Nitroxyl Hybrids with Curcumin and Stilbene Scaffolds Display Potent Antioxidant Activity, Remodel the Amyloid Beta Oligomer, and Reverse Amyloid Beta-Induced Cytotoxicity

Abstract

The disorder and heterogeneity of low-molecular-weight amyloid-beta oligomers (AβOs) underlie their participation in multiple modes of cellular dysfunction associated with the etiology of Alzheimer's disease (AD). The lack of specified conformational states in these species complicates efforts to select or design small molecules to targeting discrete pathogenic states. Furthermore, targeting AβOs alone may be therapeutically insufficient, as AD progresses as a multifactorial, self-amplifying cascade. To address these challenges, we have screened the activity of seven new candidates that serve as Paramagnetic Amyloid Ligand (PAL) candidates. PALs are bifunctional small molecules that both remodel the AβO structure and localize a potent antioxidant that mimics the activity of SOD within live cells. The candidates are built from either a stilbene or curcumin scaffold with nitroxyl moiety to serve as catalytic antioxidants. Measurements of PAL AβO binding and remolding along with assessments of bioactivity allow for the extraction of useful SAR information from screening data. One candidate (HO-4450; PMT-307), with a six-membered nitroxyl ring attached to a stilbene ring, displays the highest potency in protecting against cell-derived Aβ. A preliminary low-dose evaluation in AD model mice provides evidence of modest treatment effects by HO-4450. The results for the curcumin PALs demonstrate that the retention of the native curcumin phenolic groups is advantageous to the design of the hybrid PAL candidates. Finally, the PAL remodeling of AβO secondary structures shows a reasonable correlation between a candidate's bioactivity and its ability to reduce the fraction of antiparallel β-strand.

Keywords: Alzheimer’s disease; Aβ oligomer; EPR; amyloid beta peptide; antioxidant; bifunctional drug; electron paramagnetic resonance spectroscopy; nitroxide; oxidative stress; protein misfolding.

Figure 1

The potent radical scavenging of the nitroxide in cells. The N-oxyl cycles through alternative redox states. In the presence of the principal intracellular antioxidants glutathione (GSH) and ascorbate (ASC), the nitroxide undergoes bioreduction, enabling multiple rounds of PAL antioxidant activity.

Scheme 1

Synthesis of HO-4897.

Figure 2

The chemical structure of PAL candidates. For convenience, PAL family codes are designated as PMT. The original HO- designations, as previously published, are also provided.

Figure 3

PAL neuronal protection activity. Induced (TC–) C99 expression in the MC65 model results in amyloid beta cytotoxicity that can be eliminated with PAL titration. Error bars are the SEM from the assay of three separate measurements.

Figure 4

CD spectra of AβOs treated with a stoichiometric amount of PAL agent. CD spectra of AβOs treated for 1 h with 40 μM PAL. The control (black) was treated with an equal volume of vehicle. ∆ε represents the molar circular dichroism.

Figure 5

PALs’ effect on AβO-associated dyes. (A) The ThT fluorescence intensity of Aβ+PAL samples normalized to the sample of Aβ alone. (B) The NR fluorescence intensity of Aβ+PAL samples normalized to the sample of Aβ alone. Values represent the average intensities of triplicate samples measured at 20 h.

Figure 6

Decreases in the A11 antibody recognition of AβOs following PAL treatment. Amounts of captured A11 are reported from the oxidized luminol signal of the HRP-secondary antibody. Samples were tested in quadruplicate in each assay. The results are the average of three independent assays with errors reported as SEM.

Figure 7

Relative antioxidant capacities of PAL candidates. The oxidation of DCFH generates a strong fluorescence reflective of ROS levels. (A) shows the DCF intensities following peroxidase-dependent DCFH oxidation (by O₂•−), in the presence of PALs, relative to the ACN control. The effect of PALs on DCFH oxidation (relative to the ACN control) in the presence of •OH radical generation is shown in (B). A PAL concentration of 20 μM was used to compare the superoxide oxidation of 20 μM DCFH. A PAL concentration of 40 μM was used to compare the hydroxyl radical oxidation of 50 μM DCFH. All assays were performed in quadruplicate.

Figure 8

Effect of ROS on PAL nitroxide signal as measured by EPR spectroscopy. Shown are center line amplitudes of PAL nitroxide (160 μM) following incubation with either O₂•− or •OH radical relative to PAL in buffer. Reduction in EPR amplitude is indicative of oxidation of N-oxyl moiety to diamagnetic state. We included 4 mM GSH in •OH radical samples to diminish differential production of Cu(II)-catalyzed •OH radical generation in stilbene PALs.