MUTAGENESIS OF THE GENERAL AMYLOID INTERACTION MOTIF (GAIM) REVEALS A STRUCTURE-ACTIVITY RELATIONSHIP FOR MISFOLDED BETA-AMYLOID AND TAU AGGREGATES

Abstract

The general amyloid interaction motif (GAIM) derived from the M13 phage tip protein g3p binds a wide variety of amyloid aggregates in a conformation-dependent manner (Krishnan 2014). Dimeric GAIM-Ig-fusions robustly bind and remodel Aβ42 amyloid aggregates and inhibit tau aggregate propagation in primary neuronal cells. In transgenic models of AD and tauopathy, GAIM-fusion treatment reduces Aβ plaque load, phospho-tau levels and improves cognition (Levenson 2016). In this study, we explored the mechanism of GAIM-mediated remodeling of amyloid aggregates by mutagenesis. Using this data, we designed a next generation Ig-fusion, NPT189, which in addition to showing improved binding potency to multiple aggregates, has reduced potential for immunogenicity after removal of potential T-cell epitopes. Data obtained from H/D exchange NMR studies, GAIM-peptide-based inhibition assays, TolA binding, and computational modeling was used for targeted mutagenesis of the GAIM scaffold. Stable, high expressing variants were then screened for binding to Aβ42 and Tau-K18 fibers using SPR and binding ELISA assays. High binding variants were further screened for Aβ42 fiber remodeling as well as tau and α-synuclein transmission inhibition activities. An independent mutagenesis study was carried out to sequentially eliminate four potential T-cell epitopes of GAIM. Analysis of binding and remodeling data collected from GAIM variants illuminated a novel mechanism for GAIM activity. Amyloid binding is mediated by residues facing the interdomain groove of N1 and N2 domains of GAIM. Binding specificity depends on the stability of both domains and the melting temperature for domain separation. Over-stabilization of N1 or N2 domains leads to reduced binding activity, while destabilization of the domains leads to increased non-specific binding. We hypothesize progressive binding and rearrangement of GAIM β-strands enables GAIM to remodel amyloid aggregates into non-fibrous and non-pathogenic aggregates. GAIM-Ig-fusions represent a novel class of therapeutics to treat protein misfolding disorders. Systemic administration of these molecules slows down the accumulation of amyloid plaque and intracellular tau tangles and provides cognitive benefits in transgenic animals.