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.
