Pyruvate dehydrogenase kinase (PDK) controls the activity of pyruvate decarboxylase complex (PDC) by phosphorylating key serine residues on the E1 subunit, which leads to a decreased oxidative phosphorylation in mitochondria. Inhibition of PDK activity by natural/synthetic compounds has been shown to reverse the Warburg effect, a characteristic metabolism in cancer cells. PDK-PDC axis also has been associated with diabetes and heart disease. Therefore, regulation of PDK activity has been considered as a promising strategy to treat related diseases. Here we present the X-ray crystal structure of PDK2 complexed with a recently identified PDK4 inhibitor, compound 8c, which has been predicted to bind at the lipoyl-binding site and interrupt intermolecular interactions with the E2-E3bp subunits of PDC. The co-crystal structure confirmed the specific binding location of compound 8c and revealed the remote conformational change in the ATP-binding pocket. In addition, two novel 4,5-diarylisoxazole derivatives, GM10030 and GM67520, were synthesized and used for structural studies, which target the ATP-binding site of PDK2. These compounds bind to PDK2 with a sub-100nM affinity as determined by isothermal titration calorimetry experiments. Notably, the crystal structure of the PDK2-GM10030 complex displays unprecedented asymmetric conformation of human PDK2 dimer, especially in the ATP-lids and C-terminal tails. (C) 2020 Elsevier Inc. All rights reserved.