Small molecules targeting RhoA for platelet cold storage in cancer care

靶向 RhoA 的小分子用于癌症护理中的血小板冷藏

基本信息

批准号：
9763067
负责人：
Jose A Cancelas
金额：
$ 63.55万
依托单位：
CINCINNATI CHILDRENS HOSP MED CTR
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-04-01 至 2023-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9763067
关键词：
Accelerated Executive Review Actomyosin Allogenic Aspirin Autologous Blood Transfusion Blood Circulation Blood Platelets Cancer Patient Cells Cellular biology Chemicals Clathrin Coagulation Process Complex Cryopreservation Cytoskeleton Data Development Dreams Drug Combinations Drug Kinetics Enzymes Excision Family Generations Genetic Genetic study Glycoprotein Ib Glycoproteins Goals Guanosine Triphosphate Phosphohydrolases Hemorrhage Hemostatic Agents Hepatocyte Human Immunodeficient Mouse Integrins Intervention Knock-out Lead Lesion Life Location Longevity Macaca mulatta Mediating Membrane Membrane Lipids Membrane Microdomains Methods Molecular Monkeys Mus Patient Care Patients Phagocytosis Pharmaceutical Chemistry Pharmaceutical Preparations Pharmacology Platelet Activation Platelet Glycoproteins Platelet Transfusion Play Post-Translational Protein Processing Preparation Prevention Process Program Reviews Radiation Radiation therapy Receptor Activation Refrigeration Regimen Risk Role Signal Transduction Specificity Structure-Activity Relationship Supportive care Technology Temperature Therapeutic Transferase Transfusion Translating Vesicle analog base cancer care chemotherapy cytotoxicity drug discovery efficacy testing enantiomer glycosylation in vivo inhibitor/antagonist innovation macrophage member mouse model new technology nonhuman primate platelet function platelet preservation platelet storage pre-clinical preservation prevent protein protein interaction receptor rho rho GTP-Binding Proteins small molecule small molecule inhibitor structural biology three dimensional structure transfusion medicine von Willebrand Factor

项目摘要

Platelet transfusion in supportive care of cancer patients has seen a significant increase since 1980s, but a safe, long-term platelet storage method remains missing. Current FDA-mandated practice allows platelets to be stored at 20 to 24°C after preparation with a limited lifetime up to 5 days due to concerns about bacterial contamination. Refrigerated storage drastically reduces platelet life-span because it causes glycoprotein-Ib (GPIb) receptors to cluster on specific microdomains of the platelet membrane. Recognition of specific de-glycated/de-syalylated residues on clustered glycoproteins by von Willebrand factor, macrophage β2 integrins and hepatocyte Ashwell-Morell receptors results in platelet phagocytosis by the host and removal from circulation. Thus, prevention of glycoprotein clustering represents a useful way for chemical intervention. Platelet glycoproteins are intimately associated with intracellular cytoskeleton and their glycosylation depends on the location and activity of specific glycosyl-transferases. Their clustering depends on the formation of lipid raft in the platelet membrane which in turn depends on the dynamics of the highly regulated processes of cytoskeletal rearrangements. RhoA is the founding member of the Rho GTPase family that are central regulators of cytoskeletal dynamics, and has been shown to control lipid raft formation and composition. Therefore, changes in RhoA activity may influence platelet membrane lipid raft assembly and glycoprotein composition. Our preliminary studies by genetic and pharmacological means show that cold receptor upregulates RhoA activity, which in turn induces platelet activation. We have discovered a lead RhoA inhibitor, Rhosin/G04, that is specific to RhoA activation and is able to mimic RhoA knockout to prevent clathrin-independent internalization of lipid raft enriched in glycosyl transferase and cold-induced GPIb clustering. We hypothesize that RhoA inhibition can prevent platelet cold-induced GPIb clustering and consequent clearance by host upon transfusion, and that development of a reversible RhoA inhibitor can be translated to a new regimen of platelet cold storage. In this proposal, we will determine the mechanism of action by the lead RhoA inhibitor, Rhosin/G04, and demonstrate the therapeutic benefits of Rhosin/G04 and analogues for efficacious refrigerated long-term platelet storage in mouse models and non-human primates. The combination of drug discovery, medicinal chemistry and structural biology, cell biology, and platelet transfusion expertise as demonstrated by the co-PIs led to this highly innovative, outside-the-box method for intervention in platelet storage technology. Small molecule targeting of RhoA GTPase through the interference of protein-protein interaction is a revolutionary approach allowing for reversible inhibition of RhoA and help preserve cold stored platelet function. Our innovation through the preclinical drug discovery effort will benefit cancer patient care.

自 20 世纪 80 年代以来，用于癌症患者支持治疗的血小板输注显着增加，但目前 FDA 规定的做法仍然缺乏安全、长期的血小板储存方法。血小板制备后需保存在 20 至 24°C 下，由于担心其使用寿命有限，最多可达 5 天关于细菌污染，冷藏会大大缩短血小板的寿命。导致糖蛋白 Ib (GPIb) 受体聚集在血小板膜的特定微域上。冯·维勒布兰德 (von Willebrand) 识别聚集糖蛋白上的特定去糖基/去硫基化残基因子、巨噬细胞 β2 整合素和肝细胞 Ashwell-Morell 受体导致血小板被宿主吞噬并从循环中去除，从而防止糖蛋白聚集。代表了化学干预的一种有用方法。细胞内的细胞骨架及其糖基化取决于特定的位置和活性它们的聚集取决于血小板膜中脂筏的形成。这反过来又取决于细胞骨架高度调控过程的动态 RhoA 是 Rho GTPase 家族的创始成员，是 Rho GTPase 的核心调节因子。细胞骨架动力学，并已被证明可以控制脂筏的形成和组成，因此， RhoA活性的变化可能影响血小板膜脂筏组装和糖蛋白我们通过遗传和药理学手段进行的初步研究表明，冷受体。上调 RhoA 活性，进而诱导血小板活化我们发现了一种先导 RhoA。抑制剂，Rhosin/G04，对 RhoA 激活具有特异性，能够模拟 RhoA 敲除以防止富含糖基转移酶和冷诱导 GPIb 的脂筏的不依赖于网格蛋白的内化我们认为抑制 RhoA 可以防止血小板冷诱导的 GPIb 聚集。输血时宿主随后清除，以及可逆 RhoA 抑制剂的开发可以转化为血小板冷藏的新方案。在本提案中，我们将确定主要 RhoA 抑制剂 Rhosin/G04 的作用机制，并证明了治疗效果 Rhosin/G04 及其类似物在小鼠模型中有效冷藏长期储存血小板和非人类灵长类动物的药物发现、药物化学和结构的结合。联合 PI 所证明的生物学、细胞生物学和血小板输注专业知识导致了这一高度用于干预血小板储存技术的创新、开箱即用的方法。通过干扰蛋白质-蛋白质相互作用来靶向 RhoA GTPase 是一项革命性的技术我们的方法允许对 RhoA 进行可逆抑制，并有助于保持冷藏血小板功能。通过临床前药物发现工作进行的创新将有利于癌症患者的护理。