In this review, Kamalinia et al. discuss mRNA display and its role in peptide and protein design.

In recent decades it has become increasingly clear that induction of autophagy plays an important role in the development of treatment resistance and dormancy in many cancer types. Unfortunately, chloroquine (CQ) and hydroxychloroquine (HCQ), two autophagy inhibitors in clinical trials, suffer from poor pharmacokinetics and high toxicity at therapeutic dosages. This has prompted intense interest in the development of targeted autophagy inhibitors to re-sensitize disease to treatment with minimal impact on normal tissue. We utilized Scanning Unnatural Protease Resistant (SUPR) mRNA display to develop macrocyclic peptides targeting the autophagy protein LC3. The resulting peptides bound LC3A and LC3B—two essential components of the autophagosome maturation machinery—with mid-nanomolar affinities and disrupted protein–protein interactions (PPIs) between LC3 and its binding partners in vitro. The most promising LC3-binding SUPR peptide accessed the cytosol at low micromolar concentrations as measured by chloroalkane penetration assay (CAPA) and inhibited starvation-mediated GFP-LC3 puncta formation in a concentration-dependent manner. LC3-binding SUPR peptides re-sensitized platinum-resistant ovarian cancer cells to cisplatin treatment and triggered accumulation of the adapter protein p62 suggesting decreased autophagic flux through successful disruption of LC3 PPIs in cell culture. In mouse models of metastatic ovarian cancer, treatment with LC3-binding SUPR peptides and carboplatin resulted in almost complete inhibition of tumor growth after four weeks of treatment. These results indicate that SUPR peptide mRNA display can be used to develop cell-penetrating macrocyclic peptides that target and disrupt the autophagic machinery in vitro and in vivo.

Iterative in situ click chemistry provides a route to novel mutli-peptide ligands with antibody-like affinity and specificity.

Autophagy is a bulk catabolic process that modulates tumorigenesis, therapeutic resistance, and dormancy. The tumor suppressor ARHI (DIRAS3) is a potent inducer of autophagy and its expression results in necroptotic cell death in vitro and tumor dormancy in vivo. ARHI is down-regulated or lost in over 60 % of primary ovarian tumors yet is dramatically up-regulated in metastatic disease. The metabolic changes that occur during ARHI induction and their role in modulating death and dormancy are unknown. We employed Nuclear Magnetic Resonance (NMR)-based metabolomic strategies to characterize changes in key metabolic pathways in both cell culture and xenograft models of ARHI expression and autophagy. These pathways were further interrogated by cell-based immunofluorescence imaging, tracer uptake studies, targeted metabolic inhibition, and in vivo PET/CT imaging. Induction of ARHI in cell culture models resulted in an autophagy-dependent increase in lactate production along with increased glucose uptake and enhanced sensitivity to glycolytic inhibitors. Increased uptake of glutamine was also dependent on autophagy and dramatically sensitized cultured ARHI-expressing ovarian cancer cell lines to glutaminase inhibition. Induction of ARHI resulted in a reduction in mitochondrial respiration, decreased mitochondrial membrane potential, and decreased Tom20 staining suggesting an ARHI-dependent loss of mitochondrial function. ARHI induction in mouse xenograft models resulted in an increase in free amino acids, a transient increase in [18F]-FDG uptake, and significantly altered choline metabolism. ARHI expression has previously been shown to trigger autophagy-associated necroptosis in cell culture. In this study, we have demonstrated that ARHI expression results in decreased cellular ATP/ADP, increased oxidative stress, and decreased mitochondrial function. While this bioenergetic shock is consistent with programmed necrosis, our data indicates that the accompanying up-regulation of glycolysis and glutaminolysis is autophagy-dependent and serves to support cell viability rather than facilitate necroptotic cell death. While the mechanistic basis for metabolic up-regulation following ARHI induction is unknown, our preliminary data suggest that decreased mitochondrial function and increased metabolic demand may play a role. These alterations in fundamental metabolic pathways during autophagy-associated necroptosis may provide the basis for new therapeutic strategies for the treatment of dormant ovarian tumors.

Autophagy can protect cancer cells from acute starvation and enhance resistance to chemotherapy. Previously, we reported that autophagy plays a critical role in the survival of dormant, drug resistant ovarian cancer cells using human xenograft models and correlated the up-regulation of autophagy and DIRAS3 expression in clinical samples obtained during “second look” operations. DIRAS3 is an imprinted tumor suppressor gene that encodes a 26 kD GTPase with homology to RAS that inhibits cancer cell proliferation and motility. Re-expression of DIRAS3 in ovarian cancer xenografts also induces dormancy and autophagy. DIRAS3 can bind to Beclin1 forming the Autophagy Initiation Complex that triggers autophagosome formation. Both the N-terminus of DIRAS3 (residues 15–33) and the switch II region of DIRAS3 (residues 93–107) interact directly with BECN1. We have identified an autophagy-inhibiting peptide based on the switch II region of DIRAS3 linked to Tat peptide that is taken up by ovarian cancer cells, binds Beclin1 and inhibits starvation-induced DIRAS3-mediated autophagy.