NOVEL PHENOTHIAZINES FOR DECONTAMINATION OF RED CELLS

该提案的总体目标是开发一种技术， 灭活浓缩红细胞中的病毒和其他病原体 （PRBC）。这将通过化学合成和生物合成来实现 光化学降解的新型试剂的表征 核酸在吸收红光时的修饰。起始 点是一类化合物被称为吩噻嗪，一组 具有已证实的抗病毒活性的三环杂原子染料。 吩噻嗪通过嵌入结合到DNA和RNA，并且它们已经被 用直接测序法显示了裸露脊髓灰质炎病毒RNA和质粒DNA 光化学改性已知的吩噻嗪如亚甲蓝， 然而，对于PRBC治疗有几个缺陷：吩噻嗪结合到 并通过产生单线态氧不加选择地破坏红细胞膜 （1-O2）。此外，已知的吩噻嗪类药物不能抑制与HIV相关的 在红细胞功能受损的情况下， 树立政治意识本提案中概述的化合物旨在 通过分子设计改进克服这些限制：（1） 核酸选择性通过添加化学试剂而增加。 增强与DNA和RNA结合的功能。(2)膜分配 化合物通过消除侧链基团而减少， 疏水性环境。(3)细胞内病毒的灭活是 通过合成具有增加的渗透到细胞中的化合物而增强。 (4)提出侧链修饰以减缓 红细胞中化合物的酶促还原，这是一个消耗 光活性剂的浓度。(5)杂原子取代是 建议优化化合物的吸收特性， 在PRBC。(6)吩噻嗪骨架的电子修饰是 提出促进激发态光化学氧化还原反应， 促进吩噻嗪与核酸的直接结合。（七） 研究了分子内猝灭基团，以减少非特异性 红细胞膜损伤。病毒灭活将通过以下方式进行评估： HlV、鸭乙型肝炎病毒、HCV和几种噬菌体灭活 模型系统化合物将在各种生化筛选 测定1-O2产生、DNA结合亲和力、酶促 减少红细胞，和光化学修饰的核酸， 体外PCD后的红细胞功能将通过多种方法进行测量， 体外和体内试验，包括输血后恢复和存活 在两个模型动物系统中。一个实用的净化系统模型将 使用最好的新型化合物开发，该系统将 使用全尺寸PRBC单元进行验证。

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.