Hydrocarbon stapled interfacial peptide inhibitors of HIV -1 integrase activity and dimerization and branched polyguanidinium transporters for cellular delivery

Stefan J Hershberger, Purdue University

Abstract

HIV integrase, a vital enzyme in the life cycle of the human immunodeficiency virus (HIV), is responsible for the integration of viral DNA into the host genome. The essential nature of HIV integrase in viral replication has made it the focus of numerous inhibition efforts. Peptides derived from the interfacial region of dimeric HIV integrase have previously been evaluated as inhibitors of integrase dimerization and 3'-endonuclease activity and three peptides were found to be moderately potent inhibitors. While each interfacial peptide is known to assume a distinct a-helical secondary structure in the context of the integrase protein, the peptides adopted a random coil conformation in aqueous media. Therefore, an investigation was undertaken to determine the role of secondary structure on the dimerization inhibition of integrase. To this end, a hydrocarbon staple was employed to constrain the random coil α5 peptide into an α-helical conformation. This staple was formed by the synthesis and incorporation of two unnatural amino acids containing alkene side chains into the peptide sequence. The amino acid side chains were then coupled via olefin metathesis to afford a hydrocarbon staple. Peptide design, monomer synthesis, and solid phase peptide synthesis of the constrained peptides as well as the appropriate control peptides were undertaken and resulted in increased helical character and enhanced 3'-processing inhibitory potency for all constrained moieties. Cell penetrating peptides (CPPs), short peptide sequences typically rich in cationic amino acids, are well studied and have proven beneficial in the cellular delivery of therapeutic agents. Research on the structural features of CPPs led to the conclusion that a linear peptide backbone is not required for cellular internalization. In this project, a series of branched polyguanidinium molecules, containing 2, 4, or 8 guanidinium groups were designed and synthesized as potential prodrug molecules. An alcohol linker was added for the versatile attachment of therapeutic agents via an ester linkage; thus creating a cationic prodrug molecule. In addition, the carbon chain length of each branch of the dendrimer-like molecule was designed to be varied in an effort to probe the effect of branch length on cellular uptake. These compounds were analyzed for cellular uptake in human cancer cells as well as their effect on cell viability to determine their potential as prodrug molecules. The compound containg four guanidinium moieties demonstrated a 5-fold increase in cellular uptake in comparison to the well-studied Tatp. A cell viability assay confirmed the utility of these compounds as molecular transporters as they were found to impart little cytotoxicity to MCF-7 cells at low concentrations for a sustained time period.

Degree

Ph.D.

Advisors

Chmielewski, Purdue University.

Subject Area

Organic chemistry

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