Multi-Drug Resistant Bacteria represent a global emergency, limiting the effective treatment of bacterial infections. The World Health Organization considers bacterial resistance among the three main risks for human health, and the development of new strategies for fighting bacterial infections strongly desirable [1]. In 2014 report, WHO evokes a post-antibiotic era in which "common infections and minor injuries can kill" [2]. In bacteria, the SOS response is mainly orchestrated by two proteins: RecA and LexA [3, 4]. As a consequence of damage to the DNA, RecA promotes self-cleavage of the transcriptional repressor LexA, inducing the expression of more than 40 genes involved in DNA repair and mutagenesis [5]. The inactivation of the SOS regulators, by deletion of RecA and/or by engineering a noncleavable LexA into bacteria, reduces de facto the acquisition and integration of new resistance genes and the ability of bacteria to mutate, slowing the appearance of new drug resistance phenotypes [6-8]. The development of resistant bacteria is delayed at the level of vertical and horizontal transfer of resistance genes [9]. LexA represents therefore, an attractive target: blocking its action and reducing bacterial ability to evolve antibiotic resistance, would extend the long-term viability of both known and new antibiotics. This study is focused on the development of potential inhibitors of the SOS activation, through the inhibition of LexA self-cleavage. The autoproteolysis of LexA was experimentally induced by alkaline pH, or in presence of RecA protein, ATP and ssDNA. A first structure-based in silico screening has been performed and few boronic acid derivatives (Fig 1a) have been identified as potential LexA inhibitors. In our hand, boronic acids represented a potent tool to probe, for the first time, LexA catalytic site. We have developed a quantitative UPLC-MS method to efficiently follow the kinetics of LexA self-cleavage and the inhibitory effect on autoproteolysis of boronic acids. While the first order rate constant was calculated from the time course of autohydrolysis, to quantify the “potency” of inhibition a novel kinetic model has been developed (Fig 1b). The in vivo efficacy of boronic acids was evaluated by observing the reduction of filamentation induced by levofloxacin in E. coli (Fig 1c). The results coming from this first screening were encouraging, providing critical information on the catalytic pocket and on the binding requirements necessary for the improved design of high affinity ligands. Our preliminary data suggest that boronic acids can efficaciously inhibit the autoproteolysis of LexA.

The SOS response in bacteria and the LexA transcriptional repressor. Boronic acids derivatives: withstanding drug resistance by inhibiting the bacterial mechanisms of adaptation to antimicrobials / Celenza, Giuseppe; Tondi, Donatella; Cendron, Laura; Spyrakis, Francesca; Bellio, Pierangelo; Mancini, Alisia; Di Pietro, Letizia; Vicario, Mattia; Perilli, Mariagrazia; Amicosante, Gianfranco. - (2017). ((Intervento presentato al convegno 13th beta-lactamases Meeting tenutosi a Santo Stefano di Sessanio nel 16-19 June 2017.

The SOS response in bacteria and the LexA transcriptional repressor. Boronic acids derivatives: withstanding drug resistance by inhibiting the bacterial mechanisms of adaptation to antimicrobials

TONDI, Donatella;SPYRAKIS, FRANCESCA;
2017

Abstract

Multi-Drug Resistant Bacteria represent a global emergency, limiting the effective treatment of bacterial infections. The World Health Organization considers bacterial resistance among the three main risks for human health, and the development of new strategies for fighting bacterial infections strongly desirable [1]. In 2014 report, WHO evokes a post-antibiotic era in which "common infections and minor injuries can kill" [2]. In bacteria, the SOS response is mainly orchestrated by two proteins: RecA and LexA [3, 4]. As a consequence of damage to the DNA, RecA promotes self-cleavage of the transcriptional repressor LexA, inducing the expression of more than 40 genes involved in DNA repair and mutagenesis [5]. The inactivation of the SOS regulators, by deletion of RecA and/or by engineering a noncleavable LexA into bacteria, reduces de facto the acquisition and integration of new resistance genes and the ability of bacteria to mutate, slowing the appearance of new drug resistance phenotypes [6-8]. The development of resistant bacteria is delayed at the level of vertical and horizontal transfer of resistance genes [9]. LexA represents therefore, an attractive target: blocking its action and reducing bacterial ability to evolve antibiotic resistance, would extend the long-term viability of both known and new antibiotics. This study is focused on the development of potential inhibitors of the SOS activation, through the inhibition of LexA self-cleavage. The autoproteolysis of LexA was experimentally induced by alkaline pH, or in presence of RecA protein, ATP and ssDNA. A first structure-based in silico screening has been performed and few boronic acid derivatives (Fig 1a) have been identified as potential LexA inhibitors. In our hand, boronic acids represented a potent tool to probe, for the first time, LexA catalytic site. We have developed a quantitative UPLC-MS method to efficiently follow the kinetics of LexA self-cleavage and the inhibitory effect on autoproteolysis of boronic acids. While the first order rate constant was calculated from the time course of autohydrolysis, to quantify the “potency” of inhibition a novel kinetic model has been developed (Fig 1b). The in vivo efficacy of boronic acids was evaluated by observing the reduction of filamentation induced by levofloxacin in E. coli (Fig 1c). The results coming from this first screening were encouraging, providing critical information on the catalytic pocket and on the binding requirements necessary for the improved design of high affinity ligands. Our preliminary data suggest that boronic acids can efficaciously inhibit the autoproteolysis of LexA.
giu-2017
13th beta-lactamases Meeting
Santo Stefano di Sessanio
16-19 June 2017
Celenza, Giuseppe; Tondi, Donatella; Cendron, Laura; Spyrakis, Francesca; Bellio, Pierangelo; Mancini, Alisia; Di Pietro, Letizia; Vicario, Mattia; Perilli, Mariagrazia; Amicosante, Gianfranco
The SOS response in bacteria and the LexA transcriptional repressor. Boronic acids derivatives: withstanding drug resistance by inhibiting the bacterial mechanisms of adaptation to antimicrobials / Celenza, Giuseppe; Tondi, Donatella; Cendron, Laura; Spyrakis, Francesca; Bellio, Pierangelo; Mancini, Alisia; Di Pietro, Letizia; Vicario, Mattia; Perilli, Mariagrazia; Amicosante, Gianfranco. - (2017). ((Intervento presentato al convegno 13th beta-lactamases Meeting tenutosi a Santo Stefano di Sessanio nel 16-19 June 2017.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11380/1141363
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