Evaluation of Peroxisome Proliferator-activated receptor beta/delta behavior in skin regeneration processes and its modulation by ligands

Peroxisome Proliferator Activated Receptors (PPAR) are transcriptional factors regulated by ligands and members of the nuclear receptor superfamily. The PPARâ/ä is involved in metabolic processes such as energetic metabolism, adipogenesis and lipid metabolisms, which made it an interesting target for the development of drugs for metabolic disorders. In addition, this isotype is the most expressed in skin and, in this organ, it is involved in processes such as epidermal barrier maintenance, lipid synthesis, epidermal differentiation and wound healing, which made it a target for the development of dermocosmetics or drugs for skin repair. Aiming to select new activators (agonists) for PPARâ/ä, for future drug development, in this work, we developed a pipeline for screening of agonists for this receptor. The pipeline is composed of a cellular transactivation assay followed by two biophysical assays (Thermal Shift Assay and 8-Anilino-1-naphthalenesulfonic acid (ANS) fluorescence quenching assay) for characterization of the direct interaction of ligand:protein. The pipeline brings improvements if compared to traditional methods for PPAR ligand-screening: semi-automatization; parameter of apparent cytotoxicity; biophysical methodologies for confirmation of direct interaction ligand:protein; and calculation of dissociation constant between hits and the receptor. With the pipeline, we screened 121 compounds/extracts given by collaborators, 560 Brazilian plant extracts from Phytobios library, 719 drug-like compounds from NIH-NCC library, and 80 semi-synthetic natural products derivatives from TimTec NDL-3,000 library pre-selected via virtual screening. At the end of the pipeline, Extract 2 and one of its fraction (Extract 2-18) from Phytobios library showed to activate PPARâ/ä, stabilize its tertiary structure and had a dissociantion constant of, respectively, 0.011 ± 0.007 mg/mL e 0.016 ± 0.007 mg/mL, being selected as PPARâ/ä agonists. Besides the screening pipeline, methods of migration, proliferation and differentiation of keratinocytes (HaCaT cells) were also developed to characterize PPARâ/ä modulation by ligands in skin regeneration processes. These methods will be incorporated to the pipeline for agonist screening to futher characterize new PPARâ/ä ligands. The commercial agonists GW0742, GW501516 and L-165,041 increased the migration rate of HaCaT, and GW501516 and L-165,041 decreased proliferation in this cell type. Regarding HaCaT differentiation, gene expression of the analysed markers (keratin 14, keratin 1 and involucrin) were not changed after ligand treatment in the selected protocols. The RNA expression levels of extracellular matrix proteins (collagens, fibronectin and collagenases) were not altered after keratinocyte activation by PPARâ/ä ligand GW501516, nor in PPARâ/ä- knockout compared to wild type after wound healing stimuli. In summary, in this work we developed a pipeline to screening for PPARâ/ä agonists and further characterize them in skin regeneration processes (AU)