NOVEL PHENOTHIAZINES FOR DECONTAMINATION OF RED CELLS

这项提案的总体目标是开发一种技术，用于 红细胞中病毒和其他病原体的灭活 (PRBC)。这将通过化学合成和生物合成来实现。 新型光化学灭活剂的表征 核酸在吸收红光时的修饰。起跑线 POINT是一类被称为吩噻嗪的化合物，一组 经证实具有抗病毒活性的三环杂原子染料。 吩噻嗪通过插入DNA和RNA结合在一起，它们已经 显示通过直接灭活裸脊髓灰质炎病毒RNA和质粒DNA 光化学修饰。已知的吩噻嗪类药物，如亚甲蓝， 然而，治疗PRBC有几个缺陷：吩噻嗪结合到 通过产生单线态氧不分青红皂白地破坏红细胞膜 (1-O2)。此外，已知的吩噻嗪不能灭活与艾滋病毒相关的 感染的淋巴细胞在红血球功能 维护好了。本提案中概述的化合物旨在 通过分子设计修改克服这些限制：(1) 通过添加化学物质来提高核酸的选择性。 增强与DNA和RNA结合的功能。(2)膜分离 化合物通过消除侧链基团减少，有利于 疏水环境。(3)细胞内病毒灭活 通过合成具有更多渗透到细胞的化合物来增强。 (4)对侧链进行修饰，以减缓反应动力学。 红细胞中化合物的酶促还原，这一过程会耗尽 光敏剂的浓度。(5)杂原子取代为 建议优化化合物的吸光度特性以供使用 在PRBC。(6)吩噻嗪支架的电子修饰为 提出了促进激发态光化学氧化还原反应和 促进吩噻嗪与核酸的直接结合。(7) 分子内猝灭基团被研究以减少非特异性 对红细胞膜的破坏。将通过以下方式评估病毒灭活 HLV、鸭乙型肝炎病毒、丙型肝炎病毒和几种噬菌体的灭活 模型系统。化合物将在各种生化反应中进行筛选 酶促1-O2产生、DNA结合亲和力的测定 红细胞的还原和核酸的光化学修饰 体外培养。PCD后的红细胞功能将通过各种不同的指标进行测量 包括输血后恢复和存活的体外和体内检测 在两个模型动物系统中。一个实用的模型净化系统将 使用最好的新型化合物开发，该系统将被 使用全尺寸PRBC单位进行验证。

The overall goal of this proposal is to develop a technology for inactivation of viruses and other pathogens in packed red blood cells (PRBC). This will be achieved through chemical synthesis and biological characterization of novel agents that inactivate by photochemical modification of nucleic acids upon absorption of red light. The starting point is a class of compounds known as the phenothiazines, a group of tricyclic, heteroatomic dyes with proven antiviral activity. Phenothiazines bind by intercalation to DNA and RNA, and they have been shown to inactivate naked polio virus RNA and plasmid DNA by direct photochemical modification. Known phenothiazines such as methylene blue, however, have several flaws for treatment of PRBC: Phenothiazines bind to and damage red cell membranes indiscriminantly by producing singlet oxygen (1-O2). Furthermore, known phenothiazines cannot inactivate HIV associated with infected lymphocytes under conditions where red cell function is maintained. The compounds outlined in this proposal are designed to overcome these limitations through molecular design modifications: (1) Nucleic acid selectivity is increased through the addition of chemical functions to enhance binding to DNA and RNA. (2) Membrane partitioning of compounds is decreased by elimination of side chains groups which favor hydrophobic environments. (3) Inactivation of intracellular virus is enhanced by synthesis of compounds with increased penetration into cells. (4) Side chain modifications are proposed to slow the kinetics of enzymatic reduction of compounds in red cells, a process that depletes the concentration of photo active agent. (5) Heteroatom substitutions are proposed to optimize the absorbance characteristics of compounds for use in PRBC. (6) Electronic modifications of the phenothiazine scaffold are proposed to promote excited state photochemical redox reactions and promote direct binding of the phenothiazine to nucleic acid. (7) Intramolecular quenching groups are investigated to reduce non-specific damage to red cell membranes. Viral inactivation will be assessed by inactivation of HlV, duck hepatitis B virus, HCV and several bacteriophage model systems. Compounds will be screened in a variety of biochemical assays to measure 1-O2 production, DNA binding affinity, enzymatic reduction by red cells, and photochemical modification of nucleic acids in vitro. Erythrocyte function after PCD will be measured by a variety of in vitro and in vivo assays including post transfusion recovery and survival in two model animal systems. A practical model decontamination system will be developed using the best novel compound, and the system will be validated using full size units of PRBC.