Shedding Light on Protein Aggregates by Bisindolyl-Based Fluorogenic Probes: Unveiling Mechanistic Pathways and Real-Time Tracking of Protein Aggregation.

Abstract

Herein, we synthesized a pair of oxidized bisindolyl derivatives with anthracene (probe 1) and pyrene (probe 2) fluorophores for selective protein aggregate detection, crucial in disorders like Alzheimer's disease. Probe 1 exhibited a significant "turn-on" response (∼12-fold) and concomitant red shift (∼21 nm) with lysozyme aggregates, while showing ∼3-fold fluorescence enhancement with insulin aggregates, indicating high selectivity for aggregated proteins. Probe 2 showed similar responses but with less preference, as compared to probe 1. Furthermore, the thiazole orange (TO) assay confirmed the ability of probe 1 to detect protein fibrils and monitor aggregation kinetics (with distinct responses at different phases of aggregation). Molecular docking calculations demonstrated efficient binding of probes to aggregated proteins, stabilized primarily by hydrophobic interactions (π-π stacking). Additionally, density functional theory (DFT)-based global reactivity descriptors were computed to assess the reactivity and preferential docking sites. This work underscores the potential for novel therapeutic strategies targeting protein aggregates and early diagnosis of protein disorders.