P. Kocis, M. Tolar, K. Blennow, H. Fillit, J.A. Hey
OBJECTIVES: Soluble beta-amyloid oligomers play a key role in the pathogenesis of Alzheimer’s disease (AD). Our goal was to elucidate molecular mechanism of action (MOA) of tramiprosate, the active component of an optimized prodrug ALZ-801 entering confirmatory Phase 3 program in AD. We report here the discovery and characterization of a novel multi-ligand enveloping stabilizing effect of the small molecule tramiprosate: modulation of conformational flexibility of beta amyloid (Aß), prevention of Aß-seed formation, stabilization of Aß42 monomers, and resulting prevention of Aß-oligomer formation.
METHODS: Three independent molecular analytical methods including Ion Mobility Mass Spectrometry (IMS-MS), Nuclear Magnetic Resonance (NMR), and molecular dynamics were used to experimentally demonstrate the anti-oligomer effect of tramiprosate, and to characterize the concentration-dependent effects on conformational landscape of Aß42 monomers. The molecular stoichiometry of the tramiprosate vs. Aß42 interaction was further analyzed in the context of brain exposure and clinical dose exposure, as well as brain Aß42 levels in AD patients (Hey et al. AAIC poster).
RESULTS: We observed a concentration dependent multi-ligand interaction of tramiprosate with monomeric Aß42, which differs from the traditional 1:1 binding. This binding interaction resulted in stabilization of Aß42 monomers, and inhibition of oligomer formation and elongation, as demonstrated by IMS MS and molecular dynamics. Using NMR spectroscopy and molecular dynamics, we also showed that tramiprosate binds to Lys16, Lys28 and Asp23, the key amino acid side chains of Aß42 that are responsible for both amyloid seed formation and neuronal toxicity. Complete inhibition of oligomer formation was observed when tramiprosate:Aß42 ratio reached 1:1,000.
CONCLUSIONS: We have identified novel enveloping molecular mechanism of tramiprosate. This MOA indicates that it is possible to modulate and control the Aß42 conformational dynamics landscape by a small molecule, resulting in stabilization of Aß42 conformation that yields a clinically relevant amyloid anti-aggregation effects, as well as resulting inhibition of oligomer formation that may account for the clinical efficacy of tramiprosate observed in APOE4/4 homozygous AD patients. This novel enveloping MOA may be applied to development of disease-modifying therapies for AD and other neurodegenerative diseases caused by misfolded proteins.