Structural basis for the inhibition of PDK2 by novel ATP- and lipoyl-binding site targeting compounds.

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. Image 1 • A crystal structure of human PDK2 with a novel allosteric inhibitor at the lipoyl-binding site. • Crystal structures of human PDK2 with the 4,5-diarylisoxazole-derived compounds. • These inhibitors are first to be found in the crystal structure of any PDK isoform. • First structures showing the asymmetric composition of PDK2 dimer from human. Scientific Summary: We describe the structural, biophysical, and mechanistic characterization of human PDK2, which is responsible for modulating the activity of pyruvate dehydrogenase complex (PDH) through phosphorylating key serine residues of the E1 subunit. Overexpression of PDK isoforms results in the inhibition of OXPHOS in mitochondria, which at least partly contribute to the Warburg effect, a characteristic feature of cancer metabolism. PDK activities are also known to be closely related to diabetes and heart failure, hence have been extensively studied. We have determined a crystal structure of human PDK2, the most abundant isoform among four, in the presence of an allosteric inhibitor to 1.93 Å resolution. The chemical structure of this compound is based on a novel anthraquinone-scaffold, and the co-crystal structure provides a unique binding mode of the inhibitor in the lipoyl-binding pocket of the protein. Moreover, we have determined two crystal structures bound with ATP-competitive inhibitors, which are derived from 4,5-diarylisoxazole core. These structures are also the first to demonstrate detailed molecular interactions of PDK with a 4,5-diarylisoxazole derivative. We interrogated in vitro binding affinities of these compounds in solution through biophysical approaches including isothermal calorimetry (ITC) and saturation transfer difference (STD) experiments. Lastly, the anticancer activities of these inhibitors were examined via cell viability assays against HeLa cells. Highlights and significance of this manuscript include 1) co-crystal structures of PDK2 provide valuable insights into the mechanism underlying the structural basis for the inhibition of PDK by compounds targeting the ATP- and lipoyl-binding sites, 2) the asymmetric composition of conformational states within dimeric PDK2, which has not previously identified in human PDK2 structures, and 3) a potential structural-guide for designing enhanced therapeutic agents to control related diseases including diabetes, obesity, and cancer. [ABSTRACT FROM AUTHOR]