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

• 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
• 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; 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

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thalassymia是最常见的两个 单基因疾病。 中心贫血描述了一组异质的突变，插入，缺失或 -珠蛋白基因HBB的取代，导致-珠蛋白的合成降低或减少。另一方面 是一种常染色体隐性疾病，在HBB基因中具有明确定义的错义点突变， 在多肽序列中的第六位置将谷氨酸更改为Valine居住地。这种突变会导致 成人红细胞细胞以表达sanglobin并合成一种异常形式的血红蛋白，HBS，容易发生 聚合。 血红蛋白病患者无法综合正常珠蛋白，因此无法形成 通常发挥成年血红蛋白，HBA，成熟的红细胞细胞中的主要形式。升高的水平 患有血红蛋白病患者的胎儿血红蛋白HBF可以缓解临床症状。与HBA相比 由球蛋白亚基组成22，HBF由22亚基组成。出生后，造血发生变化 从胎儿肝脏中的胎儿血红蛋白表达到产生成年红细胞的骨髓， 表达成人血红蛋白。从胎儿到成年血红蛋白的过渡，从珠蛋白到珠蛋白表达， 需要几个关键转录因子（TF），即BCL11A和ZBTB7A，它们募集了核小体 重塑和脱乙酰基酶，NURD，复合物及其催化成分CHD4到球蛋白基因座， 抑制珠蛋白的表达。最近发现第三个TF ZNF410是CHD4的独特激活剂 表达。这三个TF中每一个的CRISPR/CAS9敲除或shRNA敲低诱导HBF表达， 针对这些因素的有针对性疗法可能为血红蛋白病提供新的治疗途径。 已显示免疫调节药物（IMID）招募Cys2-His2锌指（C2H2-ZF）TFS到小脑， 靶蛋白的Cullin-4 E3泛素连接酶（CRL4）复合物的底物识别成分 降解。感兴趣的三个TF都包含各种数量的串联C2H2-ZF域，因此， 我假设IMID诱导的靶向蛋白质降解是BCl11a的一个或一个组合， ZBTB7A或ZNF410将导致-珠蛋白基因的重新激活并增加HBF的水平。使用1122 Compound IMID库由机组人员实验室构建，AIM 1将确定库中哪些化合物可以使用 作为铅命中化合物，将通过迭代的结构活动关系研究和 在纤维素中以靶标TF降解为特征。 AIM 2将检查这些优化化合物的能力 在HUDEP-2和原发性红细胞祖细胞中重新激活珠蛋白的表达并诱导HBF合成。 该提案的完成将提供临床前框架，以评估目标的治疗潜力 针对TFS的蛋白质降解至关重要的tfs tfs tfs tfs to tfs tfs球蛋白开关是一种新型治疗途径 主要的 - 血红蛋白病。