Noncovalent, Electrostatic Interactions Induce Positively Cooperative Binding of Small Molecules to Alzheimer’s and Parkinson’s Disease-Related Amyloids

Abstract

Amyloids are self-assembled protein aggregates that represent a major hallmark of many neurologic and systemic diseases. Among the common features of amyloids is the presence of a high density of multiple binding sites for small molecule ligands, making them an attractive target for design of multimeric binding agents. Here, we demonstrate that noncovalent, intermolecular interactions between a 1:1 mixture of oppositely charged benzothiazole molecules enhances their binding to two different amyloid aggregates: Alzheimer's-related amyloid-β (Aβ) peptides or Parkinson's-related α-synuclein (αS) proteins. We show that this mixture leads to positively cooperative binding to amyloid targets, with up to 10-fold enhancement of binding compared to the uncharged parent compound. The observed enhancement of amyloid binding using noncovalent interactions was similar in magnitude to a benzothiazole dimer to aggregated Aβ. These results represent a novel strategy for designing amyloid-targeting molecules with enhanced affinity, which could aid in the development of new diagnostic or treatment strategies for amyloid-associated diseases.

Exploring the Effect of Aliphatic Substituents on Aryl Cyano Amides on Enhancement of Fluorescence upon Binding to Amyloid-β Aggregates

“…In order to examine the binding and fluorescence properties of ARCAM and its analogues in the presence of Aβ aggregates, we prepared a solution of aggregated Aβ(1–42) peptides using a previously reported protocol (see the Methods section for details of this preparation and the Supporting Information for the characterization) . Binding measurements revealed that all of the compounds 1 – 5 bound with similar low micromolar affinities to aggregated Aβ (Table and Supporting Information Figure S9), demonstrating that small aliphatic substituents on the ARCAM scaffold do not significantly affect the binding to amyloids.…”

“…14,16,17,25 In order to examine the binding and fluorescence properties of ARCAM and its analogues in the presence of Aβ aggregates, we prepared a solution of aggregated Aβ(1−42) peptides using a previously reported protocol (see the Methods section for details of this preparation and the Supporting Information for the characterization). 26 Binding measurements 21 revealed that all of the compounds 1−5 bound with similar low micromolar affinities to aggregated Aβ (Table 1 and Supporting Information Figure S9), demonstrating that small aliphatic substituents on the ARCAM scaffold do not significantly affect the binding to amyloids. Interestingly, all of the probes with aliphatic substituents show a trend of stronger binding to Aβ aggregates, which could be due to increased hydrophobic interactions betwen these ARCAM analogues with the binding pockets on Aβ aggregates compared to the parent ARCAM (1).…”

“…Aggregated Aβ (1−42) was prepared as previously described. 26 Briefly, Aβ (1−42) was dissolved in 100% 1,1,1,3,3,3,-hexafluoro-2propanol (HFIP) to a concentration of 1 mM and put on a shaker at room temperature (RT) for 24 h. The solution was then diluted in cold nanopure water (2:1 H 2 O/HFIP). Aliquoted fractions were lyophilized for 3 d before being dissolved in nanopure water to a concentration of 100 μM.…”

On the Binding of Congo Red to Amyloid Fibrils

“…These studies led to an alternative binding mode in the fibril groove, with a stoichiometry of n =1, in agreement with spectroscopic data (Supporting Information, Figure S4). In the same direction, it has been recently shown that non‐covalent, electrostatic interactions induce positive cooperative binding of small molecules to Alzheimer's and Parkinson's disease‐related amyloids …”

On the Binding of Congo Red to Amyloid Fibrils

“…In the same direction, it has been recently shown that non-covalent, electrostatic interactions induce positive cooperative binding of small molecules to Alzheimers and Parkinsons diseaserelated amyloids. [11] The structural model proposed for the 1:1 stoichiometric binding of CR to HET-s PFD is shown in Figure 3. This binding mode was stable in three independent MD simulations (Supporting Information, Figures S5-S8).…”