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

描述(申请人提供)：人类造血干细胞(HSC)移植目前被用作再生医学，用于治疗先天性缺陷和恶性疾病，以及癌症和其他血液和免疫系统疾病。然而，尽管人们对HSC生物学和治疗学充满热情，但基于HSC的疗法的潜力尚未完全实现。成体干细胞广泛应用的一个主要障碍是每份收获的用于治疗的HSC数量有限，以及它们对增殖刺激的扩增和分化行为知之甚少。HSC的体外扩增仍然是HSC移植广泛应用于患者的主要挑战。我们的研究表明，p18是细胞周期蛋白依赖性激酶(CDK)抑制因子(CKI)的一员，是HSC自我更新的强有力的负性调节因子。因此，我们假设p18是一个独特的药物靶点，能够阻断p18功能并干扰p18/CDK6相互作用的小分子很可能是激活HSC自我更新的有效药物。我们的目标是筛选和鉴定p18抑制剂，通过破坏p18/CDK6的相互作用，从而激活HSC自我更新，增加活跃干细胞的数量，并将它们用作HSC自我更新机制研究的化学探针。拟议的创新性研究的可行性得到了成熟的研究小组所获得的原则证明试验数据的支持，这些研究小组与拟议的研究具有互补的专门知识。考虑到目前HSC骨髓培养方案的处理能力有限，我们建议首先使用我们已建立的电子筛选方法进行初步筛选，以产生以p18为中心的铅亚库(目标1)。我们还应用核磁共振分析来筛选/验证和表征p18 HITS及其与蛋白质的结合作用，以产生p18活性亚集(目标2A)。此外，验证的p18靶向化合物的小亚集随后将通过广泛的HSC功能分析(目标2B)进行确认。通过这些，能够增加骨髓培养中活跃干细胞数量的化合物将被确定为先导。然后，发现的先导被用作特殊的化学探针，用于研究p18/CDK6相互作用和细胞周期G1期的信号机制。作为未来的计划，已确定的先导将通过化学修饰和SAR药物化学研究进一步优化，以提高效力和细胞毒性。我们的长期目标是确定/设计CKI p18特异性小分子效应器，以一种可预测的方式维持/刺激造血干细胞的自我更新，并最终开发用于HSC治疗的新药。实现这一目标将对干细胞药物研究发展产生重大影响。 公共卫生相关性：叙述：我们建议筛选和鉴定能够刺激造血干细胞自我更新的特定小分子效应器。这项研究将为造血干细胞治疗的新药设计提供一条很有前途的途径，并对未来干细胞药物的研究和开发产生重大影响。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Narrative: We propose to screen and identify specific small molecule effectors that can stimulate self-renewal of hematopoietic stem cells. The proposed research will offer a promising approach to design new drugs for hematopoietic stem cell therapies and have a significant impact on stem cell drug research and development in the future.