Synthetic and cellular methods for developing membrane-permeable antagonists of protein-protein interactions

Megan Elizabeth Bucks, Purdue University

Abstract

Aberrant protein–protein interactions are implicated in numerous disease states, necessitating discovery of low-molecular-weight inhibitors that exhibit potent interaction antagonism and facile permeation of cellular membranes. Peptides are promising candidates for inhibiting protein–protein complexes because they are capable of displaying complex epitopes composed of diverse functionalities. Poor membrane permeation generally limits the cellular utility of peptides, however, due to the high energies required to desolvate the polar amide backbone during the internalization process. Peptidomimetic structures combining peptide activity with improved cellular internalization and proteolytic stability are therefore more promising protein–protein complex modulators. Design of such structures requires detailed understanding of how structural modifications affect peptide activity and permeation. The studies presented herein describe the development of methods for converting bioactive peptides that modulate protein–protein interactions into cell-permeable probes of biological function, specifically to target the Hdm2–p53 and Hdmx–p53 interactions implicated in many cancers. Solid-phase attachment strategies for efficient synthesis of backbone-cyclic and terminally-modified linear peptides were devised and implemented for preparing novel Hdmx–p53 antagonists. A cellular internalization assay implemented in Escherichia coli cells was developed and validated for real-time assessment of relative permeation rates for small molecules and peptides derivatized as chromogenic disulfides. A series of mono-N-methylated dipeptide chromogens was assayed to evaluate the effect of removing backbone hydrogen-bond donors on peptide internalization. Similarly, N-methylated analogs of an anti-Hdmx tripeptide were synthesized using the novel solid-phase strategy and evaluated for retention of biological activity exhibited by the parent tripeptide. Furthermore, synthetic methodologies for generating substituted polyether and polyester macrocycles as mimics of cyclic peptides were investigated. Collectively, these studies yielded generic methods for developing cell-permeable probes based on bioactive peptides targeting protein complexes implicated in disease.

Degree

Ph.D.

Advisors

Savinov, Purdue University.

Subject Area

Biochemistry|Organic chemistry

COinS