Molecular characterization of biological substrates and natural/synthetic compounds as inhibitors of typical 2-Cys peroxiredoxins (AhpC) from bacteria

Abstract

Multidrug resistant (MDR) bacterial strains are a threat to global public health and the search for new biological targets is a matter of worldwide interest. The host immune system has several strategies to combat infection by pathogenic bacteria, such as oxidative burst, increase in reactive oxygen species (ROS), and nitrosative, increase in reactive nitrogen species (ERNS) by immune cells, which aim to annihilate the pathogen by preventing its establishment. Additionally, several antibiotics are also capable of generating ROS, directly or indirectly, which contributes to the control of infections. On the other hand, pathogens have highly efficient enzymes to decompose these species, neutralizing the oxidative defenses of hosts and certain antibiotics. Among the species produced by defense cells, hydroperoxides represent a diverse group of compounds whose toxicity is mitigated by enzymes called peroxidases, especially in bacteria, for peroxiredoxin (Prx) AhpC. This Prx is extremely abundant in Gram+ and Gram- bacteria and can decompose hydroperoxides with extraordinary rates reaching 107-8M-1s-1. AhpCs were identified since ”ahpc strains are highly sensitive to organic peroxides, including long-chain fatty acid derivatives, which are generated in significant amounts because of oxidative burst and treatment with certain drugs. However, no work to date has attempted to biochemically investigate the organic biological substrates of AhpCs. About biochemical aspects, AhpCs are able to decompose hydroperoxides using a highly reactive cysteine called peroxidase (CP) which during the catalytic cycle forms a disulfide with a second cysteine residue called resolution (CR). The high reactivity of AhpC is related to a catalytic triad (TC), composed of two polar residues (Thr/Ser and Arg) that keep the CP sulfur in the thiolate form (S-) and are involved in directing and stabilizing the substrate. The replacement of Thr by Ser in Prx was considered redundant, however recent works by our group revealed that this substitution leads to oligomeric differences and differences in reactivity on hydroperoxides. When hydroperoxide concentrations are very high, there is a loss of peroxidase activity due to the hyperoxidation of CP in cysteine sulfinic acid (CP-SO2H). In bacteria there is no system capable of reducing this intermediate and the enzyme is degraded. In this context, substrates that have greater affinity for AhpC could act as biological inhibitors of the enzyme due to the inactivation of the enzyme and could be an important strategy to combat pathogenic bacteria. Recently, our group identified a natural compound, called CN-ABP1, which is similar to hydroperoxides derived from long-chain lipids, which can be biological substrates of AhpC. Interestingly, CN-ABP1 is able to inhibit AhpC from P. aeruginosa (Gram- which has Thr as part of TC) but not AhpC from S. epidermidis (Gram+ with Ser in TC). The objectives of this project are divided into three fronts involving: 1) investigations of the inhibitory potential of natural, semi-synthetic and synthetic compounds; 2) peroxides derived from long chain lipids can act as inhibitors of AhpC from P. aeruginosa and S. epidermidis, through methodologies involving computational and biochemical analysis and 3) determination of crystallographic structures of PaAhpC and reduced SeAhpC and containing CN-ABP1 and selected compounds. The project already presents preliminary data.