Repurposing Triphenylmethane Dyes to Bind to Trimers Derived from Aβ

Abstract

Soluble oligomers of the β-amyloid peptide, Aβ, are associated with the progression of Alzheimer's disease. Although many small molecules bind to these assemblies, the details of how these molecules interact with Aβ oligomers remain unknown. This paper reports that crystal violet, and other C3 symmetric triphenylmethane dyes, bind to C3 symmetric trimers derived from Aβ. Binding changes the color of the dyes from purple to blue, and causes them to fluoresce red when irradiated with green light. Job plot and analytical ultracentrifugation experiments reveal that two trimers complex with one dye molecule. Studies with several triphenylmethane dyes reveal that three N, N-dialkylamino substituents are required for complexation. Several mutant trimers, in which Phe, Phe, and Ile were mutated to cyclohexylalanine, valine, and cyclohexylglycine, were prepared to probe the triphenylmethane dye binding site. Size exclusion chromatography, SDS-PAGE, and X-ray crystallographic studies demonstrate that these mutations do not impact the structure or assembly of the triangular trimer. Fluorescence spectroscopy and analytical ultracentrifugation experiments reveal that the dye packs against an aromatic surface formed by the Phe side chains and is clasped by the Ile side chains. Docking and molecular modeling provide a working model of the complex in which the triphenylmethane dye is sandwiched between two triangular trimers. Collectively, these findings demonstrate that the X-ray crystallographic structures of triangular trimers derived from Aβ can be used to guide the discovery of ligands that bind to soluble oligomers derived from Aβ.

Rational Design of Crystallization‐Induced‐Emission Probes To Detect Amorphous Protein Aggregation in Live Cells

“…Organometallic probes pioneered by Meade [11, 12] and Marti [13–16] not only broaden the chemical space for rational design but also introduce multiple detection modes other than fluorescence intensity. Kelly, [17] Nowick, [18] and Petersson [19] et al. further resolved the oligomeric state of amyloid precursors using FlAsH, triphenylmethane, and thioamide dye‐related fluorescence‐based detections.…”

Moving analytical ultracentrifugation software to a good manufacturing practices (GMP) environment

“…This approach follows in the footsteps of the openAUC architecture proposed earlier [ 16 ], and permits direct interfacing with the instrument over ethernet using alternative third-party software solutions like UltraScan. This avenue offers maximum flexibility and fosters development of new and interesting tools that can explore novel features like multi-wavelength experiments [ 19 , 20 , 21 , 22 , 23 , 24 ] and GMP capable data acquisition software, discussed here. Using the network-capable Linux tools provided with the Optima, our laboratory has extended the UltraScan-III AUC analysis software [ 13 , 14 ] and the UltraScan Laboratory Information Management Systems (UltraScan-LIMS) framework [ 15 ] to interface directly with the Optima for experimental design, data acquisition, and remote experiment monitoring.…”

Covalent Probes for Aggregated Protein Imaging via Michael Addition

“…[34] Organic dyes,l ike Thioflavin T, Congo Red, carbazoles,perylenes,and their derivatives have been demonstrated to selectively bind to amyloid precursors and fibrils. [35] In particular,T ang, [36] Zhu, [37] and Hong [38] et al pioneered the design of AIEgens to report on amyloid and unfolded proteins during protein phase separation. Materials based probes,s uch as luminescent polymers, quantum dots,a nd nanoparticles opened new avenues for amyloid sensors.…”

“…The structural information provides blueprint to design non‐covalent dyes and sensors that selectively bind to amyloid aggregated proteins [34] . Organic dyes, like Thioflavin T, Congo Red, carbazoles, perylenes, and their derivatives have been demonstrated to selectively bind to amyloid precursors and fibrils [35] . In particular, Tang, [36] Zhu, [37] and Hong [38] et al.…”