Effects of natural compounds on peroxidase activity of AhpCs and survival of pathogenic bacteria

Author(s)

Vitória Isabela Montanhero Cabrera

• Document type: Master's Dissertation
• Press: São Vicente. 2023-02-16.
• Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências. São Vicente
• Defense date: 2023-02-02
• Advisor: Marcos Antonio Oliveira

Abstract

The resistance of pathogenic bacteria to multiple antibiotics has increased worldwide in recent years, requiring the search for new antibacterial compounds. Recently, some studies have shown that distinct antibiotics with different targets have the convergent ability to produce reactive oxygen species (ROS), which contributes to the death of bacterial pathogens. Bacteria possess several antioxidant enzymes capable of breaking down ROS. Among them, the typical 2-Cys peroxiredoxins, called AhpC in bacteria, are highly reactive peroxidases that are extremely abundant in the cellular environment, and studies indicate that AhpC is associated with virulence. The high reactivity of AhpC is related to a catalytic triad, composed of two polar residues (Thr/Ser and Arg) that keep the sulfur of the catalytic cysteine, called cysteine peroxidase (CP) in the thiolate (S- ) form and are involved in the targeting and stabilization of the substrate to allow for chemical bimolecular nucleophilic substitution (SN2). Despite the importance of AhpC in bacteria, to date no inhibitors have been identified for these enzymes. On the other hand, some natural products have been identified as inhibitors of human isoforms. In general, these compounds present as common features a bulky hydrophobic skeleton and a carbonyl system capable of performing a thiol-Michael addition. In this work, we evaluated the inhibitory activity of natural compounds from the coastal biota of São Paulo state on the peroxidase activity of AhpC from Pseudomonas aeruginosa (PaAhpC) and Staphylococcus epidermidis (SeAhpC), two opportunistic Gram negative and positive bacteria, respectively, responsible for several hospital infections. The selection of compounds was made based on functional and structural characteristics of natural compounds identified for human isoforms. Through biochemical assays we identified a prenylated benzoic acid (CN-ABP1) from Piper crassinervium was able to inhibit the peroxidase activity of PaAhpC but did not inhibit SeAhpC or the human isoform (HsPrx2). Additionally we demonstrated that CN-ABP1 is not able to inhibit other thiol enzymes. The IC50 determination was 20.3 µM and by SDS PAGE we showed that the compound was not able to perform a thiol-Michael addition, indicating a mode of inhibition not yet described for this group of proteins. Computational simulations involving molecular docking, indicate that the compound is stabilized in the active site pocket by amino acids of the catalytic triad through polar and a large number of hydrophobic interactions. In this context, the results obtained in this investigation led to the identification of the first natural compound from the Brazilian coastal biota with high specificity for bacterial AhpC. As direct consequences of the investigation we initiated approaches with a series of compounds similar to CN-ABP1 aiming the identification of structural/functional characteristics involved in the inhibition promoted by CN-ABP1. Based on the elongated hydrophobic structure of the compound we hypothesized that it could mimics biological substrates such as long chain fatty acid hydroperoxides and initial results revealed that AhpC has high affinity for this type of substrate with hyperoxidation rates of 1.06 ´ 106 M-1 s-1 indicating that these compounds may act as biological inhibitors of AhpC. In order to deepen the knowledge of the structural biology of the enzyme we performed the enzyme crystallization assays and obtained crystals suitable to X-ray diffraction approaches. The determination of the structure of these proteins may help in a better understanding of the interaction between CN-ABP1- PaAhpC opening the perspective to identify other inhibitors that keep similarity with CN-ABP1 or long-chain hydroperoxides derivatives, which may, ultimately, assist the combat against pathogenic bacteria resistant to multiple antibiotics. (AU)

• FAPESP's process: 20/02868-1 - Effects of natural compounds over the peroxidase activity of AhpCs and pathogenic bacteria survival
• Grantee: Vitória Isabela Montanhero Cabrera
• Support Opportunities: Scholarships in Brazil - Master