Protein-ligand interactions: a tool for discovery of bioactive natural products from environmental mixtures Ludek Sehnal 1 , Zuzana Tousova 1 , Petra Mikusova 1 , Tomas Kotacka 1 , Garry Paul Codling 1 , Roman Grabic 2 , Klara Hilscherova 1 1 Masaryk University, Czech Republic, 2 University of South Bohemia in České Budějovice, Czech Republic Exposure to complex mixtures of chemicals in the environment can cause harmful effects on organisms, including humans. Over the last two decades, different environmental matrices such as indoor dust 1 , wastewater 2 , or freshwater algal blooms 3 , have been identified as sources of endocrine-disrupting compounds (EDCs). Some EDCs adversely affect hormonal signaling through interaction with nuclear receptors such as thyroid hormone receptor, retinoic acid receptor, or estrogen receptor. By employing current knowledge of the interaction of the nuclear receptors with their natural ligands, we developed pull-down assays for the isolation and characterization of unknown bioactive compounds from environmental mixtures. We heterologously expressed and purified the His-tagged ligand-binding domain of thyroid hormone and retinoic acid receptors and confirmed the ligand-binding activity of these recombinant proteins using pull-down assays with natural ligands triiodothyronine (T3) and all- trans retinoic acid (ATRA), respectively. Pull-down assay efficiency was verified using ICP-LC-MS/MS, LC-MS/ MS and through bioactivity testing using receptor-specific reporter cell lines. Furthermore, environmental samples that showed high thyroid receptor-mediated (wastewaters) or retinoid-like (freshwater blooms) bioactivity was used for receptor-specific pull-down assays to capture potentially unknown chemicals. These samples were run on LC-Orbitrap in full scan for non-target analysis. By comparing bound and unbound fractions along with controls and blanks, it was possible to reduce the number of potentially bioactive compounds within a matrix from many thousand features to less than one hundred. This approach can identify novel and unknown chemicals interacting with these receptors. The work was supported by the Czech Science Foundation Grant No. 20-04676X. References 1. Nováková, Z. et al. Toxicity to bronchial cells and endocrine disruptive potentials of indoor air and dust extracts and their association with multiple chemical classes. J. Hazard. Mater. 424 , 127306 (2022). 2. Kasonga, T. K., Coetzee, M. A. A., Kamika, I., Ngole-Jeme, V. M. & Benteke Momba, M. N. Endocrine-disruptive chemicals as contaminants of emerging concern in wastewater and surface water: A review. Journal of Environmental Management 277 , 111485 (2021). 3. Sehnal, L. et al. Widespread occurrence of retinoids in water bodies associated with cyanobacterial blooms dominated by diverse species. Water Res. 156 , 136–147 (2019).
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