Elucidating the Aβ42 Anti-Aggregation Mechanism of Action of Tramiprosate in Alzheimer’s Disease: Integrating Molecular Analytical Methods, Pharmacokinetic and Clinical Data

Abstract

BackgroundAmyloid beta (Aβ) oligomers play a critical role in the pathogenesis of Alzheimer’s disease (AD) and represent a promising target for drug development. Tramiprosate is a small-molecule Aβ anti-aggregation agent that was evaluated in phase III clinical trials for AD but did not meet the primary efficacy endpoints; however, a pre-specified subgroup analysis revealed robust, sustained, and clinically meaningful cognitive and functional effects in patients with AD homozygous for the ε4 allele of apolipoprotein E4 (APOE4/4 homozygotes), who carry an increased risk for the disease. Therefore, to build on this important efficacy attribute and to further improve its pharmaceutical properties, we have developed a prodrug of tramiprosate ALZ-801 that is in advanced stages of clinical development. To elucidate how tramiprosate works, we investigated its molecular mechanism of action (MOA) and the translation to observed clinical outcomes.ObjectiveThe two main objectives of this research were to (1) elucidate and characterize the MOA of tramiprosate via an integrated application of three independent molecular methodologies and (2) present an integrated translational analysis that links the MOA, conformation of the target, stoichiometry, and pharmacokinetic dose exposure to the observed clinical outcome in APOE4/4 homozygote subjects.MethodWe used three molecular analytical methods—ion mobility spectrometry–mass spectrometry (IMS–MS), nuclear magnetic resonance (NMR), and molecular dynamics—to characterize the concentration-related interactions of tramiprosate versus Aβ42 monomers and the resultant conformational alterations affecting aggregation into oligomers. The molecular stoichiometry of the tramiprosate versus Aβ42 interaction was further analyzed in the context of clinical pharmacokinetic dose exposure and central nervous system Aβ42 levels (i.e., pharmacokinetic–pharmacodynamic translation in humans).ResultsWe observed a multi-ligand interaction of tramiprosate with monomeric Aβ42, which differs from the traditional 1:1 binding. This resulted in the stabilization of Aβ42 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 bound to Lys16, Lys28, and Asp23, the key amino acid side chains of Aβ42 that are responsible for both conformational seed formation and neuronal toxicity. The projected molar excess of tramiprosate versus Aβ42 in humans using the dose effective in patients with AD aligned with the molecular stoichiometry of the interaction, providing a clear clinical translation of the MOA. A consistent alignment of these preclinical-to-clinical elements describes a unique example of translational medicine and supports the efficacy seen in symptomatic patients with AD. This unique “enveloping mechanism” of tramiprosate also provides a potential basis for tramiprosate dose selection for patients with homozygous AD at earlier stages of disease.ConclusionWe have identifie...

Structural Insight into IAPP‐Derived Amyloid Inhibitors and Their Mechanism of Action

“…Nevertheless,ithas been shown that multi-ligand interactions of tramisprosate with monomeric Ab42 can prevent amyloid oligomer formation. [25] Hydrophobic small molecules can form colloids that resemble protein liquid droplets in size. [7] These small-molecule colloids are,h owever, unspecific inhibitors,a st hey interact promiscuously with hydrophobic regions of aprotein and eventually induce protein unfolding.…”

“…Thedesign of the ISMs was based on the finding that amyloids are generally composed of a b-sheet-turn-b-sheet structural motif and that IAPP uses the same two binding regions for both its amyloid self-and its cross-amyloid hetero-assembly with Ab40/42. [1a, 2] ISMs were thus derived by linking the two hot segments IAPP (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) and IAPP (22)(23)(24)(25)(26)(27)(28) in native or N-methylated form to each other via different linkers,mostly tripeptide sequences consisting of identical amino acids;notably,these two segments are highly homologous to segments of the amyloid core of Ab. [3] High Ab40/42 anti-amyloidogenic activity was found for seven out of the 16 studied ISMs with six of them containing bulky hydrophobic/aromatic residues (e.g.L LL, III, FFF) in the linker tripeptide and one of them, termed R3-GI, the RRR tripeptide.…”

Comparative Oral Absorption of Different Citicoline and Homotaurine Formulations: A Single-Dose, Two-Period Crossover Trial in the Dog

“…This substance may interfere with several cellular pathways and exert neuroprotective and neurotropic activities through different mechanisms including effects against the oxidative damage to DNA, antifibrillogenic activity, and antinociceptive and analgesic activities, related to the activation of GABA type-A receptors. In addition, homotaurine prevents the neurotoxicity of Aβ peptide by reducing amyloid aggregation in humans [11,12].…”

Clinical Effects of Oral Tramiprosate in Apoe4/4 Homozygous Patients With Mild Alzheimer’s Disease Suggest Disease Modification

“…Tramiprosate is an oral amyloid anti-aggregation agent that reduces oligomer formation and fibrillar (plaque) amyloid deposition in transgenic animal models (1–4). Tramiprosate has been shown to inhibit β-amyloid (Aβ) fibrillation and to reduce soluble Aβ.…”

“…It has also been reported to inhibit the inflammatory response. More recently, tramiprosate has been shown to modulate “conformational flexibility of amyloid beta Aβ42, leading to the prevention of oligomer seed formation and thus aggregation” (4). …”