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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：
10119767
负责人：
Kari N Nejak-Bowen
金额：
$ 46.21万
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/10119767
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 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 therapeutics 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.

卟啉症是由于血红素生物合成途径的缺陷而引起的一类代谢性疾病酶，肝脏通常是过量卟啉的来源或目标。治疗大多数卟啉症是有限的，往往侧重于症状缓解和姑息治疗。因此，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/β-catenin信号通路作为3，5-二乙氧羰基-1，4-二氢可力丁的可调控靶点 (DDC)饮食诱导的小鼠卟啉病模型。在DDC小鼠中抑制b-连环蛋白导致由于卟啉积累减少，肝损伤显著减少。我们发现了一个关键网络血红素生物合成酶如δ-氨基乙酰丙酸（ALA）合成酶和ALA-乙酰丙酸酶，在缺乏β-连环蛋白的情况下受到抑制，导致卟啉中间体的产生减少， DDC相关蛋白聚集。自噬在缺乏b-连环蛋白的小鼠中也增加，这可能进一步有助于保护免受伤害。因此，该提案的首要假设是，在卟啉症的临床相关模型中，Wnt/β-连环蛋白信号传导将减轻损伤和进展，疾病通过减少卟啉中间体的产生和/或增加自噬。在目标1中，我们将确定通路中受β-连环蛋白抑制影响的最近步骤肝细胞与ALA和测量卟啉中间体的外观;研究机制 β-连环蛋白通过定点诱变、计算机模拟研究和染色质免疫沉淀;并通过利用靶向β-连环蛋白来证明患者中的治疗相关性免疫组化以将b-连环蛋白表达的程度与血红素酶的表达相关联。在目的2：研究Wnt/β-catenin抑制后自噬在卟啉病中的作用和调控。我们将使用RFP-EGFP-LC 3小鼠，一种pH依赖性荧光报告菌株，和一种基因敲除的谷氨酰胺合成酶，β-连环蛋白/mTOR途径的组分，以及体外测定，全面解决这一细胞过程对观察到的受保护表型的贡献。在aim中 3，我们将确定在卟啉症的遗传小鼠模型中抑制b-连环蛋白是否会减少卟啉积累和改善肝脏病理。两种特征良好的卟啉病模型：亚铁螯合酶（Fechm 1 Pas）突变小鼠，其模拟人红细胞生成性原卟啉症，具有显著的肝脏参与;和T1/T2小鼠，其是羟甲基胆烷合酶的复合杂合型，模拟急性间歇性卟啉病，在用苯巴比妥刺激后，将用于确定用β-连环蛋白抑制剂进行治疗性干预是否可以预防疾病进展或提供保护急性发作时。因此，拟议的研究将进一步了解抑制的潜力， β-连环蛋白治疗卟啉症，并将提供高度重要的信息，为治疗和翻译使用。