Periodic Reporting for period 4 - ERACHRON (Eradicating Chronic Infections)
Periodo di rendicontazione: 2022-08-01 al 2024-07-31
During the last decades, there has been an alarming increase in the recalcitrance to treatment of chronic and recurrent infections. Even more alarming has been the seemingly unstoppable advance of antimicrobial resistance, listed among the top 10 global health threats and responsible for 1,27 million deaths in 2019 alone. The World Bank estimated that up to 3.8% of the global gross domestic product could be lost due to AMR by 2050.
This situation is worrying not only from the societal and economical point of view but also from a scientific perspective. Indeed, no new antibiotic class has been discovered in the last thirty years and only few new chemical entities (i.e. modifications of known antibiotic classes) have been approved for this use. We undeniably are in a so-called “post-antibiotic era”.
In this project, we are developing a conceptually novel approach against an underrated bacterial survival mechanism that enables the upkeep of chronic infections and paves the way to the insurgence of antimicrobial resistance. This phenomenon is called persistence.
Bacterial persisters are a dormant phenotype (meaning they do not carry specific causative genetic traits), present as a small percentage of every bacterial population, that simply appear to have shut down their metabolism.
This dormant subpopulation is now insensitive to most drugs and effectively constitutes a bacterial reservoir able to survive antibiotic treatment. Surprisingly though, when persisters “awake”, they become again sensitive to the antibiotic exactly as the original population.
The exact molecular mechanism responsible for this awake-dormant switch remains to be fully unravelled and that’s where we came into play with the ERACHRON project. Our goal is to fill the knowledge gap between persisters’ formation and infection eradication, providing the community with potent and selective small molecular tools that can be used to elucidate their formation mechanism and challenge complementary survival strategies.
This situation is worrying not only from the societal and economical point of view but also from a scientific perspective. Indeed, no new antibiotic class has been discovered in the last thirty years and only few new chemical entities (i.e. modifications of known antibiotic classes) have been approved for this use. We undeniably are in a so-called “post-antibiotic era”.
In this project, we are developing a conceptually novel approach against an underrated bacterial survival mechanism that enables the upkeep of chronic infections and paves the way to the insurgence of antimicrobial resistance. This phenomenon is called persistence.
Bacterial persisters are a dormant phenotype (meaning they do not carry specific causative genetic traits), present as a small percentage of every bacterial population, that simply appear to have shut down their metabolism.
This dormant subpopulation is now insensitive to most drugs and effectively constitutes a bacterial reservoir able to survive antibiotic treatment. Surprisingly though, when persisters “awake”, they become again sensitive to the antibiotic exactly as the original population.
The exact molecular mechanism responsible for this awake-dormant switch remains to be fully unravelled and that’s where we came into play with the ERACHRON project. Our goal is to fill the knowledge gap between persisters’ formation and infection eradication, providing the community with potent and selective small molecular tools that can be used to elucidate their formation mechanism and challenge complementary survival strategies.
The project envisages a multidisciplinary approach, targeting a specific bacterial survival enzyme that, when activated, increases significantly the intracellular levels of a signalling molecule called (p)ppGpp (an “alarmone”). We aim to design small molecules ad hoc to inhibit the accumulation of the alarmone, therefore hampering bacterial persisters formation.
We initially performed computational studies and obtained a catalytically competent model of the enzyme. We performed virtual screening of large and diversified fragment libraries (imagine collections of different Lego bricks) into the catalytic site of the enzyme, identifying three preferred chemotypes (i.e. the best fitting Lego brick shapes into a pocket) that were subsequently modified to obtain the first generation of ligands (i.e. adding small pieces to the core shape). We synthesised a selected set of molecules and performed interaction studies with the target enzyme, finding several ligands with affinities comparable to those of the natural substrates or better. Affinity measurements do not reveal where the binding takes place (i.e. where the Lego brick sticks to the protein). For this reason, we can either assess their effect on the enzyme activity (its inhibition is our end goal) or adopt structural approaches to identify their binding site.
We tried several techniques to quantify the formation of the alarmone, such as nuclear magnetic resonance (NMR), liquid chromatography (HPLC), electrochemistry, and fluorescence measurements with little success. Recently, we have been implementing a coupled enzymatic assay that could provide an easy readout of the reaction.
From the structural point of view, we have been working towards the achievement of the protein-ligand complexes X-ray crystal structures (in collaboration with another research group) and towards the assignment of the protein nuclear resonances in solution.
These results have been disseminated over the years by all involved group members mostly in the form of poster or oral communication at conferences.
We initially performed computational studies and obtained a catalytically competent model of the enzyme. We performed virtual screening of large and diversified fragment libraries (imagine collections of different Lego bricks) into the catalytic site of the enzyme, identifying three preferred chemotypes (i.e. the best fitting Lego brick shapes into a pocket) that were subsequently modified to obtain the first generation of ligands (i.e. adding small pieces to the core shape). We synthesised a selected set of molecules and performed interaction studies with the target enzyme, finding several ligands with affinities comparable to those of the natural substrates or better. Affinity measurements do not reveal where the binding takes place (i.e. where the Lego brick sticks to the protein). For this reason, we can either assess their effect on the enzyme activity (its inhibition is our end goal) or adopt structural approaches to identify their binding site.
We tried several techniques to quantify the formation of the alarmone, such as nuclear magnetic resonance (NMR), liquid chromatography (HPLC), electrochemistry, and fluorescence measurements with little success. Recently, we have been implementing a coupled enzymatic assay that could provide an easy readout of the reaction.
From the structural point of view, we have been working towards the achievement of the protein-ligand complexes X-ray crystal structures (in collaboration with another research group) and towards the assignment of the protein nuclear resonances in solution.
These results have been disseminated over the years by all involved group members mostly in the form of poster or oral communication at conferences.
Several ligands for the target enzyme have been already identified, with affinities comparable or improved with respect to the few molecules reported in literature. We have obtained a catalytically competent model of the enzyme that will facilitate a more accurate design of improved ligands. We have been working intensively on the setup of a reproducible and high- to mid-throughput enzymatic assay to assess the inhibitory potency of the synthesized ligands.
We expanded the planned activities including the characterization of the binding site at atomic resolution via NMR experiments. We therefore expect to be able to fully characterize the protein-ligand interaction and to assess the inhibitory potency of our molecules in the near future.
We expanded the planned activities including the characterization of the binding site at atomic resolution via NMR experiments. We therefore expect to be able to fully characterize the protein-ligand interaction and to assess the inhibitory potency of our molecules in the near future.