Molecular docking studies of isolated marine natural products against α-chymotrypsin

Abstract The α-chymotrypsin is widely present in the digestive system of prokaryotes and eukaryotes that helps in the digestion by the hydrolysis of the peptide bond. It is serine protease enzyme (E.C. 3.4.21.1) and involves in many biological processes as well as in pancreatic disorders. In the previous study, marine red alga namely Jolyna laminariodes was used for the isolation of succinylanthranilic acid ester (2). Further, analogues were synthesised from anthranilic acid by using succinic anhydride, maleic anhydride and glutaric anhydride into corresponding dicarboxylic acids and further into acid ester including succinylanthranilic acid (1), succinylanthranilic acid ester (2), maleinylanthranilic acid (3), maleinylanthranilic acid ester (4), glutarnylanthranilic acid (5) and glutarnylanthranilic acid ester (6). For all natural products analogues percent inhibition against α-chymotrypsin have been calculated. In this study, molecular docking is used to estimate the binding energy of natural product analogues against α-chymotrypsin enzyme. The docking energies are in good agreement with experimental findings. Graphical Abstract


Introduction
The a-chymotrypsin (CHT) is a biologically important protein involved in protein digestion via hydrolysis of the peptide bond.It is a serine protease enzyme and belongs to the S1 protease family (E.C. 3.4.21.1) (Mazumdar-Leighton and Broadway 2001;Broehan et al. 2008).It is used in medicine for the treatment of a number of diseases including pharyngitis, rhinitis, lung abscess, etc (Biswas et al. 2022).In the literature, this enzyme is associated with a number of diseases and pathogenesis of multiple diseases (Beer and Sahin-Toth 2011;Zhou and Sahin-Toth 2011;Moffitt et al. 2012;Yamashita et al. 2013;Oldziej et al. 2014).The protein folding and unfolding mechanism plays an important role in enzyme stability (Farhadian et al. 2017).Xingchen Zhao used different molecular techniques including UV-vis, fluorescence, circular dichorism to study interactions between carbon nanotubes and a-chymotrypsin (Zhao et al. 2015).
In 2013, Pankaj Attri explores the stability of a-chymotrypsin enzyme by using different ammonium-based ionic liquids.Fluorescence spectroscopy and circular dichorism were used for the calculation of thermodynamic parameters to check the stability of a-chymotrypsin in different liquids (Attri and Venkatesu 2013).In 2016, Sadegh Farhadian studied the stability of a-chymotrypsin enzyme by using lactose as a solvent.It has been observed that the a-chymotrypsin enzyme showed stability in lactose and it was determined by a number of other techniques including fluorescence spectroscopy, thermodynamic parameters and circular dichorism.One other technique, molecular docking was used to check the binding mechanism of a-chymotrypsin enzyme due to the presence of lactose as a solvent.Hence negative binding energy reveals that lactose forms a bond with a-chymotrypsin active site amino acid and exists as a complex form (Farhadian et al. 2016).In 2018, Hao Xiong utilised molecular docking to discover nanopeptide probes for a-chymotrypsin (Xiong et al. 2018).
Originally, succinylanthranilic acid ester (2) was isolated from marine red alga J. laminariodes (Atta-ur-Rahman et. al. 1997).In a previous study, inhibition constant of competitive inhibitors (N-acetyl-D-tryptophan amide, N-acetyl-D-tryptophan and N-acetyl-L-tryptophan) were calculated (Foster et al. 1955;Johnson and Knowles 1966) and percent inhibitions of natural product analogues have been calculated against a-chymotrypsin enzyme (Cannell et al. 1988;Khan et al. 2006).The purpose of this study is to investigate the binding pattern of a-chymotrypsin to understand the inhibition mechanism of the enzyme.This study will further help to explore the binding mechanism of other lead compounds.

Experimental
The details are provided in the Supplementary Material (S).

Conclusions
In silico finding predicts the binding mechanism of competitive inhibitors and natural compounds with a-chymotrypsin enzyme.In this study, competitive inhibitors were docked with a-chymotrypsin enzyme to validate docking protocols.The result showed that strong binding interactions exist with active site amino acids and similarly natural product analogues also have a similar trend of binding with active site amino acid of a-chymotrypsin.Overall, inhibition activity of both competitive inhibitors and natural product analogues showed similar trends that showed a correlation between docking energies and percent inhibition.