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

Abstract

The LC3/GABARAP family of proteins is involved in nearly every stage of autophagy. Inhibition of LC3/GABARAP proteins is a promising approach to blocking autophagy, which sensitizes advanced cancers to DNA-damaging chemotherapy. Here, we report the structure-based design of stapled peptides that inhibit GABARAP with nanomolar affinities. Small changes in staple structure produced stapled peptides with very different binding modes and functional differences in LC3/GABARAP paralog selectivity, ranging from highly GABARAP-specific to broad inhibition of both subfamilies. The stapled peptides exhibited considerable cytosolic penetration and resistance to biological degradation. They also reduced autophagic flux in cultured ovarian cancer cells and sensitized ovarian cancer cells to cisplatin. These small, potent stapled peptides represent promising autophagy-modulating compounds that can be developed as novel cancer therapeutics and novel mediators of targeted protein degradation.

Semisynthetic LC3 Probes for Autophagy Pathways Reveal a Noncanonical LC3 Interacting Region Motif Crucial for the Enzymatic Activity of Human ATG3

“…We also postulated that cyclization of the LIR peptide would recapitulate its β-sheet conformation and lead to tighter binding than the linear peptide due to preorganization . This approach has recently been shown to be successful in the de novo design of GABARAP-specific peptide binders . Cyclic peptide 6 was prepared by solid-phase peptide synthesis followed by cyclization using selective cysteine alkylation (Figure S11).…”

“…37 This approach has recently been shown to be successful in the de novo design of GABARAP-specific peptide binders. 38 Cyclic peptide 6 was prepared by solid-phase peptide synthesis followed by cyclization using selective cysteine alkylation (Figure S11). 39,40 Peptide 6 inhibited the reaction of 2 with ATG3 C1 with an IC 50 of 24 μM (Figure 2c,d).…”

Curcumin-loaded graphene oxide quantum dots enhance otoprotective effects via blocking cuproptosis

“…Considerable efforts have been made to explore new drugs to reduce or prevent CIS-related ototoxicity ( Ho et al, 2022 ). Although no drugs have been approved to be completely effective to date ( Salaroglio et al, 2022 ), many drugs have achieved success in preclinical work and some of them have shown protective effects in the clinic ( Brown et al, 2022 ). In this study, we established GOQDs for loading CUR to successfully promote penetration of RWM and maintained CUR concentration in the inner ear perilymph for 24 h after a single injection.…”

Computational Prediction of Cyclic Peptide Structural Ensembles and Application to the Design of Keap1 Binders

“…Baker and colleagues have reported methods for designing well-structured head-to-tail CPs and recently extended their methodology to designing CPs with greater passive membrane permeability. , However, these efforts have been limited to well-structured head-to-tail CPs with natural amino acids and their enantiomers, and they have not yet succeeded in the routine design of PPI inhibitors. As an alternative to head-to-tail cyclization, cysteine alkylation reactions allow for improved rational design and library development for CP discovery, − and they can induce a variety of secondary structures in peptides to improve affinity and specificity . For example, cross-linking using cysteine alkylation increased α-helicity, bioactivity, and cell permeability for a series of BH3 peptide-derived MCL-1 ligands. , The Kritzer group has also used cysteine alkylation with ortho- , meta- , and para -dibromomethylbenzene linkers, originally described by Timmerman et al, for “diversity-oriented stapling” in the development of helical and nonhelical PPI inhibitors. ,, However, to date, such strategies have rarely been performed without computational methods to guide them.…”

“…As an alternative to head-to-tail cyclization, cysteine alkylation reactions allow for improved rational design and library development for CP discovery, − and they can induce a variety of secondary structures in peptides to improve affinity and specificity . For example, cross-linking using cysteine alkylation increased α-helicity, bioactivity, and cell permeability for a series of BH3 peptide-derived MCL-1 ligands. , The Kritzer group has also used cysteine alkylation with ortho- , meta- , and para -dibromomethylbenzene linkers, originally described by Timmerman et al, for “diversity-oriented stapling” in the development of helical and nonhelical PPI inhibitors. ,, However, to date, such strategies have rarely been performed without computational methods to guide them. It remains impossible to predict computationally which non-natural linker will best stabilize a desired CP conformation, particularly for nonhelical binding epitopes.…”