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

人类造血干细胞（HSC）移植目前被用作再生医学，用于治疗癌症。 治疗先天性缺陷和恶性疾病以及癌症和其他疾病， 血液和免疫系统。然而，尽管围绕HSC生物学和治疗学的所有热情， 基于HSC的治疗的潜力还没有完全实现。更广泛地使用成人的主要障碍 干细胞是每次收获的有限数量的HSC用于治疗益处，并且它们的了解很少。 扩增和分化行为的增殖刺激的反应。HSC的体外扩增仍然是一个研究热点。 这是HSC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）抑制剂（CKI）的成员，是HSC自身免疫的有效负调节剂。 退款因此，我们假设p18是一个独特的药物靶点，并且能够阻断p18的小分子可能是一个潜在的靶点。 p18功能和干扰p18/CDK 6相互作用可能是激活HSC自身免疫的有效药物。 退款我们的目标是筛选/鉴定通过破坏p18/CDK 6相互作用起作用的p18抑制剂， 激活HSC自我更新，增加活性干细胞的数量，并将其用作化学品， 探讨HSC自我更新的机制。建议的创新研究的可行性 由成熟的研究团队获得的原理验证试验数据支持， 为拟议的研究提供补充专业知识。考虑到目前有限的吞吐能力， HSC骨髓培养方案，我们建议首先使用我们建立的计算机筛选方法， 初步筛选以产生p18聚焦的前导子文库（Aim 1）。我们还将NMR分析应用于 筛选/验证和表征p18命中及其与蛋白质的结合相互作用， p18-活性亚群（Aim 2A）。此外，验证的p18靶向化合物的小子集将随后被 通过广泛的HSC功能测定证实（Aim 2B）。通过这些，能够 增加骨髓培养物中活性干细胞的数量将被确定为先导。的 然后将发现的先导物用作研究p18/CDK 6相互作用和信号传导的特异性化学探针 细胞周期G1期的机制。作为未来计划，将通过以下方式进一步优化已确定的潜在客户： 化学修饰和SAR药物化学研究，以提高效力和细胞毒性。我们长久以来- 长期目标是鉴定/设计CKI p18特异性小分子效应物， 以可预测的方式更新造血干细胞，并最终开发用于HSC的新药 治疗实现这一目标将对干细胞药物的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.