Design, synthesis, and evaluation of novel modulators of protein kinase C [electronic resource]

The theme of this work is the application of function-oriented design and synthesis to enhance our understanding of the biological effects of protein kinase C (PKC) modulators, including prostratin, ingenol esters, and bryostatin, and to apply this new understanding to further improve upon these modulators. Chapter 1 surveys the competitive landscape for therapeutic efforts focused on PKC modulation. This chapter discusses the various methods for exogenously regulating PKC, including kinase site inhibition and disruption of protein-protein interactions, with a focus on C1-domain based allosteric ligands (such as prostratin and bryostatin). There are now a number of small molecule families with natural or non-natural members that have demonstrated the ability to modulate PKC via the C1 domain. These families include the tiglianes, ingenanes, and daphnanes; and the bryostatins. Chapter 2 examines both the tigliane and ingenane scaffolds, as well as the relation between them. A prodrug strategy was applied to prostratin and ingenol analogs, resulting in a small library of prodrugs that offer a range of stability, release, and physical properties. The tigliane (prostratin) and ingenane scaffolds are related structurally, in addition to their similar biological activities, and the rearrangement from a tigiliane scaffold to the ingenane core has been pursued. Chapter 3 details an approach to prepare more accessible compounds with the same or better efficacy as bryostatin 1. This strategy centered on a designed scaffold derived from salicylic acid which was prepared in just 23 steps, nearly half of that required to prepare the natural bryostatin scaffolds. The parent salicylate system and related compounds were found to be potent inhibitors of Chikungunya virus, albeit with a likely PKC-independent mechanism. Chapter 4 presents an evaluation of PKC modulators, with a focus on binding affinity to PKC and ability to translocate a PKC-GFP fusion protein to the plasma membrane, a key step in the activation of PKC. Chapter 5 represents a slight departure from the theme of PKC modulators, but this work was initially inspired by the problem of efficient delivery of PKC-GFP plasmid DNA (pDNA) to cells and is still driven by the principles of using design and synthesis to impact therapeutically relevant systems. It details an approach to address the challenge of delivering DNA, employing charge-altering releasable transporters (CARTs) that were previously successful for the delivery of mRNA. These CARTs initially function as cationic agents that non-covalently complex DNA and transfect cells, but degrade upon cellular entry to actively release the DNA. A series of lipid-rich CARTs were prepared and evaluated for in vitro pDNA delivery. These co-oligomers were synthesized in only two steps via sequential organocatalytic ring-opening polymerization of lipid-containing cyclic carbonate monomers and morpholinone monomers. Lipid block variations were found to have substantial effects on the delivery efficiency of pDNA, with oleyl- and linoleyl-based systems showing enhanced performance relative to the commercial transfection agent Lipofectamine 2000 (Lipo). The optimized oleyl co-oligomer was carried forward to study stable transfection with a Sleeping Beauty (SB) transposase system. In this system the oleyl co-oligomer demonstrated improved stable transfection compared to Lipo through four generations. The success of these co-oligomers, especially as agents for long-term stable transfection, suggests their potential for future DNA delivery applications.

Keywords: Chemistry.
Advisor: Wender Paul A. primary advisor. Thesis advisor
Bertozzi Carolyn R.
Du Bois Justin advisor. Thesis advisor