Development of novel fetal hemoglobin inducers using targeted protein degradation

利用靶向蛋白质降解开发新型胎儿血红蛋白诱导剂

基本信息

批准号：
10605620
负责人：
Sijin Zheng
金额：
$ 3.26万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-04-16 至 2026-04-15
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10605620
关键词：
Academic skills Adult Anemia Antibodies Basic Science Biochemistry Biological Biological Assay Birth Bone Marrow CD34 gene CHD4 gene CRISPR/Cas technology Calorimetry Cell Differentiation process Cell Survival Cells Cellular biology Chemistry Clinical Clustered Regularly Interspaced Short Palindromic Repeats Complex Computer software Cullin Proteins Deacetylase Dependence Development Disease Dose Environment Erythrocytes Erythroid Cells Erythroid Progenitor Cells Fetal Hemoglobin Fetal Liver Firefly Luciferases Flow Cytometry Frequencies Gene Delivery Gene Mutation Genes Genetic Transcription Globin Glutamic Acid Goals Hematopoiesis Hemoglobin Hemoglobin concentration result Hemoglobinopathies High Pressure Liquid Chromatography Human K562 Cells Kinetics Knock-out Lead Leukopenia Libraries Luciferases Molecular Mutation Nature Neutropenia NuRD complex Nucleosomes Patients Pharmaceutical Chemistry Physicians Point Mutation Positioning Attribute Proteins Proteome Proteomics Publishing Repression Research Training Scientist Sickle Cell Anemia Sickle Cell Trait Stains Structure Structure-Activity Relationship Surface Plasmon Resonance Symptoms Techniques Testing Thalassemia Thalidomide Therapeutic Thermodynamics Thrombocytopenia Titrations Toxic effect Training Valine Western Blotting Work Zinc Fingers autosome design fetal gene therapy immune modulating agents in silico interest knock-down model building multicatalytic endopeptidase complex novel novel therapeutics operation polypeptide pomalidomide pre-clinical protein degradation recruit scaffold screening side effect small hairpin RNA small molecule stable cell line stem targeted treatment transcription factor ubiquitin-protein ligase

项目摘要

The major -hemoglobinopathies, sickle cell disease (SCD) and -thalassemia, are two of the most common monogenetic diseases. -thalassemia describes a heterogenous set of mutations, insertions, deletions, or substitutions of the -globin gene, HBB, that result in no or decreased -globin synthesis. SCD on the other hand is an autosomal recessive disorder that has a well-defined missense point mutation in the HBB gene, that changes a glutamic acid to valine residue at the sixth position in the polypeptide sequence. This mutation causes adult erythroid cells to express s-globin and synthesize an abnormal form of hemoglobin, HbS, that is prone to polymerizing. Patients with -hemoglobinopathies cannot synthesize normal -globin, and thus cannot form normally functioning adult hemoglobin, HbA, the predominant form in mature erythroid cells. Elevating levels of fetal hemoglobin, HbF, in patients with -hemoglobinopathies can alleviate clinical symptoms. Compared to HbA, which is composed of globin subunits 22, HbF is composed of 22 subunits. After birth, hematopoiesis shifts from fetal hemoglobin expression in fetal liver to the bone marrow where adult erythrocytes are produced and express adult hemoglobin. The transition from fetal to adult hemoglobin, from -globin to -globin expression, requires several critical transcription factors (TFs), namely BCL11A and ZBTB7A, that recruit the Nucleosome Remodeling and Deacetylase, NuRD, complex, and its catalytic component, CHD4, to the globin locus and repress -globin expression. A third TF, ZNF410, was recently found to be a unique activator of CHD4 expression. CRISPR/Cas9 knockout or shRNA knockdown of each of these three TFs induces HbF expression, and targeted therapies against these factors might offer a new therapeutic avenue for -hemoglobinopathies. Immunomodulatory drugs (IMiDs) have been shown to recruit Cys2-His2 zinc-finger (C2H2-ZF) TFs to cereblon, the substrate recognition component for the Cullin-4 E3 ubiquitin ligase (CRL4) complex, for targeted protein degradation. The three TFs of interest all contain various numbers of tandem C2H2-ZF domains, and therefore, I hypothesize that IMiD-induced targeted protein degradation of one or a combination of BCL11A, ZBTB7A, or ZNF410, will lead to reactivation of the -globin gene and increase levels of HbF. Using a 1122 compound IMiD library built by the Crews lab, Aim 1 will determine which compounds from the library can serve as lead hit compounds that will be optimized by iterative rounds of structure-activity relationship studies and characterized in cellulo for target TF degradation. Aim 2 will examine the ability of these optimized compounds to reactivate -globin expression and induce HbF synthesis in HUDEP-2 and primary erythroid progenitor cells. Completion of this proposal will provide the preclinical framework to assess the therapeutic potential of targeted protein degradation against TFs critical in the -globin to -globin switch as a novel treatment avenue for the major -hemoglobinopathies.

主要的  血红蛋白病、镰状细胞病 (SCD) 和  地中海贫血是两种最常见的疾病 -地中海贫血描述了一组异质性突变、插入、缺失或突变。另一方面，-珠蛋白基因 HBB 的替换导致 -珠蛋白合成缺失或减少。是一种常染色体隐性遗传病，HBB 基因中有明确的错义点突变，将多肽序列中第六个位置的谷氨酸变为缬氨酸残基，导致这种突变。成体红细胞表达 β-球蛋白并合成异常形式的血红蛋白 HbS，这很容易患有 β-血红蛋白病的患者无法合成正常的 β-珠蛋白，因此无法形成。功能正常的成人血红蛋白 HbA，是成熟红细胞中的主要形式。与 HbA 相比，β-血红蛋白病患者的胎儿血红蛋白 HbF 可以缓解临床症状。由珠蛋白亚基 α22 组成，HbF 由 α2α2 亚基组成。出生后，造血功能发生转变。从胎儿肝脏中的胎儿血红蛋白表达到产生成人红细胞的骨髓，表达成人血红蛋白。从胎儿血红蛋白到成人血红蛋白的转变，从 β-珠蛋白表达到 β-珠蛋白表达，需要几个关键的转录因子 (TF)，即 BCL11A 和 ZBTB7A，它们可招募核小体重塑和脱乙酰酶、NuRD、复合物及其催化成分 CHD4 对球蛋白基因座和最近发现第三个 TF ZNF410 是 CHD4 的独特激活剂。这三种 TF 的 CRISPR/Cas9 敲除或 shRNA 敲除都会诱导 HbF 表达，针对这些因素的靶向治疗可能为  血红蛋白病提供新的治疗途径。免疫调节药物 (IMiD) 已被证明可以将 Cys2-His2 锌指 (C2H2-ZF) TF 募集至 cereblon， Cullin-4 E3 泛素连接酶 (CRL4) 复合物的底物识别组件，针对目标蛋白感兴趣的三个 TF 均包含不同数量的串联 C2H2-ZF 结构域，因此，我注意到 IMiD 诱导 BCL11A 中的一种或多种的靶向蛋白降解，第1122章被人欺负了由 Crews 实验室构建的化合物 IMiD 库，目标 1 将确定库中的哪些化合物可以使用作为先导化合物，将通过迭代的结构-活性关系研究进行优化，目的 2 将检查这些优化化合物的能力。重新激活 HUDEP-2 和原代红系祖细胞中的 γ-珠蛋白表达并诱导 HbF 合成。该提案的完成将为评估靶向药物的治疗潜力提供临床前框架针对 TF 的蛋白质降解在 -珠蛋白到 -珠蛋白转换中至关重要，作为一种新的治疗途径主要的-血红蛋白病。