Design, Synthesis, and Evaluation of Inhibitors of Pyruvate Phosphate Dikinase

Pyruvate phosphate dikinase (PPDK) catalyzes the phosphorylation reaction of pyruvate that forms phosphoenolpyruvate (PEP) via two partial reactions: PPDK + ATP + P_i → PPDK-P + AMP + PP_i and PPDK-P + pyruvate → PEP + PPDK. Based on its role in the metabolism of microbial human pathogens, PPDK is a potential drug target. A screen of substances that bind to the PPDK ATP-grasp domain active site revealed that flavone analogues are potent inhibitors of the <i>Clostridium symbiosum</i> PPDK. In silico modeling studies suggested that placement of a 3–6 carbon-tethered ammonium substituent at the 3′- or 4′-positions of 5,7-dihydroxyflavones would result in favorable electrostatic interactions with the PPDK Mg-ATP binding site. As a result, polymethylene-tethered amine derivatives of 5,7-dihydroxyflavones were prepared. Steady-state kinetic analysis of these substances demonstrates that the 4′-aminohexyl-5,7-dyhydroxyflavone <b>10</b> is a potent competitive PPDK inhibitor (<i>K</i>_i = 1.6 ± 0.1 μM). Single turnover experiments were conducted using 4′-aminopropyl-5,7-dihydroxyflavone <b>7</b> to show that this flavone specifically targets the ATP binding site and inhibits catalysis of only the PPDK + ATP + P_i → PPDK-P + AMP PP_i partial reaction. Finally, the 4′-aminopbutyl-5,7-dihydroxyflavone <b>8</b> displays selectivity for inhibition of PPDK versus other enzymes that utilize ATP and NAD