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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：
10478241
负责人：
Kari N Nejak-Bowen
金额：
$ 43.81万
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/10478241
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 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.

卟啉症是一组由血红素生物合成途径缺陷引起的代谢紊乱。而肝脏通常是过量卟啉的来源或靶标。治疗大部分脑部疾病门静脉症是有限的，通常侧重于症状缓解和姑息治疗。因此，基于机械论的强调治疗发展的研究是迫切需要的。以前的工作已经确定了 Wnt/b-catenin信号通路在3，5-二乙氧基-1，4-二氢碰撞碱中的调控作用 (DDC)饮食诱导的小鼠卟啉中毒模型。抑制b-连环蛋白在DDC所致的小鼠中导致由于减少了卟啉蓄积，显著减少了肝脏损伤。我们确定了一个关键的网络血红素生物合成酶，如δ-氨基乙酰丙酸(ALA)合成酶和ALA-脱水酶在没有b-连环蛋白的情况下被抑制，导致卟啉中间体和 DDC相关蛋白聚集。缺乏b-连环蛋白的小鼠的自噬也会增加，这可能会进一步有助于保护身体免受伤害。因此，该提案的首要假设是抑制临床相关的门静脉高压症模型中的WNT/b-连环蛋白信号转导通路将减轻门静脉高压症的损伤和进展通过减少卟啉中间体的产生和/或增加自噬而引起的疾病。在目标1中，我们将通过治疗确定受b-连环蛋白抑制影响的通路中最近的一步。肝细胞与丙氨酸结合，检测卟啉中间体的出现；探讨其作用机制在计算机研究和染色质研究中，b-连环蛋白通过定点突变调节血红素酶。免疫沉淀；并证明靶向β-连环蛋白在患者中的治疗意义免疫组织化学方法检测b-连环蛋白表达程度与血红素酶表达的关系。在……里面目的研究Wnt/b-catenin抑制后自噬在卟啉症中的作用和调控。我们将使用RFP-EGFP-LC3小鼠，一个pH依赖的荧光报告菌株，和一个基因敲除谷氨酰胺合成酶，b-连环蛋白/mTOR途径的一个组成部分，以及体外检测，以全面阐述这一细胞过程对观察到的受保护表型的贡献。在AIM 3，我们将确定在遗传性卟啉症小鼠模型中抑制b-连环蛋白是否会减少卟啉。蓄积，改善肝脏病理。两种典型的卟啉症模型：铁络合酶 (Fechm1Pas)突变小鼠，模仿具有显著肝脏的人红细胞生成性原卟啉以及T1/T2小鼠，它是羟甲基丁烷合成酶和羟甲基丁烷合成酶的复合杂合子。在苯巴比妥刺激下模拟急性间歇性卟啉症，将被用于确定 B-连环蛋白抑制剂的治疗干预是否可以防止进展或提供保护在急性发作期间。因此，拟议的研究将进一步加深我们对抑制的潜力的理解。 B-连环蛋白治疗卟啉症，将为治疗和翻译使用提供非常有意义的信息。