Neudecker, P. | bioRxiv Science

bioRxiv2024

Exploring Arylidene-Indolinone Ligands of Autophagy Proteins LC3B and GABARAP

We report the first structure-activity studies of arylidene-indolinone compound GW5074 which was reported as a ligand of autophagy-related protein LC3B. The literature has conflicting information on the binding affinity of this compound and there is some debate regarding its use as a component of autophagy-dependent degrader compounds. We developed an AlphaScreen assay to measure competitive inhibition of the binding of known peptide ligands to LC3B and its paralog GABARAP. 18 analogs were synthesized and tested against both proteins. Inhibitory potencies were found to be in the mid- to high micromolar range. 2D-NMR data revealed the binding site on GABARAP as hydrophobic pocket 1, where native peptide ligands bind with an aromatic side chain. Our results suggest that GW5074 binds LC3B and GABARAP with micromolar affinity. These affinities could support further exploration in targeted protein degradation, but only if off-target effects and poor solubility can be appropriately addressed.

biochemistry↗

bioRxiv2020

Structural details of amyloid beta oligomers in complex with human prion protein as revealed by solid-state MAS NMR spectroscopy

Human PrP (huPrP) is a high-affinity receptor for oligomeric A{beta}. Synthetic oligomeric A{beta} species are known to be heterogeneous, dynamic and transient, rendering their structural investigation particularly challenging. Here, we used huPrP to preserve A{beta} oligomers by co-precipitating them into large hetero-assemblies to investigate the conformation of A{beta}(1-42) oligomers and huPrP in the complex by solid-state MAS NMR spectroscopy. The disordered N-terminal region of huPrP becomes immobilized in the complex and therefore visible in dipolar spectra without adopting chemical shifts characteristic of a regular secondary structure. Most of the well-defined C-terminal part of huPrP is part of the rigid complex, and solid-state NMR spectra suggest a loss in regular secondary structure in the last two -helices. For A{beta}(1-42) oligomers in complex with huPrP, secondary chemical shifts reveal a substantial {beta}-strand content. Importantly, not all A{beta}(1-42) molecules within the complex have identical conformations. Comparison with the chemical shifts of synthetic A{beta} fibrils suggests that the A{beta} oligomer preparation represents a heterogeneous mixture of {beta}-strand-rich assemblies, of which some have the potential to evolve and elongate into different fibril polymorphs, reflecting a general propensity of A{beta} to adopt variable {beta}-structure conformers.

molecular biology↗