Directed evolution of imaging agents and therapeutics targeting LC3 and autophagy

• Published in: The Journal of nuclear medicine (1978) Vol. 59; no. 2; p. 357
• Main Authors: Gray, Joshua P, Kelderhouse, Lindsay E, Lu, Zhen, Bast, Robert C, Millward, Steven W
• Format: Journal Article
• Language: English
• Published: New York Society of Nuclear Medicine 01.02.2018
• Subjects: Adaptor proteins; Affinity; Alanine; Amino acid sequence; Amino acids; Apoptosis; Autophagy; Binding; Cancer; Cell death; Chloroquine; Clinical trials; Conserved sequence; Cytosol; Directed evolution; Displays; Eukaryotes; Evolution; Gene sequencing; Homeostasis; Hydroxychloroquine; Hypoxia; Inhibitors; Medical imaging; Medical research; mRNA; Organelles; Ovarian cancer; Peptides; Permeability; Phagocytosis; Phagosomes; Protease; Protein interaction; Proteinase; Proteins; Targeted cancer therapy; Toxicity; Tumorigenesis; Ubiquitin
• ISSN: 0161-5505; 1535-5667

Autophagy is a homeostatic cellular process highly conserved among eukaryotes in which damaged proteins and organelles in the cytosol are sequestered by double-membraned autophagosomes and shepherded to the lysosome for degradation. Dysregulation of autophagy has been observed in cancer both as a mechanism to avoid programmed cell death and as a means to survive the hypoxic, nutrient deprived conditions often found in the tumor microenvironment. Study of the role of autophagy on tumorigenesis has been hampered by the lack of selective autophagy inhibitors. Chloroquine and hydroxychloroquine, the only autophagy inhibitors to enter clinical trials, are non-specific lysosomotropic agents and have generally shown poor efficacy and high toxicity. LC3, an 18 kDa ubiquitin-like protein, plays a critical role in the maturation of autophagosomes and the selective recruitment of cargo to the autophagosome interior. Novel ligands that selectively bind to LC3 could be of immense value for tracking autophagy in living cells and for disrupting protein-protein interactions critical for autophagosome function. We have utilized SUPR peptide mRNA display to design LC3-targeted macrocyclic peptides for inhibition and molecular imaging of autophagy. SUPR peptide mRNA display is a directed evolution process in which translated peptides are covalently bound to their encoding mRNA allowing trillions of unique peptide sequences to be iteratively sieved for binding to a target protein. By incorporating unnatural, N-methyl amino acids and posttranslational cyclization, peptides with extraordinary protease resistance and nanomolar affinities can be obtained. These Scanning Unnatural Protease Resistant (SUPR) peptides bind target proteins with antibodylike affinities while potentially maintaining the tumor- and cell-penetrating properties of small molecules. SUPR peptide mRNA display selections incorporating N-methyl alanine were performed against recombinant LC3. After 7 rounds of selection, library convergence was observed by binding and PCR analysis. Subsequent sequencing of the library revealed that the final pool was dominated by two families of peptides which each show a consensus amino acid sequence analogous to the LC3 Interacting Motif (LIM) observed in natural LC3 adaptor proteins. Moreover, N-methyl alanine was found to be incorporated at a single position within the macrocycle in all sequences, suggesting that this unnatural residue plays a key role in enforcing cyclic peptide conformation and function. Binding analysis of individual clones confirmed the presence of LC3-binding sequences and indicated several residues within and surrounding the modified LIM that play a significant role in peptide affinity. The most promising LC3-binding SUPR peptides have been chemically synthesized and tested for cell-permeability, binding affinity, and serum-stability to identify candidates for further testing in starvation- and DIRAS3-induced models of ovarian cancer autophagy.