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

项目摘要/摘要：直接控制造血主转录因子PU1 造血，即所有血细胞来源的过程，是由一种 转录因子的限制性组。目前，因子特异性控制在很大程度上依赖于遗传方法，如 如RNA干扰和CRISPR/Cas9，改变目的转录因子的表达。虽然高度 选择性的、基于基因的方法与显著的延迟(几个小时到几天)相关，因此 无法在每分钟到每小时的报告中访问关键的细胞动力学。此外，细胞毒性和 病毒和非病毒基因传递造成的遗传毒性仍然是突出的问题，特别是在治疗中。直接 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 药物化学的内源性配体和广泛的结构同源性对药物发现提出了挑战。相遇 在这一挑战中，我们将因子/DNA识别中分子水化的处置转化为 文库筛选的正交选择标准。作为概念验证，我们开发了一种渗透驱动噬菌体 显示屏幕以获得增强或抑制主要转录因子PU.1与DNA结合的短肽 在造血干细胞的动态平衡和分化方面。PU1的去调节代表了一个主要的分子 几种血液系统恶性肿瘤(如急性髓系白血病、多发性骨髓瘤和何杰金氏病)的损害 疾病)以及肺、肝和肾的纤维化。这项提案的目标是：验证 PU.1靶向多肽的生物学和分子性质，并采用渗透筛选技术 靶向与PU1协同发挥作用的其他转录因子。为了达到这些目标，我们建议 三个具体目标。1)我们将确定PU1靶向多肽在培养的造血细胞中的功能 模型，以及原代小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靶向多肽进入细胞核并调节AS主要PU1靶基因的表达 短短30分钟，发病时间远低于目前通过基因操作所能达到的极限。我们建议的研究 目的是描述它们的转录特征以及伴随而来的细胞和疾病的变化 表型。2)我们将确定调节因子/DNA识别的多肽的分子性质。 详细的研究旨在剖析多肽/复合体的各种结构和机制基础。 互动。3)我们将扩大渗透筛选，以靶向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，AS 以及通过低亲和力PU1结合协作招募的合作伙伴，如C/EBPα。总而言之，这是 该提案有望通过新的靶向试剂促进转录因子药理学的发展，特别是 激活剂，并演示了结构和物理化学询问的组合(库平移+ 渗透压)作为一种易处理、可推广的解决方案，以克服目前化学控制中的瓶颈。

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.