Small molecules targeting RhoA for platelet cold storage in cancer care

自20世纪80年代以来，用于癌症患者支持护理的血小板输注显著增加， 但一种安全、长期的血小板储存方法仍然缺乏。目前FDA规定的做法允许 由于担心，准备后的血小板将在20至24°C下保存，有限寿命最长为5天 关于细菌污染。冷藏大大缩短了血小板的寿命，因为它 使糖蛋白Ib(GPIB)受体聚集在血小板膜的特定微域上。 Von Willebrand对簇状糖蛋白上特定脱糖/脱糖残基的识别 因子、巨噬细胞β-2整合素和肝细胞Ashwell-Morell受体导致血小板 宿主的吞噬作用和循环中的清除。因此，防止糖蛋白聚集 代表了一种有用的化学干预方式。血小板膜糖蛋白与 细胞内细胞骨架及其糖基化依赖于特定的位置和活性 糖基转移酶。它们的聚集依赖于血小板膜上脂筏的形成。 而这又取决于高度调控的细胞骨架过程的动力学 重新安排。RhoA是Rho GTPase家族的创始成员，该家族是 细胞骨架动力学，并已被证明控制脂筏的形成和组成。因此， RhoA活性变化可能影响血小板膜脂筏组装和糖蛋白 组成。我们通过遗传学和药理学手段进行的初步研究表明，寒冷受体 上调RhoA活性，进而诱导血小板活化。我们发现了一条线索RhoA Rhosin/G04，它是RhoA激活的特异性抑制剂，能够模拟RhoA基因敲除来防止 富含糖基转移酶的脂筏与冷诱导的GPIb的网状蛋白非依赖性内化 集群化。我们推测，抑制RhoA可以阻止血小板冷诱导的GPIB聚集和 输血后宿主清除以及可逆RhoA抑制剂的开发 可以转化为一种新的血小板冷藏方案。在这项提案中，我们将确定 RhoA先导抑制剂Rhosin/G04的作用机制及治疗效果 Rhosin/G04及其类似物在小鼠模型中长期冷冻保存血小板的研究 以及非人类灵长类动物。药物发现、药物化学和结构化学的结合 共同PI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 创新的、开箱即用的干预血小板储存技术的方法。小分子 通过蛋白质-蛋白质相互作用干扰RhoA GTP酶靶向是一项革命性的工作 这种方法允许可逆地抑制RhoA，并有助于保存冷藏的血小板功能。我们的 通过临床前药物发现工作进行的创新将使癌症患者的护理受益。

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.