Ziaul Hasan

Conference 2023 Presentation

Project title

Therapeutic potential of rutin trihydrate against Aspergillus aspartic protease through spectroscopic and docking investigation

Authors and Affiliations

Ziaul Hasan1,2#, Asimul Islam2 , Luqman Ahmad Khan1*

1. Department of Biosciences, Jamia Millia Islamia (Central University), New Delhi 110025, India
2. Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia (Central University), New Delhi 110025, India

Abstract

Background

Systemic fungal infections like Aspergillosis and Candidiasis can be fatal if not treated well on time. However, increasing antimicrobial resistance and increasing side effects of existing antifungals exert challenges in treatment strategies. Pathogenic fungi such as Aspergillus spp. often secretes several virulence factors responsible for host infection. Secretary proteases are important among them and can be served as a biomarker for inhibitor identification. Secondary metabolites especially the class of flavonoids possess proven antimicrobial potential. Rutin trihydrate (RT) which is a flavanol glycoside is known for its antifungal properties. We have checked the structural and functional changes undergone in Aspergillus aspartic protease (PepA) upon interaction with RT.

Methods

Binding interaction and structural changes were studied using fluorescence, UV-Vis and CD spectroscopy. Binding interaction is further validated with synchronous, three-dimensional fluorescence, extrinsic fluorescence and isothermal titration calorimetric experiments. Enzyme activity and inhibition assay was performed to calculate the IC50 value. Molecular docking was used to validate the spectroscopic results.

Results

Fluorescence spectra showed clear quenching of PepA and strong static binding with Ka of 2.7 x 107 M-1. UV-Vis spectroscopy suggested ground state complex formation followed by CD spectroscopy which showed mark changes in the secondary structure with a decrease in α-helical content at 100 µM of RT. The activity of PepA was found to be drastically reduced on increasing RT concentration with an IC50 value of 132.41 µM of RT. Molecular docking studies confirmed the binding results and showed negative binding energy of -10 kcal/mol. Human serum albumin (HSA), bovine serum albumin (BSA) and Lysozyme (LYZ) showed strong binding and ground state complex formation with RT. HSA was found to lose its secondary structure upon RT binding but the structure of BSA and LYZ were conserved and not much affected. Docking analysis showed binding of these proteins with RT with a binding energy of -8.6, -9.2 and -8.7 kcal/mol which was weaker when compared to PepA.

Conclusions

The present spectroscopic and in-silico analysis suggests rutin as a potential inhibitor of Aspergillus aspartic protease and as prospective phytotherapy for devastating fungal infections.