Stapled Peptide Inhibitors of Autophagy Adapter LC3B

Abstract

A growing body of evidence suggests that autophagy inhibition enhances the effectiveness of chemotherapy, especially in difficult-to-treat cancers. Existing autophagy inhibitors are primarily lysosomotropic agents. More specific autophagy inhibitors are highly sought-after. The microtubule-associated protein 1A/1B light chain 3B protein, LC3B, is an adapter protein that mediates key protein-protein interactions at several points in autophagy pathways. In this work, we used a known peptide ligand as a starting point to develop improved LC3B inhibitors. We obtained structure-activity relationships that quantify the binding contributions of peptide termini, individual charged residues, and hydrophobic interactions. Based on these data, we used artificial amino acids and diversity-oriented stapling to improve affinity and resistance to biological degradation, while maintaining or improving LC3B affinity and selectivity. These peptides represent the highest-affinity LC3B-selective ligands reported to date, and they will be useful tools for further elucidation of LC3B's role in autophagy and in cancer.

Structure-Based Design of Stapled Peptides That Bind GABARAP and Inhibit Autophagy

“…Covalent linking of side chains, or “peptide stapling,” is a promising strategy to minimize these limitations . While most stapled peptides are α-helical, we and others have shown that appropriately designed staples can improve the properties of peptides in other conformations as well. ,− Based on the crystal structure of K1 bound to GABARAP, we designed peptides Cys5, Cys4, and Cys3, which stapled K1 in ( i , i + 3), ( i , i + 4), and ( i , i + 5) positions, respectively (Figure a). We stapled each peptide using dithiol bis-alkylation with ortho- , meta- , and para- dimethylbenzene linkers, allowing for a “diversity-oriented stapling” approach, where the staple geometry was varied (Figure a). ,, Stapling in ( i , i + 3) positions (the Cys5 peptides) reduced binding to both GABARAP and LC3B compared to K1.…”

“…23−25 In previous work, we described structure-activity relationships for an LC3specific LIR motif derived from FYCO1, a Rab7 effector protein that mediates autophagosome trafficking. 26 We also described peptides with side-chain-to-side-chain crosslinks, or "stapled" peptides, with improved affinity, selectivity for LC3B over GABARAP, proteolytic stability, and cytosolic delivery. In this work, we report two new classes of stapled peptides that inhibit LC3/GABARAP proteins with nanomolar affinity, one GABARAP-selective class and another that inhibits both subfamilies.…”

“…LIR motifs bind Atg8-family proteins in an extended conformation anchored by conserved hydrophobic side chains. ,, Several studies have explored structural determinants of selectivity for LC3 and GABARAP proteins, and generally, it was observed that GABARAPs are able to accommodate a wider variety of LIR ligands. − In previous work, we described structure-activity relationships for an LC3-specific LIR motif derived from FYCO1, a Rab7 effector protein that mediates autophagosome trafficking . We also described peptides with side-chain-to-side-chain crosslinks, or “stapled” peptides, with improved affinity, selectivity for LC3B over GABARAP, proteolytic stability, and cytosolic delivery.…”

Dapagliflozin Inhibits Ventricular Remodeling in Heart Failure Rats by Activating Autophagy through AMPK/mTOR Pathway

“…Thus, we attempted to clarify whether DAPA activated autophagy pathway and the AMPK/mTOR signaling. LC3B is an adapter protein mediating vital protein-protein interactions in autophagy pathways [ 21 ]. Thus, we examined LC3B expression changes in myocardial tissue of rats using IHC.…”

Stapled β-Hairpins Featuring 4-Mercaptoproline

“…One of the potential advantages of incorporating non-proteinogenic amino acids and covalent staples is resistance to proteolytic degradation. To test whether the designed β-hairpins were stable to degradation, we adapted a cell lysate stability assay recently applied for development of olefin-stapled α-helices. − Because HeLa cell lysate contains all cellular proteases including lysosomal proteases, this represents a relatively rigorous test for metabolic stability of peptide therapeutics. Control peptides HP7 and ΔHP7 were degraded rapidly, with roughly 0% and 19%, respectively, remaining after only 2 h (Figure a).…”