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: 10540346
• 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 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10540346
• 关键词: Acute; Acute Myelocytic Leukemia; Address; Affinity; Area; Bacteriophages; Binding; Biological; Biophysics; Blood; Blood Cells; 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; Lymphoma; Malignant Neoplasms; Maps; Mediating; Metabolic; Methods; Modeling; Molecular; Molecular Mechanisms of Action; Multiple Myeloma; Mus; Myelogenous; Myeloid Cells; New Agents; Nuclear Receptors; Osmosis; Osmotic Pressure; Pathway interactions; Patients; Peptides; Phage Display; Pharmaceutical Chemistry; Pharmacology; Phenotype; Process; Property; Proteins; Pulmonary Fibrosis; RNA Interference; Reagent; Research; Role; Selection Criteria; Site; Source; Specific qualifier value; Specificity; Steroids; Structural Models; Structure; Surface; Techniques; Therapeutic; 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; member; non-viral gene delivery; novel; novel therapeutics; pharmacologic; programs; proto-oncogene protein Spi-1; recruit; screening; stem cell homeostasis; success; tool; transcription factor; viral gene delivery

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.

项目总结/摘要：造血主转录因子PU.1的直接控制 造血，即所有血细胞谱系的衍生过程，是在一个协调控制下， 限制性转录因子群。目前，因子特异性控制严重依赖于遗传方法，例如 如RNA干扰和CRISPR/Cas9，以改变感兴趣的转录因子的表达。虽然高度 选择性的、基于基因的方法与显著的潜伏期（数小时至数天）相关，因此 无法在分钟到小时的时间尺度上访问关键的细胞动力学。此外，细胞毒性和 由于病毒和非病毒基因递送引起的遗传毒性仍然是突出的问题，特别是在治疗中。直接 化学控制特定的转录因子可以解决这些机会，但普遍缺乏， 用于药物化学的内源性配体和广泛的结构同源性挑战药物发现。满足 在这一挑战中，我们已经将因子/DNA识别中的分子水合作用的处置转化为 正交选择标准进行文库筛选。作为概念验证，我们开发了一种免疫驱动的噬菌体 显示屏幕，以获得增强或抑制主转录因子PU.1与DNA结合的短肽 造血干细胞的稳态和分化。PU.1的失调代表了一个主要分子 几种造血系统恶性肿瘤中的病变（例如，急性骨髓性白血病、多发性骨髓瘤和霍奇金淋巴瘤 疾病）以及肺、肝和肾的纤维化。本提案的目标是： 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靶向肽在培养的造血细胞中的功能概况。 模型，以及原代鼠和患者来源的白血病和促纤维化细胞。初步数据显示， PU.1靶向肽进入细胞核并调节细胞中主要PU.1靶基因的表达， 短至30分钟，远低于目前通过遗传操作可实现的极限的起始。我们建议的研究 目的是表征它们的转录谱和伴随的细胞和疾病的变化， 表型2)我们将确定因子/DNA识别的肽调制的分子特性。 详细的研究旨在剖析肽/复合物的不同结构和机制基础 交互. 3)我们将扩大渗透筛选的目标谱系特异性转录因子，共调节 与PU.1，包括与PU.1协同结合DNA的干扰素调节因子IRF 4和IRF 8，如 以及通过低亲和力PU.1结合协同募集的配偶体，如C/EBPα。总之，这 该提案有望用新的靶向试剂推进转录因子药理学，特别是 激活剂，并证明结构和物理化学询问的组合（文库淘选+ 渗透压）作为一个易于处理的，可推广的解决方案，以克服目前的瓶颈，在化学控制。