title: Inhibition of human ornithine decarboxylase activity by enantiomers of difluoromethylornithine.
abstract: Racemic difluoromethylornithine (D/L-DFMO) is an inhibitor of ODC (ornithine decarboxylase), the first enzyme in eukaryotic polyamine biosynthesis. D/L-DFMO is an effective anti-parasitic agent and inhibitor of mammalian cell growth and development. Purified human ODC-catalysed ornithine decarboxylation is highly stereospecific. However, both DFMO enantiomers suppressed ODC activity in a time- and concentration-dependent manner. ODC activity failed to recover after treatment with either L- or D-DFMO and dialysis to remove free inhibitor. The inhibitor dissociation constant (K(D)) values for the formation of enzyme-inhibitor complexes were 28.3+/-3.4, 1.3+/-0.3 and 2.2+/-0.4 microM respectively for D-, L- and D/L-DFMO. The differences in these K(D) values were statistically significant ( P <0.05). The inhibitor inactivation constants (K(inact)) for the irreversible step were 0.25+/-0.03, 0.15+/-0.03 and 0.15+/-0.03 min(-1) respectively for D-, L- and D/L-DFMO. These latter values were not statistically significantly different ( P >0.1). D-DFMO was a more potent inhibitor (IC50 approximately 7.5 microM) when compared with D-ornithine (IC50 approximately 1.5 mM) of ODC-catalysed L-ornithine decarboxylation. Treatment of human colon tumour-derived HCT116 cells with either L- or D-DFMO decreased the cellular polyamine contents in a concentration-dependent manner. These results show that both enantiomers of DFMO irreversibly inactivate ODC and suggest that this inactivation occurs by a common mechanism. Both enantiomers form enzyme-inhibitor complexes with ODC, but the probability of formation of these complexes is 20 times greater for L-DFMO when compared with D-DFMO. The rate of the irreversible reaction in ODC inactivation is similar for the L- and D-enantiomer. This unexpected similarity between DFMO enantiomers, in contrast with the high degree of stereospecificity of the substrate ornithine, appears to be due to the alpha-substituent of the inhibitor. The D-enantiomer may have advantages, such as decreased normal tissue toxicity, over L- or D/L-DFMO in some clinical applications.
authors.affiliation: Arizona Cancer Center, The University of Arizona, Tucson, AZ 85724, USA.
authors.forename: Ning
authors.initials: N
authors.lastname: Qu
authors.forename: Natalia A
authors.initials: NA
authors.lastname: Ignatenko
authors.forename: Phillip
authors.initials: P
authors.lastname: Yamauchi
authors.forename: David E
authors.initials: DE
authors.lastname: Stringer
authors.forename: Corey
authors.initials: C
authors.lastname: Levenson
authors.forename: Patrick
authors.initials: P
authors.lastname: Shannon
authors.forename: Scott
authors.initials: S
authors.lastname: Perrin
authors.forename: Eugene W
authors.initials: EW
authors.lastname: Gerner
mesh_terms: D004305:Dose-Response Relationship, Drug; D000518:Eflornithine / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D009955:Ornithine Decarboxylase / Q000378:metabolism; D065108:Ornithine Decarboxylase Inhibitors; D013237:Stereoisomerism; D013997:Time Factors
chemical_list: D004791:Enzyme Inhibitors; D065108:Ornithine Decarboxylase Inhibitors; D009955:Ornithine Decarboxylase; D000518:Eflornithine