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Staphylococcus aureus and monocytogenes is reduced by the small molecule inhibitor AV73 Jan Vomacka,1 Vadim S. Korotkov,1 Megan C. Jennings,2 Matthias Stahl,1 William M. Wuest,2 Katrin Lorenz-Baath1 and Stephan A. Sieber1 1 Technische Universität München, Department of Chemistry, Lichtenbergstraße 4, 85748 Garching, Germany 2 Temple University, Department of Chemistry, 448 Beury Hall, Philadelphia, PA 19086, United States

Background: Persistent forming The spread of resistant bacteria poses a by the for pose a major threat in nosocomial major threat to public health. In addition to establishment and propagation (3). in other organism pathogenesis treatment with , strategies are emerging Inhibition of virulence pathways settings. In particular in combination with cytolysis in other organism multi , toxin-secreting that target the pathogenicity, i.e. the virulence, of disarms bacteria without directly bacteria, instead of inhibiting growth (1, 2). affecting their viability, presumably killing of cells of other organism cause life-threatening . Bacterial virulence is determined by a diverse set of thus leading to less selective pressure cytolysis multi-organism cellular process molecules, including toxins and adhesins, needed and slower resistance development disruption of Material / methods: We screened a library of cell killing cells of other cellular process (4-8). organism over 200 potential anti-virulence compounds Searching for potent virulence modification of morphology or against aureus hemolysis. The inhibitors we screened a library of physiology of other organism screening of > 200 potential compounds over 200 potential anti-virulence mode of action of the most potent compound in phenotypic hemolysis assay with S. aureus NCTC8325 was studied by quantitative proteomics compounds against S. aureus regulation of biological quality HO multi-organism process experiments. hemolysis. As one of the best hits we obtained AV73, a simple small biological regulation 7 molecule with a diol core structure Results: One of the best compounds (AV73) OH AV73 (Fig. 1). In our recently published biological_process from the anti-virulence screening did not only work we show, that a close derivative reduce hemolytic toxin α- in further analysis of AV73 exhibits a broad inhibition of S. aureus supernatant to less then 20% but to of biological Fig. 2. GO term hierarchical network analysis of 5% most down- effect on bacteria S. aureus virulence (9). We therefore inhibition of... regulated in S. aureus supernatant, grown under hemolysis our surprise also impeded in vitro anticipated an effect of AV73 also on assay growth conditions, treated with 50 µM AV73. Network formation by S. aureus strain NCTC8325 to 1. 2. 3. other virulence phenotypes such as generation with the BiNGO plugin for Cytoscape. S. aureus S. aureus L. monocytogenes biofilm formation. Indeed we could less than 25%. At the same time bacterial NCTC8325 NCTC8325 EGD-e growth was not affected. Furthermore Listeria hemolysis biofilm formation biofilm formation measure a clear effect on S. aureus but also on L. monocytogenes biofilms. monocytogenes biofilms could be reduced by DMSO DMSO and fibriniogen-binding proteins was observed in S. aureus However the desired retention of AV73. In addition, the effect on bacterial DMSO supernatants which corresponds to the anti-hemolytic and anti- AV73 growth was not observed with (50 µM) biofilm formation activity of AV73. A GO term analysis of the most proteomes and also extracellular levels L. monocytogenes. Here AV73 treated 0 50 100 down-regulated proteins in S. aureus supernatant, grown under was analyzed. Subsequent proteomics cultures reduced growth to 60% of hemolysis in % AV73 (50 µM) AV73 (50 µM) hemolysis assay conditions, demonstrates a clear cluster of virulence experiments will provide a direct target profile DMSO control which implies a higher 10 8 2 related terms like ‚pathogenesis‘, ‚cytolysis‘ and ‚cell via chemical probe based target identification / 8 6 risk for resistance development. 600 6 600 600 killing‘ (Fig. 2). Future studies will include the synthesis of a deconvolution in whole bacterial cells. 4 4 1 To explain the phenotypic effect of OD OD OD photoactivatable chemical tool compound that can be used to further 2 2 AV73 on a protein level, we 0 0 0 deconvolute the protein target and elucidate the cellular performed quantitative (dimethyl- Conclusions: The simple small molecule mechanisms that lead to the desired anti-virulence effect.

AV73 AV73 AV73 labeling) mass spectrometric analysis

AV73 is a potential anti-virulence (anti-biofilm DMSO DMSO DMSO of cellular but also extracellular and anti-hemolysis) agent addressing S. aureus proteins. For those proteomic In conclusion, AV73 has the potential to guide efforts towards a and L. monocytogenes in an in vitro setting. A Fig. 1. Hemolysis assay was performed with a experiments bacteria were cultured better understanding of bacterial virulence mechanisms. This lack of growth inhibition implies low selective suspension of sheep erythrocytes in PBS added to knowledge will enable the development of evolution-proof bacterial supernatants. Biofilms of bacteria grown under the same conditions as in the pressure and therefore presumably low under static conditions in 96-well plates were hemolysis and biofilm assays. A drugs (8), that address hemolysis, biofilm formation and other resistance development in S. aureus. stained with . strong down-regulation of virulence phenotypes.

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