PDHK1 is a non-canonical Ser/Thr kinase that negatively regulates the pyruvate dehydrogenase complex (PDC), restricting entry of acetyl-CoA into the tricarboxylic acid (TCA) cycle and downregulating oxidative phosphorylation. In many glycolytic tumors, PDHK1 is overexpressed to suppress activity of the PDC and cause a shift in metabolism toward an increased reliance on glycolysis (the Warburg effect). Genetic studies have shown that knockdown or knockout of PDHK1 reverts this phenotype and inhibits tumor growth in vitro and in vivo, but chemical tools to pharmacologically validate and build upon these data are lacking. We used AtomNet®, a deep convolutional neural network bioactivity predictor, to identify compound 7 as a potential inhibitor of PDHK1. During the process of hit validation, the active species was determined to be isomeric compound 10. Structure-activity studies based on 10 identified 17 as a low μM inhibitor of PDHK1 (IC50 = 1.5 ± 0.3 μM) that is selective against the other PDHK isoforms in both biochemical and cell-based assays. In A549 epithelial lung carcinoma cells, compound 17 inhibits phosphorylation of PDC E1α Ser232, a site that is specifically phosphorylated only by PDHK1, while minimally suppressing phosphorylation of Ser293, a site that is phosphorylated by all four PDHK isoforms. Altogether, these data identify 17 as a selective PDHK1 chemical probe useful for biochemical and cell-based studies.
