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 μM respectively for D-, L- and D/L-DFMO. The differences in these KD values were statistically significant (P < 0.05). The inhibitor inactivation constants (Kinact) 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 ∼ 7.5 μM) when compared with D-ornithine (IC50 ∼ 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 α-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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 465-470 |
| Number of pages | 6 |
| Journal | Biochemical Journal |
| Volume | 375 |
| Issue number | 2 |
| DOIs | |
| State | Published - Oct 15 2003 |
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Keywords
- Difluoromethylornithine enantiomer
- Enzyme inactivation parameter
- Ornithine decarboxylase
- Polyamine
ASJC Scopus subject areas
- Biochemistry
Cite this
Inhibition of human ornithine decarboxylase activity by enantiomers of difluoromethylornithine. / Ning, Q. U.; Ignatenko, Natalia; Yamauchi, Phillip; Stringer, David E.; Levenson, Corey; Shannon, Patrick; Perrin, Scott; Gerner, Eugene W.
In: Biochemical Journal, Vol. 375, No. 2, 15.10.2003, p. 465-470.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Inhibition of human ornithine decarboxylase activity by enantiomers of difluoromethylornithine
AU - Ning, Q. U.
AU - Ignatenko, Natalia
AU - Yamauchi, Phillip
AU - Stringer, David E.
AU - Levenson, Corey
AU - Shannon, Patrick
AU - Perrin, Scott
AU - Gerner, Eugene W.
PY - 2003/10/15
Y1 - 2003/10/15
N2 - 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 μM respectively for D-, L- and D/L-DFMO. The differences in these KD values were statistically significant (P < 0.05). The inhibitor inactivation constants (Kinact) 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 ∼ 7.5 μM) when compared with D-ornithine (IC50 ∼ 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 α-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.
AB - 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 μM respectively for D-, L- and D/L-DFMO. The differences in these KD values were statistically significant (P < 0.05). The inhibitor inactivation constants (Kinact) 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 ∼ 7.5 μM) when compared with D-ornithine (IC50 ∼ 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 α-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.
KW - Difluoromethylornithine enantiomer
KW - Enzyme inactivation parameter
KW - Ornithine decarboxylase
KW - Polyamine
UR - http://www.scopus.com/inward/record.url?scp=0142169526&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0142169526&partnerID=8YFLogxK
U2 - 10.1042/BJ20030382
DO - 10.1042/BJ20030382
M3 - Article
VL - 375
SP - 465
EP - 470
JO - Biochemical Journal
JF - Biochemical Journal
SN - 0264-6021
IS - 2
ER -