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bioRxiv · 10.64898/2026.03.09.710535

Minimal N-methylated and stapled peptide ligands for the autophagy protein GABARAP

McDonald, I.·Wilms, J.·Cardi, N.·Engstrom, A.·Miao, J.·Willbold, D.·Lin, Y.-S.·Lokey, S.·Weiergraber, O.·Kritzer, J.
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Abstract

The LC3/GABARAP protein family is a promising target for selective inhibition of autophagy. Further, LC3/GABARAP ligands have been used as targeted degraders of soluble proteins, protein aggregates, mitochondria, lipid droplets, and RNA. However, the small molecules used for such applications have poor binding affinity and known off-target effects. LC3/GABARAP proteins are challenging targets for small-molecule drug development due to their long, shallow binding grooves. In this work, we evaluate multiple approaches to stabilizing the extended structure of the native binding motif, producing N-methylated peptides and stapled peptides with low nanomolar affinity. A crystal structure and molecular dynamics simulations support a model where the N-methylation pre-organizes the motif into an extended, strand-like structure. N-methylation allowed minimization of the binding motif to a tetrapeptide that retained sub-micromolar affinity while minimizing charge and overall molecular weight. The truncated, N-methylated tetrapeptide showed passive permeability in artificial membrane and cell-based transwell assays. These results highlight new drug-like space for LC3/GABARAP ligands with high affinity and subfamily selectivity. Table of Contents GraphicDrawn and developed by Mollie McGibbon and Joshua Kritzer O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/710535v2_ufig1.gif" ALT="Figure 1"> View larger version (68K): org.highwire.dtl.DTLVardef@1b74a1org.highwire.dtl.DTLVardef@861c09org.highwire.dtl.DTLVardef@9abc2corg.highwire.dtl.DTLVardef@b7f745_HPS_FORMAT_FIGEXP M_FIG C_FIG

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McDonald, I., Wilms, J., Cardi, N., Engstrom, A., Miao, J., Willbold, D., Lin, Y.-S., Lokey, S., Weiergraber, O., Kritzer, J.. 2026-03-11. Minimal N-methylated and stapled peptide ligands for the autophagy protein GABARAP. https://doi.org/10.64898/2026.03.09.710535

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