Investigation of molecular interactions between AhpC from Staphylococcus epidermidis with organic hydroperoxides and inhibitors

• Grant number: 21/00209-3
• Support type: Scholarships in Brazil - Scientific Initiation
• Effective date (Start): March 01, 2021
• Effective date (End): December 31, 2021
• Field of knowledge: Biological Sciences - Biochemistry - Metabolism and Bioenergetics
• Principal researcher: Marcos Antonio de Oliveira
• Grantee: Luiz Fernando Ribeiro
• Home Institution: Instituto de Biociências (IB-CLP). Universidade Estadual Paulista (UNESP). Campus Experimental do Litoral Paulista. São Vicente , SP, Brazil
• Associated research grant: 13/07937-8 - Redoxome - Redox Processes in Biomedicine, AP.CEPID

Abstract

Reactive oxygen (ROS) and nitrogen species (RNS) are produced by the defense repertoire of the hosts' immune system to combat microbial pathogens and by the antibiotics administration. Among ROS, organic hydroperoxides (OHPs), including lipid hydroperoxides, are potent microbicidal agents. 2-Cys peroxiredoxins (2-Cys Prx) represent an important line of defense capable to decompose a high variety of substrates, including OHPs. In bacteria, the typical 2-Cys Prx are so called alkyl hydroperoxide reductase C (AhpC), and studies show their importance for pathogens infection and maintenance in their hosts. AhpCs use a reactive cysteine residue (peroxidatic cysteine - CP) to decompose the substrates, and the high CP reactivity is achieved by interactions with the amino acids Thr, in some cases replaced by Ser, and Arg, which together form the catalytic triad (CT). Despite that the Thr to Ser substitution was considered redundant, we demonstrated this substitution leads to functional and structural differences in 2-Cys Prx and that the substitution is frequently found in prokaryotes but almost nonexistent in eukaryotes. Another difference resides in a C-terminal extension that projects to the active site, which occurs almost exclusively in eukaryotic isoforms. In this context, it's reasonable to assume that these differences in the microenvironment of the active site may lead to differences in the interaction with substrates and inhibitors. Our research group demonstrated that a natural compound (Adenanthin), initially identified as an inhibitor of human Prx 1 and 2, can inhibit AhpC from Escherichia coli more efficiently than the human isoforms, presenting IC50 = 0.481 ± 0.024 ¼M which is 4-30 x lower than the determined for human Prx1 and Prx2, what motivated an intellectual property application recently submitted to the INPI (BR10 20200162454). We also identified two natural compounds from São Paulo's biota named as CN-LS1 and CN-AB1, and the determination of IC50 for CN-LS1 revealed IC50 = 0.46 ± 0.025 ¼M. It is important to note that all three inhibitors described for 2-Cys Prx have in common an extensive hydrophobic skeleton, which remits to bulky organic peroxides, and an Q, µ-unsaturated carbonyl system capable of to perform a Michael addition to AhpC cysteines. However, the molecular interactions between AhpC with biological substrates, such as organic hydroperoxides or inhibitors, have not yet been investigated. This project aims to a better understanding of the molecular the interactions between AhpCs and ligands as inhibitors and organic hydroperoxides using AhpC from Staphylococcus epidermidis (SeAhpC), which has Ser on CT, as a model for the studies due to the medical importance of infections caused by this bacterium and the absence of studies in the literature. To reach theses aims, bioinformatics approaches will be performed to build a theoretical structural model and perform docking procedures with ligands, such as synthetic organic hydroperoxides (lipid and nitrogenous bases derivatives) and the AhpC inhibitors identified by our research group (Adn, CN-LS1, and CN -AB1). Approaches involving molecular biology and biochemical assays will be performed to evaluate the recombinant enzyme activity over OHPs of biological importance and the inhibitors previously mentioned. This project is already in progress and the SeAhpC conditions for expression and purification have already been established, being possible to obtain recombinant proteins with high yields (285.0 ¼M/250 mL cell culture), purity (>95%), and the assay of NADPH oxidation was standardized. A structural model of SeAhpC was also built and analyzes of molecular interactions between AhpC and CN-LS1 were carried out. We expected that the results of this project should favor a better understanding of molecular interactions between AhpCs with ligands, such as natural substrates and inhibitors.