Effect of Antihypertensive Drug (Chlorothiazide) on Fibrillation of Lysozyme: A Combined Spectroscopy, Microscopy, and Computational Study

Amyloid fibrils abnormally accumulate together in the human body under certain conditions, which can result in lethal conditions. Thus, blocking this aggregation may prevent or treat this disease. Chlorothiazide (CTZ) is a diuretic and is used to treat hypertension. Several previous studies suggest that diuretics prevent amyloid-related diseases and reduce amyloid aggregation. Thus, in this study we examine the effects of CTZ on hen egg white lysozyme (HEWL) aggregation using spectroscopic, docking, and microscopic approaches. Our results showed that under protein misfolding conditions of 55 degrees C, pH 2.0, and 600 rpm agitation, HEWL aggregated as evidenced by the increased turbidity and Rayleigh light scattering (RLS). Furthermore, thioflavin-T, as well as trans electron microscope (TEM) analysis confirmed the formation of amyloid structures. An anti-aggregation effect of CTZ is observed on HEWL aggregations. Circular dichroism (CD), TEM, and Thioflavin-T fluorescence show that both CTZ concentrations reduce the formation of amyloid fibrils as compared to fibrillated. The turbidity, RLS, and ANS fluorescence increase with CTZ increasing. This increase is attributed to the formation of a soluble aggregation. As evidenced by CD analysis, there was no significant difference in alpha-helix content and beta-sheet content between at 10 mu M CTZ and 100 mu M. A TEM analysis of HEWL coincubated with CTZ at different concentrations validated all the above-mentioned results. The TEM results show that CTZ induces morphological changes in the typical structure of amyloid fibrils. The steady-state quenching study demonstrated that CTZ and HEWL bind spontaneously via hydrophobic interactions. HEWL-CTZ also interacts dynamically with changes in the environment surrounding tryptophan. Computational results revealed the binding of CTZ to ILE98, GLN(57), ASP(52), TRP108, TRP63, TRP63, ILE58, and ALA(107) residues in HEWL via hydrophobic interactions and hydrogen bonds with a binding energy of -6.58 kcal mol(-1). We suggest that at 10 mu M and 100 mu M, CTZ binds to the aggregation-prone region (APR) of HEWL and stabilizes it, thus preventing aggregation. Based on these findings, we can conclude that CTZ has antiamyloidogenic activity and can prevent fibril aggregation.

Automated summary

Amyloid fibrils accumulation can cause lethal conditions, but the use of Chlorothiazide (CTZ) can prevent or treat this disease. In this study, the effects of CTZ on hen egg white lysozyme (HEWL) aggregation were examined using various methods. The results showed that CTZ can reduce the formation of amyloid fibrils and prevent aggregation.