Structure-Based Design of Selective Fat Mass and Obesity Associated Protein (FTO) Inhibitors.

FTO catalyzes the Fe(II) and 2-oxoglutarate (2OG)-dependent modification of nucleic acids, including the demethylation of N6-methyladenosine (m6A) in mRNA. FTO is a proposed target for anti-cancer therapy. Using information from crystal structures of FTO in complex with 2OG and substrate mimics, we designed and synthesized two series of FTO inhibitors, which were characterized by turnover and binding assays, and by X-ray crystallography with FTO and the related bacterial enzyme AlkB. A potent inhibitor employing binding interactions spanning the FTO 2OG and substrate binding sites was identified. Selectivity over other clinically targeted 2OG oxygenases was demonstrated, including with respect to the hypoxia-inducible factor prolyl and asparaginyl hydroxylases (PHD2 and FIH) and selected JmjC histone demethylases (KDMs). The results illustrate how structure-based design can enable the identification of potent and selective 2OG oxygenase inhibitors and will be useful for the development of FTO inhibitors for use in vivo.

The Derivation of Primary Human Epicardium-Derived Cells.

To develop therapeutic strategies for the regeneration of lost heart muscle after myocardial infarction (MI), a source of functional new muscle cells and associated coronary vessels must be identified. The epicardium is a source of several cardiovascular cell types during heart development and is widely regarded as a resident progenitor population, which becomes dormant during adulthood. In adult mice, MI induces epicardial reactivation characterized by an upregulation of fetal genes and subsequent epicardium derived cell (EPDC) proliferation, migration, and differentiation. Determining whether the epicardium can be therapeutically targeted following cardiovascular disease requires an in vitro system for the study of adult human EPDCs (hEPDCs). This protocol describes techniques to establish and maintain human epicardium explant cultures from patient-derived right atrial appendage biopsies and documents methods to probe the resultant outgrowth of hEPDCs. The model facilitates a high-throughput approach to either genetic or chemical phenotypic screening for drug-like modifiers of hEPDC activation and potential cell fate.