Structure-Function Investigation of the Interaction of 1- and 2-Substituted 3-Hydroxypyridin-4-ones with 5-Lipoxygenase and Ribonucleotide Reductase

Abstract

The structural and physiochemical properties of 3-hydroxypyridin-4-one chelators (HPOs) which influence inhibition of the iron-containing metalloenzymes ribonucleotide reductase (RR) and 5-lipoxygenase (5-LO) have been investigated. HPOs with substituents at the 1-and 2-positions of the pyridinone ring have been synthesized, and their inhibitory properties compared with those of desferrioxamine (DFO). Varying the alkyl substituents does not affect the affinity constant of these ligands for iron(III), but permits a systematic investigation of the effect of hydrophobicity and molecular shape on inhibitory properties. The inhibition of RR was monitored, indirectly by measuring tritiated thymidine incorporation into DNA and directly by the quantification of the EPR signal of the enzyme tyrosyl radical. 5-LO inhibition was examined spectrophotometrically, measuring the rate of linoleic hydroperoxide formation by soybean lipoxygenase. The results indicate that the substituent size introduced at the 2-position of the HPO ring is critical for determining inhibition of both enzymes. Large substituents on the 2-position, introduce a steric factor which interferes with accessibility to the iron centers. These studies have identified chelators such as 1,6-dimethyl-2-(N-4,N-propylsuccinamido) methyl-3-hydroxypyridin-4-one (CP358), which causes only a 10% inhibition of 5-LO after 24 h of incubation at 110 M IBE (iron-binding equivalents) in comparison to simple dialkyl HPOs such as Deferiprone (CP20) which cause up to 70% inhibition. Using EPR spectroscopy, CP358 inhibits RR at a slower rate than CP20, while chelating intracellular iron(III) at a similar rate, a finding consistent with an indirect inhibition of the tyrosyl radical. However, hepatocellular iron is mobilized at a faster rate by CP358 (P < 0.001). These findings demonstrate that it is possible to design bidentate HPOs which access intracellular iron pools rapidly while inhibiting non-heme iron-containing enzymes relatively slowly, at rates comparable to DFO. It is anticipated that such compounds will possess a superior therapeutic safety margin to currently available bidentate HPOs.The rate at which iron chelators inhibit non-heme iron-containing metalloenzymes is predicted to have a critical influence on their cellular effects. Iron chelators in current clinical use are the hexadentate chelator desferrioxamine (DFO) 1 and the smaller orally active bidentate hydroxypyridinone deferiprone (CP20) (see Fig. 1, Table I) (1). Both are known to be antiproliferative (2) and to induce apoptosis in a variety of cells (3, 4). Additionally CP20 induces bone marrow hypoplasia in laboratory animals (5) and agranulocytosis in a proportion of humans (6). To limit antiproliferative activity and to have the widest possible therapeutic safety margin, such chelators must chelate excess iron without removing iron that is essential for normal cellular homeostasis.We have shown previously that the key non-heme iron containing enzymes 5-lipoxygenase (5-LO) and ribonucleotide reductase...

An Increase in the Expression of Ribonucleotide Reductase Large Subunit 1 Is Associated with Gemcitabine Resistance in Non-Small Cell Lung Cancer Cell Lines

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