Direct chemical control of the hematopoietic master transcription factor PU.1

造血主转录因子 PU.1 的直接化学控制

• 批准号: 10322390
• 负责人: Gregory Man Kai Poon
• 金额: $ 39万
• 依托单位: GEORGIA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10322390
• 关键词: Acute; Acute Myelocytic Leukemia; Address; Affinity; Area; Bacteriophages; Binding; Biological; Biophysics; Blood; Blood Cells; CCAAT-Enhancer-Binding Protein-alpha; CRISPR/Cas technology; Cell Nucleus; Cells; Chemicals; Clinic; Clinical; Collaborations; Complex; DNA; DNA Binding; Data; Development; Disease; Drug Targeting; ETS Family Protein; Event; Exercise; Family; Fibrosis; Gene Expression; Gene Expression Profile; Genes; Genetic; Genetic Transcription; Genomics; Goals; Hematopoiesis; Hematopoietic; Hematopoietic Neoplasms; Hematopoietic stem cells; Hodgkin Disease; Homeostasis; Hour; Hydration status; IRF4 gene; Interferons; Investigation; Kidney Diseases; Laboratory Study; Lesion; Libraries; Ligands; Liver Fibrosis; Liver diseases; Lung diseases; Lymphoid; Malignant Neoplasms; Mediating; Metabolic; Methods; Modeling; Molecular; Molecular Mechanisms of Action; Multiple Myeloma; Mus; Myelogenous; Myeloid Cells; New Agents; Nuclear Receptors; Osmotic Pressure; Pathway interactions; Patients; Peptides; Phage Display; Pharmaceutical Chemistry; Pharmacology; Phenotype; Process; Property; Proteins; Pulmonary Fibrosis; RNA Interference; Reagent; Regulation; Research; Role; Selection Criteria; Site; Source; Specificity; Steroids; Structural Models; Structure; Surface; Techniques; Therapeutic; Time; Tissues; Toxic effect; Transcriptional Regulation; Transgenes; Translating; Translations; Variant; Viral; Water; Work; base; cofactor; cytotoxicity; disease phenotype; drug discovery; genetic manipulation; genotoxicity; improved; innovation; insight; interest; kidney fibrosis; leukemia; leukemia/lymphoma; member; non-viral gene delivery; novel; novel therapeutics; programs; recruit; screening; stem cell homeostasis; success; tool; transcription factor

PROJECT SUMMARY/ABSTRACT: Direct control of the hematopoietic master transcription factor PU.1 Hematopoiesis, the process by which all lineages of blood cells are derived, is under coordinate control by a restricted group of transcription factors. Currently, factor-specific control relies heavily on genetic methods, such as RNA interference and CRISPR/Cas9, to alter the expression of transcription factors of interest. While highly selective, gene-based approaches are associated with significant latency (many hours to days) and therefore cannot access critical cellular dynamics at timescales in the minute-to-hour régime. Moreover, cytotoxicity and genotoxicity due to viral and non-viral gene delivery remain outstanding issues, particularly in therapy. Direct chemical control of specific transcription factors could address these opportunities, but a general lack of endogenous ligands for medicinal chemistry and broad structural homology challenge drug discovery. To meet this challenge, we have translated the disposition of molecular hydration in factor/DNA recognition into an orthogonal selection criterion to library screening. As proof of concept, we developed an osmotically driven phage display screen to obtain short peptides that enhance or inhibit DNA binding by PU.1, a master transcription factor in hematopoietic stem cell homeostasis and differentiation. De-regulation of PU.1 represents a major molecular lesion in several hematopoietic malignancies (e.g., acute myeloid leukemia, multiple myeloma, and Hodgkin's disease) as well as fibrosis of the lungs, liver, and kidneys. The objectives of this proposal are: to validate the biological and molecular properties of PU.1-targeted peptides, and adapting the osmotic screening technique to target other transcription factors that function in concert with PU.1. To achieve these objectives, we propose three specific aims. 1) We will define the functional profiles of PU.1-targeted peptides in cultured hematopoietic models, as well as primary murine and patient-derived leukemic and pro-fibrotic cells. Preliminary data show that PU.1-targeted peptides enter the cell nucleus and modulate the expression of major PU.1 target genes in as little as 30 min, an onset well below currently achievable limits by genetic manipulations. Our proposed studies are aimed at characterizing their transcriptional profiles and the attendant changes in cellular and disease phenotypes. 2) We will determine the molecular properties of peptide modulation of factor/DNA recognition. Detailed studies are aimed at dissecting the diverse structural and mechanistic bases of peptide/complex interactions. 3) We will expand osmotic screening to target lineage-specific transcription factors that co-regulate with PU.1, including the interferon regulatory factors IRF4 and IRF8 that bind DNA cooperatively with PU.1, as well as partners such as C/EBPα that are recruited collaboratively by low-affinity PU.1 binding. In summary, this proposal is expected to advance transcription factor pharmacology with novel targeted reagents, particularly activators, and demonstrate the combination of structural and physicochemical interrogation (library panning + osmotic pressure) as a tractable, generalizable solution to overcome current bottlenecks in chemical control.

项目摘要/摘要：直接控制造血主转录因子 PU.1 造血是所有血细胞谱系产生的过程，受到一个系统的协调控制。 目前，因子特异性控制很大程度上依赖于遗传方法，例如 例如 RNA 干扰和 CRISPR/Cas9，以高度改变感兴趣的转录因子的表达。 基于基因的选择性方法与显着的潜伏期（数小时至数天）相关，因此 无法在每分钟的时间范围内获取关键的细胞动态。 病毒和非病毒基因传递引起的遗传毒性仍然是突出的问题，特别是在直接治疗中。 对特定转录因子的化学控制可以解决这些机会，但普遍缺乏 药物化学的内源配体和广泛的结构同源性挑战药物发现。 为了应对这一挑战，我们将因子/DNA 识别中分子水合的配置转化为 文库筛选的正交选择标准作为概念证明，我们开发了渗透压驱动的噬菌体。 显示屏以获得增强或抑制主转录因子 PU.1 与 DNA 结合的短肽 PU.1 的失调是造血干细胞稳态和分化中的一个主要分子。 多种造血系统恶性肿瘤（例如急性髓系白血病、多发性骨髓瘤和霍奇金淋巴瘤）的病变 疾病）以及肺、肝和肾的纤维化 该提案的目标是：验证 PU.1 靶向肽的生物学和分子特性，并采用渗透筛选技术 靶向与 PU.1 协同作用的其他转录因子 为了实现这些目标，我们建议。 三个具体目标 1) 我们将定义 PU.1 靶向肽在培养造血系统中的功能特征。 模型以及原代小鼠和患者来源的白血病细胞和促纤维化细胞的初步数据表明。 PU.1 靶向肽进入细胞核并调节主要 PU.1 靶基因的表达 短至 30 分钟，远低于目前通过基因操作可实现的极限。 旨在表征它们的转录谱以及随之而来的细胞和疾病变化 2) 我们将确定因子/DNA 识别的肽调节的分子特性。 详细研究旨在剖析肽/复合物的不同结构和机制基础 3）我们将扩大渗透筛选，以针对共同调节的谱系特异性转录因子。 与PU.1，包括与PU.1协同结合DNA的干扰素调节因子IRF4和IRF8，如 以及通过低亲和力 PU.1 结合协同招募的伙伴，例如 C/EBPα。 该提案预计将通过新型靶向试剂推进转录因子药理学，特别是 激活剂，并展示结构和物理化学询问的组合（文库淘选+ 渗透压）作为一种易于处理、通用的解决方案来克服当前化学控制的瓶颈。