Bioactive molecules induce changes in the biomolecular landscape of the cell that can encode key information on compound action (Dubuis et al., 2018; Zampieri et al., 2018). In recent years, molecular profiling technologies like metabolomics have matured and now provide the necessary throughput to supply data rich in mechanistic information already in early stages of drug discovery. Drug-induced changes in pathogen metabolism can be characteristic for their mode of action (Zampieri et al., 2018), while metabolic signatures in host cells can inform on systemic effects and even host defense mechanisms. In this project, we will combine metabolome profiling with phenotypic readouts (proliferation, cytotoxicity, cytokine secretion) to identify metabolic effects linked to the action of candidate anti-infectives in pathogens (P. aeruginosa and Candida spp.) as well as in host cells, including epithelial cells. By adding molecular context to conventional phenotypic readouts, our platform will accelerate in-depth characterization of candidate anti-infectives (Ortmayr et al., 2022), including effects both on pathogens and host cells. Our results will strengthen the selection of the most promising candidates and complement proteome-level data in order to identify modes of action.

We will test the effect of natural compounds endowed with distinct anti-pseudomonal or anti-candida activity and available through AIDD consortium members on the metabolic state of cultured pathogens and in selected human cell models, including lung epithelial cells (Calu-3 cells) and cell proliferation (multiple cell lines available). Metabolic changes will be analyzed by mass spectrometry-based metabolomics (Sciex X500R QTOF-MS). In human cell models, phenotypes will be monitored by automated brightfield microscopy (TECAN Spark Cyto), resazurin assay (cytotoxicity) and ELISA (cytokines). All procedures are established in a 96-well format (Dubuis et al., 2018; Ortmayr et al., 2019), allowing efficient parallelization (up to 60 conditions/experiment).