Bleomycins (BLMs) are a class of water soluble, glycopeptide-derived antitumor antibiotics consisting of a structurally complicated unnatural hexapeptide and a disaccharide, clinically used as an anticancer chemotherapeutic agent at an exceptionally low therapeutic dose. The efficiency of BLM is likely achieved both by selective localization within tumor cells and selective binding to DNA followed by efficient double-strand cleavage. The disaccharide moiety is responsible for the tumor cell targeting properties of BLM. A recent study showed that both BLM and its disaccharide, conjugated to the cyanine dye Cy5**, bound selectively to cancer cells. Thus, the disaccharide moiety alone recapitulates the tumor cell targeting properties of BLM. Work presented here describes the synthesis of the fluorescent carbohydrate conjugates. A number of dye-labeled modified disaccharides and monosaccharides were synthesized to study the nature of the participation of the carbamoyl moiety in the mechanism of tumor cell recognition and uptake by BLM saccharides. It was demonstrated that the carbamoylmannose moiety of BLM is the smallest structural entity capable for the cellular targeting and internalization, and the carbamoyl functionality is indispensible for tumor cell targeting. It was also confirmed that BLM is a modular molecule, composed of a tumor cell targeting moiety (the saccharide) attached to a cytotoxic DNA cleaving domain (the BLM aglycone). These finding encouraged us to further synthesize carbohydrate probes for PET imaging and to conjugate the saccharide moiety with cytotoxins for targeted delivery to tumor cells.
The misacylated suppressor tRNA technique has enabled the site-specific incorporation of noncanonical amino acids into proteins. The focus of the present work was the synthesis of unnatural lysine analogues with nucleophilic properties for incorporation at position 72 of the lyase domain of human DNA polymerase beta, a multifunctional enzyme with dRP lyase and polymerase activity.
Illustrated in Chapter 1 is the general history of research on the interaction of DNA and anticancer drugs, most importantly different congener of bleomycin (BLM). Additionally, several synthetic analogues of bleomycin, including the structural components and functionalities, are discussed.
Chapter 2 describes a new approach to study the double-strand DNA lesion caused by antitumor drug bleomycin. The hairpin DNA library used in this study displays numerous cleavage sites demonstrating the versatility of bleomycin interaction with DNA. Interestingly, some of those cleavage sites suggest a novel mechanism of bleomycin interaction, which has not been reported before.
Cytidine methylation has generally been found to decrease site-specific cleavage of DNA by BLM, possibly due to structural change and subsequent reduced bleomycin-mediated recognition of DNA. As illustrated in Chapter 3, three hairpin DNAs known to be strongly bound by bleomycin, and their methylated counterparts, were used to study the dynamics of bleomycin-induced degradation of DNAs in cancer cells. Interestingly, cytidine methylation on one of the DNAs has also shown a major shift in the intensity of bleomycin induced double-strand DNA cleavage pattern, which is known to be a more potent form of bleomycin induced cleavages.
DNA secondary structures are known to play important roles in gene regulation. Chapter 4 demonstrates a structural change of the BCL2 promoter element as a result of its dynamic interaction with the individual domains of hnRNP LL, which is essential to facilitate the transcription of BCL2. Furthermore, an in vitro protein synthesis technique has been employed to study the dynamic interaction between protein domains and the i-motif DNA within the promoter element. Several constructs were made involving replacement of a single amino acid with a fluorescent analogue, and these were used to study FRET between domain 1 and the i-motif, the later of which harbored a fluorescent acceptor nucleotide analogue.
The majority of trust research has focused on the benefits trust can have for individual actors, institutions, and organizations. This “optimistic bias” is particularly evident in work focused on institutional trust, where concepts such as procedural justice, shared values, and moral responsibility have gained prominence. But trust in institutions may not be exclusively good. We reveal implications for the “dark side” of institutional trust by reviewing relevant theories and empirical research that can contribute to a more holistic understanding. We frame our discussion by suggesting there may be a “Goldilocks principle” of institutional trust, where trust that is too low (typically the focus) or too high (not usually considered by trust researchers) may be problematic. The chapter focuses on the issue of too-high trust and processes through which such too-high trust might emerge. Specifically, excessive trust might result from external, internal, and intersecting external-internal processes. External processes refer to the actions institutions take that affect public trust, while internal processes refer to intrapersonal factors affecting a trustor’s level of trust. We describe how the beneficial psychological and behavioral outcomes of trust can be mitigated or circumvented through these processes and highlight the implications of a “darkest” side of trust when they intersect. We draw upon research on organizations and legal, governmental, and political systems to demonstrate the dark side of trust in different contexts. The conclusion outlines directions for future research and encourages researchers to consider the ethical nuances of studying how to increase institutional trust.