Small molecules targeting RhoA for platelet cold storage in cancer care

自20世纪80年代以来，在癌症患者的支持性护理中输注血小板的情况显著增加， 但目前仍缺乏安全、长期的血小板储存方法。目前FDA规定的做法允许 血小板制备后在20 - 24°C下储存，由于担心寿命有限，最长可达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区上。 von Willebrand对糖蛋白簇上特异性去糖基化/去糖基化残基的识别 巨噬细胞β2整合素和肝细胞Ashwell-Morell受体导致血小板聚集 被宿主吞噬并从循环中排出。因此，防止糖蛋白聚集 是一种有效的化学干预方法。血小板糖蛋白与 细胞内细胞骨架及其糖基化依赖于特异性糖基化酶的位置和活性。 糖基转移酶。它们的聚集依赖于血小板膜脂筏的形成 这反过来又取决于细胞骨架的高度调节过程的动力学 重新安排RhoA是Rho GTTRUNK家族的创始成员，该家族是 细胞骨架动力学，并已显示控制脂筏的形成和组成。因此，我们认为， RhoA活性的变化可能影响血小板膜脂筏组装和糖蛋白 混合物.我们通过遗传学和药理学手段的初步研究表明，冷受体 上调RhoA活性，进而诱导血小板活化。我们发现了一个铅RhoA 抑制剂Rhosin/G 04，其对RhoA活化具有特异性，并且能够模拟RhoA敲除以防止 富含糖基转移酶和冷诱导GPIb的脂筏的网格蛋白非依赖性内化 聚类我们假设RhoA抑制可以防止血小板冷诱导的GPIb聚集， 随后在输血时被宿主清除，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。在本建议中，我们将确定 RhoA的主要抑制剂Rhosin/G 04的作用机制，并证明了治疗益处 Rhosin/G 04和类似物用于小鼠模型中有效冷藏长期血小板储存 和非人类灵长类动物。药物发现、药物化学和结构 生物学，细胞生物学和血小板输注专业知识，如共同PI所证明的，导致了这一高度 创新的，开箱即用的方法，用于干预血小板储存技术。小分子 通过干扰蛋白质-蛋白质相互作用靶向RhoA GT3是一个革命性的 这种方法允许可逆地抑制RhoA，并有助于保存冷藏血小板功能。我们 通过临床前药物发现工作的创新将有益于癌症患者的护理。

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.