Periodic Reporting for period 1 - CLEAR (“CLEAR”: Cell Envelope Antibacterials)
Periodo di rendicontazione: 2024-01-01 al 2025-12-31
Antimicrobial resistance (AMR) has escalated into a global health crisis, with more than one million estimated deaths in 2019 attributable to infections with antibiotic resistant pathogens. Gram-positive bacteria are responsible for a large share of these and among them, the methicillin resistant Staphylococcus aureus (MRSA) is one of the biggest challenges. MRSA strains are resistant to β lactams, which by far are the most widely used class of antibiotic that kill bacteria by inhibiting the synthesis of the bacterial cell wall. Bacteria are surrounded by a cell envelope that is complex and in addition to the cell wall also is composed of a cell membrane and abundant amounts of glycopolymers including the teichoic acids. The hypothesis of the CLEAR training project is that even though resistance has developed to major classes of antibiotics targeting the cell envelope, this compartment of Gram positive bacteria still represents valuable targets for development of new antibacterial therapeutics.
CLEAR aims to revitalize the antibiotic development pipeline by advancing three innovative strategies focused on the Gram positive cell envelope. The objective of the first is to inhibit “intrinsic” resistance factors namely bacterial components required for expressing resistance, but not classical resistance genes and thereby potentially restoring β lactam susceptibility. The objective of the second is to identify new drug candidates through pathway directed screening and structural analyses of essential cell wall biosynthesis proteins. The third objective is to explore alternative therapeutic avenues such as exploiting previously unrecognized susceptibility of MRSA to combinations of penicillins and β lactamase inhibitors, by applying bacteriophages or endolysins and by repurposing of approved drugs with the antiplatelet drug ticagrelor as an example. Together, these complementary approaches form the research and training backbone of CLEAR, aiming to generate effective solutions against some of the most challenging Gram positive pathogens driving the AMR crisis.
CLEAR aims to revitalize the antibiotic development pipeline by advancing three innovative strategies focused on the Gram positive cell envelope. The objective of the first is to inhibit “intrinsic” resistance factors namely bacterial components required for expressing resistance, but not classical resistance genes and thereby potentially restoring β lactam susceptibility. The objective of the second is to identify new drug candidates through pathway directed screening and structural analyses of essential cell wall biosynthesis proteins. The third objective is to explore alternative therapeutic avenues such as exploiting previously unrecognized susceptibility of MRSA to combinations of penicillins and β lactamase inhibitors, by applying bacteriophages or endolysins and by repurposing of approved drugs with the antiplatelet drug ticagrelor as an example. Together, these complementary approaches form the research and training backbone of CLEAR, aiming to generate effective solutions against some of the most challenging Gram positive pathogens driving the AMR crisis.
To address the nature of intrinsic resistance in S. aureus, one focus has been to study if MRSA strains can become sensitive to β lactams. This has been demonstrated to be the case based on key experiments where central coordination of cell wall synthesis has been perturbed at the points of teichoic acid synthesis and separation of daughter cells. These findings reveal intrinsic bacterial factors that are required for MRSA strains to be β lactam resistant and thus can serve as targets for novel therapeutics that sensitizes resistant strains. Such a candidate has potentially been identified in extracts of Actinomyces and future analysis will pinpoint active compound. Another approach is to revitalize β lactams in resistant strains is to use combinations β lactamase inhibitors such as clavulanic acids. To understand the mode of action the structure of MRSA PBP2a was determined bound to amoxicillin, both with and without clavulanic acid, offering valuable insights into β lactam interactions with this resistance associated protein.
New drug candidates and targets have been sought through pathway and structural analysis involving firstly examination of genes that affect cell wall porosity. To this end transposon mutagenesis combined with FACS based sorting revealed two genes representing new potential targets for enhancing antibiotic susceptibility. To assess the potential of these hits, work is in progress to screen inhibitors both virtually and experimentally. Inhibitors of cell wall synthesis has been investigated by virtual compound screening guided by AlphaFold structural predictions of the L,D transpeptidases and from a successful crystal structure of the L,D-transpeptidase from Clostridioides difficile. Further this work identified isatin as a promising hit. A synthetic route for generating multiple isatin based variants was successfully developed, demonstrating that the isatin scaffold is highly amenable to chemical modification. For screening purposes, a novel data analysis pipeline has been developed to identify promising natural product candidates based on mass spectrometry dataset. This approach has led to several new molecular hits, including a purified compound tentatively identified as a novel cyclic pentapeptide with a long fatty acid chain.
To examine the potential of repurposing of drugs, patented ticagrelor derived compounds devoid of antiplatelet activity were engineered and tested for potency against MRSA and other Gram-positive bacteria. All derivatives retained a multi target antimicrobial mode of action involving membrane disruption and inhibition of peptidoglycan biosynthesis. MRSA strains evolved for ticagrelor resistance showed increased resistance to most derivatives, and notable differences were observed in how the transcriptional regulator Spx contributes to resistance across the compound series. Further 39 novel ticagrelor-derived compounds analogues designed to retain strong antibacterial activity while eliminating unwanted antiplatelet effects and been synthesized. Mode of action studies have shown that three representative compounds act similarly to ticagrelor by targeting the bacterial cell envelope, binding to membrane lipids, and disrupting membrane potential.
As another alternative approach to target antibiotic resistant cells a high throughput screening system has been developed to test combinations of phages and antibacterial compounds and against a library of 103 compounds, resulting in the discovery of several promising phage compound combinations. Also, novel phage encoded, antimicrobial endolysins have been examined and structurally determined and activity studies are ongoing.
New drug candidates and targets have been sought through pathway and structural analysis involving firstly examination of genes that affect cell wall porosity. To this end transposon mutagenesis combined with FACS based sorting revealed two genes representing new potential targets for enhancing antibiotic susceptibility. To assess the potential of these hits, work is in progress to screen inhibitors both virtually and experimentally. Inhibitors of cell wall synthesis has been investigated by virtual compound screening guided by AlphaFold structural predictions of the L,D transpeptidases and from a successful crystal structure of the L,D-transpeptidase from Clostridioides difficile. Further this work identified isatin as a promising hit. A synthetic route for generating multiple isatin based variants was successfully developed, demonstrating that the isatin scaffold is highly amenable to chemical modification. For screening purposes, a novel data analysis pipeline has been developed to identify promising natural product candidates based on mass spectrometry dataset. This approach has led to several new molecular hits, including a purified compound tentatively identified as a novel cyclic pentapeptide with a long fatty acid chain.
To examine the potential of repurposing of drugs, patented ticagrelor derived compounds devoid of antiplatelet activity were engineered and tested for potency against MRSA and other Gram-positive bacteria. All derivatives retained a multi target antimicrobial mode of action involving membrane disruption and inhibition of peptidoglycan biosynthesis. MRSA strains evolved for ticagrelor resistance showed increased resistance to most derivatives, and notable differences were observed in how the transcriptional regulator Spx contributes to resistance across the compound series. Further 39 novel ticagrelor-derived compounds analogues designed to retain strong antibacterial activity while eliminating unwanted antiplatelet effects and been synthesized. Mode of action studies have shown that three representative compounds act similarly to ticagrelor by targeting the bacterial cell envelope, binding to membrane lipids, and disrupting membrane potential.
As another alternative approach to target antibiotic resistant cells a high throughput screening system has been developed to test combinations of phages and antibacterial compounds and against a library of 103 compounds, resulting in the discovery of several promising phage compound combinations. Also, novel phage encoded, antimicrobial endolysins have been examined and structurally determined and activity studies are ongoing.
Beyond state-of-the-art the CLEAR project has made several contributions. By uncovering that native penicillin binding proteins control splitting of cells at division via controlling teichoic acid synthesis explains the synergy observed between β lactams and teichoic acid synthesis-inhibitors in MRSA strains providing fundamental knowledge of the β lactam killing processes as well as a novel drug target. This target has already been explored by identifying natural compounds that sensitizes MRSA strains to β lactams.
The detailed structural information of LD-transpeptidase involved in intrinsic resistance of C. difficile towards β-lactams provides novel insights into the mode of action of these types of cell wall synthesis proteins and forms a very important foundation for docking studies to identify novel inhibitors.
The approach of repurposing of already approved drugs has provided novel ticagrelor derivatives that are devoid of their anti-platelet activity while maintaining antimicrobial activity against drug-resistant Gram-positive bacteria. These results show that repurposing drugs may be a promising approach to novel antimicrobials in a faster and easier way partially short cutting the need for extensive clinical tests. Another novel approach of impact is the combination of bacterial viruses (phages) or phage encoded products with antimicrobial compounds. These studies shows that there are combinations of very high efficacy in killing bacteria and calls for detailed analysis of the mode of action.
The detailed structural information of LD-transpeptidase involved in intrinsic resistance of C. difficile towards β-lactams provides novel insights into the mode of action of these types of cell wall synthesis proteins and forms a very important foundation for docking studies to identify novel inhibitors.
The approach of repurposing of already approved drugs has provided novel ticagrelor derivatives that are devoid of their anti-platelet activity while maintaining antimicrobial activity against drug-resistant Gram-positive bacteria. These results show that repurposing drugs may be a promising approach to novel antimicrobials in a faster and easier way partially short cutting the need for extensive clinical tests. Another novel approach of impact is the combination of bacterial viruses (phages) or phage encoded products with antimicrobial compounds. These studies shows that there are combinations of very high efficacy in killing bacteria and calls for detailed analysis of the mode of action.