Development of novel fetal hemoglobin inducers using targeted protein degradation

主要的β-血红蛋白病，镰状细胞病（SCD）和β-地中海贫血，是两种最常见的 单基因疾病β-地中海贫血描述了一组异质性的突变，插入，缺失，或 β-珠蛋白基因HBB的取代，导致β-珠蛋白合成减少或不合成。另一方面，SCD 是一种常染色体隐性遗传疾病，在HBB基因中存在明确的错义点突变， 将多肽序列中第六位的谷氨酸残基变为缬氨酸残基。这种突变导致 成人红系细胞表达β-珠蛋白并合成一种异常形式的血红蛋白HbS， 聚合。β-血红蛋白病患者不能合成正常的β-珠蛋白， 正常功能的成人血红蛋白，HbA，成熟红系细胞中的主要形式。不断升高的 β-血红蛋白病患者的胎儿血红蛋白HbF可以减轻临床症状。与HbA相比， HbF由α 2 β 2亚基组成。出生后，造血转移 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。从胎儿到成人血红蛋白的转变，从β-珠蛋白到β-珠蛋白的表达， 需要几个关键的转录因子（TF），即BCL 11 A和ZBTB 7A，它们募集核小体 重塑和脱乙酰酶，NuRD，复合物，及其催化组分，CHD 4，对球蛋白基因座和 抑制β-珠蛋白表达。第三个TF，ZNF 410，最近被发现是CHD 4的独特激活剂。 表情这三种TF中的每一种的CRISPR/Cas9敲除或shRNA敲低诱导HbF表达， 针对这些因子的靶向治疗可能为β-血红蛋白病提供新的治疗途径。 已显示免疫调节药物（IMiD）将Cys 2-His 2锌指（C2 H2-ZF）TF募集到cereblon， Cullin-4 E3泛素连接酶（CRL 4）复合物的底物识别组分，用于靶向蛋白 降解三种感兴趣的TF都含有不同数目的串联C2 H2-ZF结构域，因此， 我假设IMiD诱导的BCL 11 A， ZBTB 7A或ZNF 410将导致β-珠蛋白基因的重新激活并增加HbF水平。使用1122 由Crews实验室建立的化合物IMiD库，Aim 1将确定库中的哪些化合物可以使用 作为先导化合物，这些化合物将通过反复的结构-活性关系研究进行优化， 在细胞内表征用于靶TF降解。目标2将检查这些优化的化合物的能力 在HUDEP-2和原代红系祖细胞中重新激活β-珠蛋白表达并诱导HbF合成。 该提案的完成将提供临床前框架，以评估靶向药物的治疗潜力。 针对TF的蛋白质降解在β-珠蛋白到β-珠蛋白的转换中起关键作用，作为治疗糖尿病的新途径。 严重的β-血红蛋白病。

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.