Screen and Design p18 Chemical Probes for Hematopoietic Stem Cell Self-Renewal

人类造血干细胞(HSC)移植目前正被用作再生医学 先天缺陷和恶性疾病以及癌症和其他疾病的治疗 血液和免疫系统。然而，尽管人们对HSC生物学和治疗学充满热情， 基于HSC的治疗的潜力还没有完全实现。成年人更广泛使用的一个主要障碍 干细胞是每一次收获的有限数量的HSC，用于治疗的益处以及它们的知之甚少 对增殖刺激作出反应的扩增和分化行为。HSC的体外扩增仍是一种 造血干细胞移植在患者中广泛应用的主要挑战。我们的研究表明，p18，a 细胞周期蛋白依赖性激酶(CDK)抑制因子(CKI)是HSC自身的一种强有力的负性调节因子。 更新。因此，我们假设p18是一个独特的药物靶点，而能够阻止 P18功能和干扰p18/CDK6相互作用可能是激活HSC自身的强效药物。 更新。我们的目标是筛选/识别通过破坏p18/CDK6相互作用而起作用的p18抑制剂，从而 激活HSC自我更新，增加活跃干细胞数量，并将其用作化学物质 HSC自我更新机制研究的探讨。拟议的创新研究的可行性是 由久负盛名的研究团队获得的原则验证试点数据支持，这些研究团队 为拟议的研究提供补充专业知识。考虑到目前的吞吐能力有限 HSC骨髓培养方案，我们建议首先使用我们已建立的电子筛查方法 初步筛选以产生以p18为中心的铅亚库(目标1)。我们还将核磁共振分析应用于 筛选/验证和表征p18 HITS及其与蛋白质的结合作用，以便生成 P18--活动子集(目标2A)。此外，验证的p18靶向化合物的小子集将是 经广泛的肝星状细胞功能分析确认(目标2B)。通过这些，这些化合物能够 在骨髓培养中增加活跃干细胞的数量将被确定为先导。这个 然后，发现的铅被用作研究p18/CDK6相互作用和信号转导的特定化学探针 细胞周期中G1期的作用机制。作为未来计划，已确定的销售线索将通过 化学修饰和SAR药物化学研究，以提高效力和细胞毒性。我们的长- 学期目标是确定/设计CKI p18特异性小分子效应器，它可以维持/刺激自我 以可预测的方式更新造血干细胞，并最终开发治疗HSC的新药 治疗。实现这一目标将对干细胞药物研究发展产生重大影响。

Human hematopoietic stem cells (HSC) transplantation is currently being used as regenerative medicine for the treatment of congenital deficiencies and malignant diseases as well as cancers and other disorders of the blood and immune systems. However, despite all the enthusiasm surrounding HSC biology and therapeutics, the potential of HSC-based therapies has yet to be fully realized. A major roadblock of broader use of the adult stem cells is the limited number of HSC per harvest for therapeutic benefit and their poorly understood expansion and differentiation behavior in response to proliferative stimuli. In vitro expansion of HSC remains a major challenge for wide applications of HSC transplantation for patients. Our studies show that p18, a member of the cyclin-dependent kinase (CDK) inhibitors (CKI), is a potent negative regulator of HSC self- renewal. Thus, we hypothesize that p18 is a unique drug target, and that small molecules capable of blocking p18 function and interfering with p18/CDK6 interactions are likely to be potent drugs for activating HSC self- renewal. Our objective is to screen/identify p18 inhibitors that act by disrupting p18/CDK6 interactions, thus activating HSC self-renewal and increasing the quantity of active stem cells, and to use them as chemical probes for mechanism studies of HSC self-renewal. The feasibility of the proposed innovative research is supported by the proof-of-principle pilot data obtained by well-established research teams that have complementary expertise for the proposed research. Considering the limited throughput capacity of the current HSC bone-marrow culture protocol, we propose first to use our established in silico screening approach for initial screening to generate p18-focused lead sublibraries (Aim 1). We also apply NMR assays to screen/validate and characterize the p18 hits and their binding interactions with the protein in order to generate p18-active subsets (Aim 2A). Also, the small subsets of validated p18-targeting compounds will then be confirmed by extensive HSC functional assays (Aim 2B). Through these, the compounds that are capable of increasing the number of active stem cells in the bone-marrow culture will be identified as leads. The discovered leads are then used as specific chemical probes for studies of p18/CDK6 interactions and signaling mechanisms of the G1-phase of the cell cycle. As a future plan, the identified leads will be further optimized by chemistry modification and SAR medicinal chemistry studies to improve the potency and cell toxicity. Our long- term goal is to identify/design CKI p18-specific small molecule effectors that can either maintain/stimulate self- renewal of hematopoietic stem cells in a predictable manner, and ultimately to develop new drugs for HSC therapies. Achieving this goal will have a significant impact on stem cell drug research development in general.