Original Article
Small molecule NPT-440-1 inhibits ionic flux through Aβ1 -42 pores: Implications for Alzheimer's disease therapeutics

https://doi.org/10.1016/j.nano.2016.06.001Get rights and content

Abstract

Increased levels of soluble amyloid-beta (Aβ) oligomers are suspected to underlie Alzheimer's disease (AD) pathophysiology. These oligomers have been shown to form multi-subunit Aβ pores in bilayers and induce uncontrolled, neurotoxic, ion flux, particularly calcium ions, across cellular membranes that might underlie cognitive impairment in AD. Small molecule interventions that modulate pore activity could effectively prevent or ameliorate their toxic activity. Here we examined the efficacy of a small molecule, NPT-440-1, on modulating amyloid pore permeability. Co-incubation of B103 rat neuronal cells with NPT-440-1 and Aβ1 -42 prevented calcium influx. In purified lipid bilayers, we show that a 10-15 min preincubation, prior to membrane introduction, was required to prevent conductance. Thioflavin-T and circular dichroism both suggested a reduction in Aβ1 -42 β-sheet content during this incubation period. Combined with previous studies on site-specific amino acid substitutions, these results suggest that pharmacological modulation of Aβ1 -42 could prevent amyloid pore-mediated AD pathogenesis.

Graphical Abstract

Increased levels of soluble amyloid-beta (Aβ) oligomers are suspected to underlie Alzheimer's disease (AD) pathophysiology through the formation of uncontrolled multi-subunit Aβ pores in cellular membranes. In this study, the efficacy of small molecule NPT-440-1 modulation of Aβ1 -42 pore permeability was examined. We show that co-incubation of B103 rat neuronal cells with NPT-440-1 and Aβ1 -42 prevented calcium influx. In purified lipid bilayers, preincubation prior to membrane introduction was required to prevent conductance despite the presence of pore structures. The results point to compound-induced structural modulation leading to collapsed pores and suggest that pharmacological modulation of Aβ1 -42 could prevent AD pathogenesis.

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Section snippets

Methods

Aβ1 -42 was purchased from Bachem (Torrance, CA), Anaspec (Fremont, CA), and American Peptide Company (Sunnyvale, CA). Phospholipids 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) were purchased from Avanti Polar Lipids (Alabaster, AL). The compound NPT-440-1 (Figure 1, A), which upon initial solubilization in DMSO becomes water soluble, was provided by Neuropore Therapies, Inc. (San Diego, CA). All other chemicals were purchased

NPT-440-1 inhibits Aβ1-42 aggregation in solution

We first observed and validated the effect of NPT-440-1 on the aggregation of Aβ1 -42 in solution. Our SDS-PAGE data (Figure 1, B/C) show that NPT-440-1 effectively reduces the formation of aggregates even at compound concentrations ten times smaller than that of the peptide. Co-incubation of NPT-440-1 with Aβ1 -42 reduced the formation of dimers, trimers, tetramers and higher order oligomers (Figure 1, B). There is a concentration-dependent inhibition of overall oligomer formation, though this

Discussion

We present data showing that 1) NPT-440-1 inhibits Aβ1 -42 aggregation (Figure 1), and 2) incubation of NPT-440-1 with Aβ1 -42 effectively normalizes oligomer induced increases in intracellular Ca2 + levels (Figure 2) and eliminates Aβ1 -42 pore activity in DOPS/POPE (1:1) membranes (Figure 3). A detailed molecular explanation of pore modulation is difficult to determine because of the numerous potential mechanisms and missing knowledge about aspects of pore formation, structure, and conduction

Acknowledgments

The authors would like to thank Neuropore Therapies Inc. for generously providing NPT-440-1. Thanks are also given to the UCSD Biophysics Instrumentation Facility for the use of the Aviv410 CD spectrometer used in this study.

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      Citation Excerpt :

      The experimental conditions used for the incubation step (target and ligand concentrations, buffer composition, pH, temperature, presence of aggregation-inducing co-factors) have a huge and defining impact on the kinetics of amyloid misfolding. Unfortunately, a wide variety of incubation conditions have been reported in the literature: CD studies on Aβ1–42 were carried out with protein concentrations ranging from 15 μM [81] to 75 μM [87] and incubation times ranging from 15 minutes [75] to 40 days [86]. The lack of a widely recognized and standardized protocol to perform CD assays on Aβ and tau leads to the low degree of reproducibility observed across studies: for instance, the precipitation of insoluble aggregates is a common but frequently overlooked issue that gives rise to strong light scattering artifacts and greatly affects the quality of CD spectra.

    Funding Source Information: This work was supported by National Institutes of Health Grants R01 AG028709 (R.L.) and R37 AG18440 (E.M.).

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