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 造血是血细胞所有谱系的衍生过程的过程 受限的转录因子组。目前，特定于因子的控制在很大程度上取决于遗传方法，例如 作为RNA干扰和CRISPR/CAS9，以改变感兴趣的转录因子的表达。虽然很高 选择性，基于基因的方法与显着的潜伏期有关（几个小时到几天），因此 无法在分钟到小时的régime中访问时间尺度的关键细胞动力学。而且，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罕见的问题，尤其是在治疗方面。直接的 特定转录因素的化学控制可能会解决这些机会，但总体上缺乏 用于医学化学和广泛结构同源性药物发现的内源性配体。见面 这项挑战，我们已经将分子水合在因子/DNA识别中的处置转化为 库筛选的正交选择标准。作为概念证明，我们开发了一个渗透驱动的噬菌体 显示屏幕以获得短肽，以增强或抑制由主转录因子PU.1的DNA结合 在造血干细胞稳态和分化中。 pu.1的脱位代表一个主要的分子 几种造血恶性肿瘤的病变（例如急性髓性白血病，多发性骨髓瘤和霍奇金 疾病）以及肺，肝脏和肾脏的纤维化。该提案的目标是：验证 PU.1靶向肽的生物学和分子特性，并将渗透筛选技术适应为 目标其他转录因子与PU.1一起起作用。为了实现这些目标，我们提出 三个具体目标。 1）我们将定义培养的造血的PU.1靶向肽的功能分布 模型，以及原发性鼠和患者衍生的白血病和促纤维细胞。初步数据显示 PU.1靶向肽进入细胞核，并调节主要PU.1靶基因在AS中的表达 仅30分钟，远低于目前通过遗传操作达到的限制。我们提出的研究 旨在表征其转录曲线以及伴随的细胞和疾病变化 表型。 2）我们将确定因子/DNA识别的肽调节的分子特性。 详细的研究旨在剖析胡椒/复合物的潜水员结构和机械基础 互动。 3）我们将将渗透筛选扩展到靶向谱系特异性转录因子，以共同调节 使用PU.1，包括与PU.1合作结合DNA的干扰素调节因子IRF4和IRF8 以及像C/EBPα这样的合作伙伴，由低亲和力PU.1结合进行合作招募。总而言之，这 预计提案将通过新颖的靶向试剂推进转录因子药理学，尤其是 激活剂，并演示结构和物理审讯的组合（库Panning + 渗透压）是一种可易于概括的解决方案，可克服化学控制中的电流瓶颈。