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

用于造血干细胞自我更新的 p18 化学探针的筛选和设计

• 批准号: 8174548
• 负责人: Xiang-Qun Xie
• 金额: $ 22.53万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8174548
• 关键词: Affect; Behavior; Binding; Biochemical; Biological Assay; Bone Marrow; CDK4 gene; CDK6-associated protein p18; Cell Cycle; Cell Therapy; Cells; Chemicals; Chemistry; Clinic; Collaborations; Colony-Forming Units Assay; Commit; Computer Simulation; Cyclin-Dependent Kinase Inhibitor; Data; Databases; Disease; Drug Delivery Systems; Ectopic Expression; Eligibility Determination; Equilibrium; Figs - dietary; Future; G1 Phase; Goals; Grant; Harvest; Hematological Disease; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Human; Immune system; In Vitro; Laboratories; Lead; Libraries; Malignant - descriptor; Malignant Neoplasms; Mediating; Modification; Molecular Target; Mus; Nature; Outcome; Patients; Pharmaceutical Chemistry; Pharmaceutical Preparations; Protein Binding; Proteins; Protocols documentation; Published Comment; Publishing; Recommendation; Regenerative Medicine; Research; Research Design; Research Proposals; Retroviridae; Screening procedure; Signal Transduction; Site-Directed Mutagenesis; Staging; Stem Cell Research; Stem cell transplant; Stem cells; Stimulus; Study models; Synthesis Chemistry; Techniques; Testing; Therapeutic; Therapeutic Uses; Toxic effect; Umbilical Cord Blood; Work; adult stem cell; base; design; high risk; human stem cells; improved; in vitro Bioassay; in vivo; inhibitor/antagonist; innovation; leukemogenesis; meetings; member; novel; novel strategies; research and development; research study; response; self-renewal; small molecule; stem cell biology; stem cell therapy; virtual

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.

描述（由申请人提供）：人造血干细胞（HSC）移植目前被用作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的再生医学。然而，尽管围绕HSC生物学和治疗学的所有热情，基于HSC的治疗的潜力尚未完全实现。更广泛地使用成体干细胞的主要障碍是每次收获的用于治疗益处的HSC数量有限，以及它们响应于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）抑制剂（CKI）的一员，是HSC自我更新的有效负调节剂。因此，我们假设p18是一个独特的药物靶点，并且能够阻断p18功能并干扰p18/CDK 6相互作用的小分子可能是激活HSC自我更新的有效药物。我们的目标是筛选/鉴定p18抑制剂，其作用是通过破坏p18/CDK 6相互作用，从而激活HSC自我更新并增加活性干细胞的数量，并将其用作HSC自我更新机制研究的化学探针。拟议的创新研究的可行性得到了具有拟议研究补充专业知识的成熟研究团队获得的原理验证试验数据的支持。考虑到目前HSC骨髓培养方案的有限通量能力，我们建议首先使用我们建立的计算机筛选方法进行初始筛选，以生成p18聚焦的先导子文库（目标1）。我们还应用NMR分析来筛选/验证和表征p18命中及其与蛋白质的结合相互作用，以产生p18活性子集（Aim 2A）。此外，验证的p18靶向化合物的小子集随后将通过广泛的HSC功能测定来确认（Aim 2B）。通过这些，能够增加骨髓培养物中活性干细胞数量的化合物将被鉴定为先导物。然后，发现的线索被用作研究p18/CDK 6相互作用和细胞周期G1期信号传导机制的特异性化学探针。作为未来计划，将通过化学修饰和SAR药物化学研究进一步优化已确定的先导化合物，以提高效价和细胞毒性。我们的长期目标是鉴定/设计CKI p18特异性小分子效应物，其可以以可预测的方式维持/刺激造血干细胞的自我更新，并最终开发用于HSC疗法的新药。实现这一目标将对总体干细胞药物研究开发产生重大影响。 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异性小分子效应物。该研究将为造血干细胞治疗新药的设计提供一种有前途的方法，并对未来干细胞药物的研究和开发产生重大影响。