A cocktail of Cu2+- and Zn2+-peptoid-based chelators can stop ROS formation for Alzheimer's disease therapy

Abstract

The formation of reactive oxygen species (ROS) in the brain is a major cause of neuropathologic degradation associated with Alzheimer's Disease (AD). It has been suggested that the copper (Cu)-amyloid-β (Aβ) peptide complex can lead to ROS formation in the brain. An external chelator for Cu that can extract Cu from the CuAβ complex should inhibit the formation of ROS, making Cu chelation an excellent therapeutic approach for AD. Such a chelator should possess high selectivity for Cu over zinc (Zn), which is also present within the synaptic cleft. However, such selectivity is generally hard to achieve in one molecule due to the similarities in the binding preferences of these two metal ions. As an alternative to monotherapy (where Cu extraction is performed using a single chelator), herein we describe a variation of combination therapy - a novel cocktail approach, which is based on the co-administration of two structurally different peptidomimetic chelators, aiming to target both Cu²⁺ and Zn²⁺ ions simultaneously but independently from each other. Based on rigorous spectroscopic experiments, we demonstrate that our peptidomimetic cocktail allows, for the first time, the complete and immediate inhibition of ROS production by the CuAβ complex in the presence of Zn²⁺. In addition, we further demonstrate the high stability of the cocktail under simulated physiological conditions and its resistance to proteolytic degradation by trypsin and report the water/n-octanol partition coefficient, initially assessing the blood-brain barrier (BBB) permeability potential of the chelators.

Scheme 1. Schematic representation of a cocktail of two structurally different peptoid-based chelators, designed to selectively bind Cu or Zn ions from the Aβ-peptide complex, stopping the production of ROS in the context of AD.

Fig. 1. (A) Chemical structures of peptoid oligomers AD1–AD2 and PT1. (B) Solubilities of peptoid AD1 and AD2 in un-buffered water (pH = 7.0) and HEPES buffer (50 mM, pH = 7.4).

Fig. 2. (A) UV/Vis titration of AD2 (33 μM) with Cu²⁺ ions in HEPES buffer (50 mM, pH = 7.4). Inset: Job-plot of AD2 with Cu²⁺ (26 μM total concentration). (B) UV-Vis spectra of AD2 (33 μM), it's Cu²⁺ and Zn complexes, and the complexes formed upon addition of a mixture of 1 equiv. of Cu²⁺ and 5 equiv. of Zn²⁺.

Fig. 3. EPR spectra of Cu²⁺ + AD2 (green), Aβ_1–16 + Cu²⁺ + Zn²⁺ (black), Aβ_1–16 + Cu²⁺ + Zn²⁺ + AD2 + PT1 EPR tube frozen asap (blue), Aβ_1–16 + Cu²⁺ + Zn²⁺ + AD2 + PT1 EPR tube frozen after four hours (red), Cu²⁺ + PT1 (cyan). Conditions: [PT1] = [AD2] = [Aβ_1–16] = [Zn²⁺] = 280 μM, [Cu²⁺] = 250 μM, [HEPES] = 50 mM (pH = 7.4). Recording conditions: T = 203 K, ν = 9.5 GHz, modulation amplitude = 3 G, microwave power: 20 mW.

Fig. 4. Stability of (A) AD2 and (B) PT1 under physiological conditions (pH = 7.4 and T 37 °C), as monitored by UV\Vis. Conditions: [PT1] = [AD2] = 100 μM, [PBS] = 50 mM. Proteolysis experiments: overlap of the retention peaks of (C) AD2 and (D) PT1 from the corresponding HPLC chromatograms (λ = 214 nm, a linear gradient of 5–95% ACN/H₂O 0.1% TFA over 10 min, at a flow rate of 0.7 min mL⁻¹ on the analytical C18 column), taken at different times from the proteolytic stability experiment. Conditions: [PT1] = [AD2] = 0.1 mg mL⁻¹, [trypsin] = 0.1 μM, [PBS] = 50 mM (pH = 7.4), at the constant temperature of 37 °C.