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 Clostridium symbiosum 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 10 is a potent competitive PPDK inhibitor (K(i) = 1.6 ± 0.1 μM). Single turnover experiments were conducted using 4′-aminopropyl-5,7-dihydroxyflavone 7 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 8 displays selectivity for inhibition of PPDK versus other enzymes that utilize ATP and NAD.