Design, synthesis, and mechanistic studies of Sansalvamide A derivatives as anti-cancer agents

Includes bibliographical references (p. 203-214)Sansalvamide A (SanA) is a cyclic depsipeptide that was isolated from a marine fungus and demonstrates mid-micromolar anti-cancer activity in the NCI 60-cell line panel. Our laboratory has synthesized over 100 peptide derivatives of this molecule, 5 of which were contributed by the author of this dissertation. The design and solution-phase synthesis of these derivatives is described in Chapter 2. The author was also responsible for attaching PEG-biotin and fluorescein tags to lead SanA derivatives to be used in mechanism of action studies. Chapter 3 describes the mechanism of action studies that were completed with the lead derivatives, both untagged and tagged with PEG-biotin or fluorescein. The untagged compounds are tested in cell proliferation assays against the pancreatic cancer cell line PL-45 and the colon cancer cell line HCT-116. Two of the lead compounds are tested in caspase 3 apoptosis assays and PARP fragmentation analysis. The biotin-tagged compounds are used in pull down assays and it is determined that they bind to the N-middle domain of Hsp90, a well-established oncogenic protein. Hsp90 is responsible for regulating over 200 client proteins, many of which are oncogenic and involved in cancer cell growth. We show that SanA disrupts the binding between Hsp90 and four of these client proteins (Akt, Her2, Hif-1??, and IP6K2) in pure protein binding assays. Chapter 4 investigates SanA's affect proteins that bind to Hsp90's C-domain via their tetratricopeptide repeats (TPRs). Using client protein binding assays, the author determines that the lead SanA derivative disrupts binding between Hsp90 and five TPRcontaining proteins. Finally, Chapter 5 describes the synthesis and preliminary mechanism of action studies of dimerized SanA derivatives (Di-SanA). These compounds are cyclic decapeptides with C-2 rotational symmetry. The author contributed 12 decapeptides, based on one lead derivative from the first generation, which investigated how the placement of D-amino acids around the ring would affect cytotoxicity. These derivatives were synthesized via solid-phase peptide chemistry. The author also investigated mechanism of action of this class of compounds with cell proliferation assays, pull-down assays, and a client protein binding assay