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.

Keywords: LC3B; autophagy; cancer; protein-protein interactions; stapled peptides.

Figure 1.

Protein-protein interactions of Atg8 proteins involving the LC3-interacting region (LIR) motif. a) LIR-dependent protein-protein interactions are involved in several stages of autophagy, including phosphatidylethanolamine conjugation to Atg8 proteins^[51], ULK1 initiation complex tethering and early phagophore formation^[49,50], selective cargo recruitment including mitophagy adapters^[54] and ubiquitinylated aggregate adapter p62^[52], autophagosome transport^[53], and autophagosome-lysosome fusion.^[55,56] b-d) Crystal structure of a LIR motif-containing peptide from FYCO1 bound to LC3B (PDB: 5CX3).^[59] These images highlight key positions investigated in this study, including hydrophobic pockets (b), charged residues (c), and positions in close proximity suitable for stapling (d).

Figure 2.

Binding affinities of FYCO1 and K1 peptides with the human Atg8 proteins LC3B and GABARAP. a,b) Fluorescence polarization binding assays for fluorescein-labeled FYCO1 peptide (flu-FYCO1) with recombinant human GABARAP and LC3B proteins. Peptide K1, a previously described GABARAP-selective peptide, is used as a positive control. Curve fitting was performed using KaleidaGraph graphing software as described.^[60] Average K_d values and standard error were calculated from the individual curve fits of three independent replicates, each with three technical replicates (individual replicates with raw polarization values shown in Fig. S1,2). c,d) Biolayer interferometry data (BLI) for biotinylated FYCO1 peptide (FYCO1) with recombinant human GABARAP and LC3B. BLI was performed with 0.5 μg/mL of biotinylated peptides loaded onto streptavidin-coated biosensors with serial dilutions of protein at 30 °C. Association and dissociation steps were measured for 200 seconds each. Curve fitting was performed using the Octet DataAnalysis software (FortéBio) and K_d values were calculated as described in Methods. Shown are primary data (dark blue, orange and light blue traces) and curve fits (red curves) for incubation with 1.5, 0.75, and 0.375 μM for LC3, and 4, 2, and 1 μM for GABARAP. Average K_d values and standard errors of the mean (Tables 1 and 2) were calculated from the individual curve fits of three independent replicates (Tables S2–7).

Figure 3.

Proteolytic stability of peptides in HeLa cell lysates. Peptides FYCO1, Comb1 and Comb2 were incubated for selected time points in HeLa cell lysate at 37 °C.^[75] Samples were quenched in methanol and supernatant analyzed by analytical HPLC. Areas under each peptide chromatogram peak were normalized to the area under the zero timepoint chromatogram peak. HPLC chromatograms are shown in Fig S4. Average values and standard errors of the mean were calculated from four independent replicates (representative primary data shown in Fig. S4).