Merocyanine 540 (MC540) is a polymethine dye that was originally developed as a sensitizing agent for photographic emulsions. We and others have subsequently shown that MC540 is also an effective and versatile agent for the extracorporal purging of autologous bone marrow and peripheral blood stem cell grafts, the sterilization of blood components contaminated with enveloped viruses or protozoan parasites, the identification of leukemia cells in peripheral blood, the quantitation of apoptotic cells, the recording of transmembrane potential changes, and the identification of developmentally regulated membrane constituents. In collaboration with our laboratory, W.H.H. Gunther has synthesized a panel of structural analogues of MC540 specifically designed for biomedical applications. Some of these analogues are orders of magnitude more potent than MC540 as anti- leukemic and antiviral agents but retain most or all desirable properties of the parent compound. The mechanism of action of MC540 is poorly understood. Even less is known about second generation merocyanines and the basis of the dramatically improve antineoplastic and anti-viral activity. We recently discovered that the most potent structural analogues of MC540 generate a photocompound that covalently binds to serum albumin (but not to other plasma proteins) to form stable adducts that are more cytotoxic leukemia cells than to normal hematopoietic progenitor cells. We hypothesize that these photoproducts play a crucial role in the enhanced antineoplastic activity of second generation merocyanines. To test our hypotheses we propose 1) to identify the structures of the photoproduct and cytotoxic albumin adducts formed by one of our most potent second generation merocyanines (MC54), 2) to analyze the binding, cellular uptake, and subcellular localization of photoproduct-albumin adducts and to investigate the molecular basis of their differential cytotoxicity, 3) to define the role of cytotoxic adducts in merocyanine-mediated photodynamic therapy, and 4) to explore the potential utility of merocyanine-derived photoproduct-albumin adducts for systemic chemotherapy. This application focuses on photoproducts and albumin adducts generated by a single second generation merocyanine dye. However, preliminary data already indicate that similar photoproducts and cytotoxic adducts are also generated by other merocyanine and non-merocyanine photosensitizers. The proposed investigations into the nature and mechanism of action of MC54- derived photoproducts and cytotoxic adducts thus have broad theoretical and practical implications for major segments of photochemotherapy and chemotherapy in general.
