Periodic Reporting for period 2 - PP-MAGIC ((Photo-)Control of Persisters: Targeting the Magic Spot) Reporting period: 2021-09-01 to 2023-02-28 Summary of the context and overall objectives of the project The current Covid pandemic highlights the vulnerabilities of society to pathogens. While currently a viral infection is spreading, also bacterial infections are on the rise and increasingly difficult to address, as bacteria are evolving into more and more drug resistant strains. Currently, only very few antibiotics are being developed and novel mechanisms of action are rare. Besides the development of resistance to treatments, also persistence is an issue, in which the bacteria simply reduce or shut-down their metabolism to survive antibiotic treatment. Since antibiotics often target an active metabolism, this is an efficient evasion strategy and persistent infections are a great threat to patients. We want to develop new compounds that help to better understand and ultimately avoid bacterial persistence, thereby improving antibiotic treatments that target active metabolism.The development of persistence is regulated amongst others by small molecules which are called the magic spot nucleotides. We want to investigate, how magic spot nucleotides regulate bacterial metabolism and identify ways to interfere with this regulation so that metabolic shutdown of bacteria will not work anymore. Under such conditions, either additional antibiotics or even the patients immune system alone might clear the infection.Overall, we address an important societal problem, which is the threat by multidrug-resistant and persistent bacteria. We want to address this problem by developing a chemical biology medicinal chemistry approach to understand and manipulate the magic spot nucleotides. This requires combination of synthetic chemisrtry, analytical chemistry, biochemistry, and microbiology, which we aim to achieve in this project. Ultimately, we are aiming for a deeper understanding of how the magic spot nucleotides operate and how we can manipulate them to discover new antibiotics for society. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far We have been able to set up efficient chemical syntheses of magic spot nucleotides and developed chemistry to modify the compounds. We have been able to implement analytical approaches to track magic spot nucleotide concentrations, isolated from bacterial samples, such as E. coli and several mycobacteria. We have implemented capillary electrophoresis using stable isotope labeled reference compounds for their absolute quantitation. We have synthesized a small library of magic spot nucleotide analogues as potential inhibitors of the enzymes involved in magic spot turnover. In these instances, we have removed negative charges from the structure to improve cellular activity as opposed to simple enzyme inhibition. Moreover, we have started to construct photoswitchable and photoactivatable magic spot nucleotides, that will enable us to manipulate magic spot nucleotide concentrations in living bacterial cells. We have been able to identify a facilitator of cell uptake of Magic Spot Nculeotides and Photocaged Analogues, which can be released on demand. These compounds will enable us to study direct effects of MSN on different cellular processes simply by feeding them to bacteria followed by irradiation. Moreover, we have been able to delineate interactomes (protein interaction partners) of different MSN based on novel photoaffinity pull-down probes. These probes continue to provide insights into MSN signalling and are already applied in other laboratories. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) We have been particularly successful with magic spot nucleotide analyses using capillary electrophoresis mass spectrometry. These results will be published soon. The sensitivity is so high, that we have additionally been able to track these molecules also in plants, in which they occur in much lower concentrations. We studied, how plants react to bacterial infections with regards to magic spot nucleotide production but also apply this technology to relevant pathogens, such as Staphylococcus aureus and Mycobacterium tuberculosis. We will now start to study the efficiency of our new synthetic inhibitors and see if they affect bacterial growth, metabolic shutdown, and viability. This should lead to new structures for potential antibiotics that could be developed as new pharmaceutical entities. We have also developed a direct uptake system for MSN into bacteria. This will enable a plethora of experiments by simply incubating bacteria with compounds of interest. So far, MSN uptake was precluded due to the highly polar nature of MSN. Our pull-down studies with modified photoaffinity pull-down MSN has enabled us to delineate new protein interactors, which are currently under evaluation. This will provide insights into how MSN are regulated and how (and which) cellular processes are regulated by them. overview-pp-magic.png
