Introduction

The persistence of dormant, drug resistant ovarian cancer has been shown to be driven by the up-regulation of autophagy, a bulk-catabolic process in which proteins, lipids, and organelles are engulfed by autophagosomes and degraded in autolysosomes. Although the molecular mechanics of autophagy have been studied in great detail, targeted inhibition of autophagy in the clinic has been hampered by the lack of reagents available that selectively bind to components of the autophagic machinery and modulate the protein-protein interactions (PPIs) that govern this process. The focus of our project is the development of cell-permeable, macrocyclic peptides that selectively and potently inhibit the PPIs involved in autophagosome initiation and maturation. 

Methods

We employed a directed evolution strategy known as Scanning Unnatural Protease Resistance (SUPR) mRNA Display to develop high affinity macrocyclic peptides that selectively target the autophagy proteins LC3 and Beclin1. SUPR mRNA Display libraries are highly complex (10¹² - 10¹⁴ unique sequences), contain non-proteogenic N-methyl amino acids for protease resistance, and are covalently cyclized for structural rigidity. We carried out SUPR peptide mRNA selections against immobilized LC3A and Beclin1 and obtained lead peptide sequences from the final selection pools. These peptides were chemically synthesized and target binding was measured by fluorescence polarization anisotropy (FP). The most promising peptides were tested for their effect on autophagy biomarkers, autophagic flux, and cell viability in a panel of ovarian and pancreatic cancer cell lines. In vivo efficacy in combination with cisplatin was measured in an orthotopic mouse model of ovarian cancer. 

Results

LC3A is a small, ubiquitin-like protein involved in autophagosome formation and maturation. Initial selections against LC3A were carried out with the MX8K cyclic library (~10¹² sequences) and resulted in the identification of the lead peptide SUPR4B1W. SUPR4B1W bound to LC3A and LC3B with mid-nanomolar affinity and inhibited autophagic flux in both ovarian and pancreatic cell lines at low micromolar concentrations. SURP4B1W was highly cell-permeable by the chloroalkane penetration assay (CAPA) and this permeability was found to be dependent on both cyclization and backbone N-methylation. SUPR4B1W, in combination with carboplatin, dramatically inhibited tumor growth in vivo with no apparent toxicity at 10 m.p.k. three times per week. To further enhance affinity, additional selections against LC3A were carried out using the larger MX10K library (10¹³ - 10¹⁴ sequences).

Full-length Beclin1 was expressed, purified, and immobilized on streptavidin resin. Selections against the target were carried out using the MX8K library under both reducing and non-reducing conditions. The final round pools from all selections were sequenced and lead cyclic peptides identified.

Conclusion

SUPR peptide mRNA Display was employed to successfully identify cyclic peptides that bind to LC3A and Beclin1. The most promising LC3A-binding SUPR peptide inhibited autophagic flux and sensitized cells in culture to cisplatin treatment. The cell permeability and protease stability of SUPR peptides was found to depend on both macrocyclization and N-methylation suggesting that these characteristics may represent general solutions to impart bioactivity to peptide-based lead compounds. The combination of carboplatin and LC3-binding SUPR peptides resulted in nearly complete eradication of tumor burden in a mouse model of ovarian cancer indicating that the stability, affinity, and cell permeability of SUPR peptides is maintained in vivo. These results validate SUPR peptide mRNA display as a discovery platform for cell-permeable, protease stable lead compounds for the disruption of protein-protein interactions (PPIs) in the autophagy pathway.