步有助于保护伤害。那就是提案的总体假设是抑制 Wnt/b-catenin信号传导在临床上与卟啉症模型相关的模型将减轻伤害和进展通过减少卟啉中间体的产生和/或自噬增加的疾病。在AIM 1中，我们将确定途径中最近端的步骤，该步骤受到B-catenin抑制的影响带有ALA的肝细胞并测量卟啉中间体的外观；研究机制 B-catenin通过位置定向的诱变，在硅酸盐研究和染色质中调节血红素酶免疫沉淀；并通过利用患者在患者中靶向β-catenin的热相关性免疫组织化学将B-catenin表达的程度与血红素酶的表达相关。 AIM 2，我们将表征Wnt/B-catenin抑制后自噬的作用和调节。我们将使用RFP-EGFP-LC3小鼠，pH依赖性的荧光报告菌株和遗传基因敲除谷氨酰胺合成酶，B-catenin/mTOR途径的成分以及体外测定总体上解决了该细胞过程对观察到的保护表型的贡献。目标 3，我们将确定在卟啉症遗传小鼠模型中抑制B-catenin是否会降低卟啉积累并改善肝脏病理。两个特征良好的卟啉症模型：铁螯合酶（FECHM1PAS）突变小鼠，这些突变小鼠模仿人类红细胞原生质具有明显的肝脏参与；和T1/T2小鼠，该小鼠是羟甲基合酶的复合杂合子和用苯巴略酚刺激后，模仿急性间歇性斑岩，将用于确定使用B-catenin抑制剂的热干预是否可以防止进展或提供保护在急性攻击中。这是拟议的研究将进一步了解我们对抑制潜力的理解 B-catenin治疗斑岩，并将为治疗和翻译使用提供非常重要的信息。