Development of new antibiotics targeting histidine kinases regulating virulence mechanisms.

Antimicrobial resistance (AMR) is the cause of 700,000 deaths worldwide every year. Alarmingly, the pace at which we are discovering novel antibiotics has slowed while antibiotic resistance emergence has been increasing worldwide. It has been proposed that if efforts to control AMR are not increasing, AMR infections might lead to 10 million people dying every year by 2050. This thesis research project that is part of the CARTNET (Combating Antimicrobial Resistance Network) network, will focus on the design and development of compounds that target bacterial two-component systems (TCSs), promising targets for novel antimicrobials and anti-infectives.

TCSs, involved in bacterial adaptation to the environment, virulence and antibiotic resistance, consist of a sensor histidine kinase (HK) and a cognate response regulator (RR) that modulates gene expression in response to the stimulus.

The sensing module of the (HK) senses the external stimulus and undergoes an autophosphorylation reaction catalysed by the catalytic and ATP binding domain (CA). The phosphorylation allows dimerization and conformational changes in the response module of the RR leading to regulation of target gene expression.

From a drug discovery perspective, the most attractive domain to target TCS is the HK CA domain, a highly conserved domain among TCSs. We and our collaborators design compounds that bind CA domains and inhibit autophosphorylation. We hypothesize that i) such compounds can inhibit several HKs simultaneously, ii) the inhibition of multiple targets would hinder the emergence of resistance to the inhibitors, and iii) these inhibitors have broad-spectrum applications since TCS are present in nearly all bacteria.

The current most potent inhibitors of bacterial HK autophosphorylation in vitro are also bactericidal in vivo.  In this project we aim to:

• Provide biological information, including inhibitory concentrations, in vitro toxicity, potential of resistance development, and off-target activity, to support and guide structure-based development and synthesis of more potent antibacterials or anti-infectives targeting HK autophosphorylation;
• Establish an in vivo reporter gene assay for QseC-regulated genes in enteropathogenic E. coli (EPEC). QseC is a relevant TCS involved in the regulation of virulence. This reporter gene assay will be used to screen for new inhibitors and gain better understanding of the regulation of EPEC virulence;
• Test the absorption of the most promising compounds using intestinal organoids; and
• Assess the efficacy and toxicity of lead compounds in a mammalian infection model with E. faecium, a relevant Gram-positive pathogen that frequently causes hospital-associated infections.

Project leader: Prof. Jerry Wells

Email: jerry.wells@wur.nl