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A high throughput screen identifies potent and selective inhibitors to human epithelial 15-lipoxygenase-2.

Jameson JB, Kantz A, Schultz L, Kalyanaraman C, Jacobson MP, Maloney DJ, Jadhav A, Simeonov A, Holman TR - PLoS ONE (2014)

Bottom Line: MLS000545091 is a mixed-type inhibitor of 15-LOX-2 with a Ki of 0.9+/-0.4 µM and has a 20-fold selectivity over 5-LOX, 12-LOX, 15-LOX-1, COX-1, and COX-2.MLS000536924 is a competitive inhibitor with a Ki of 2.5+/-0.5 µM and also possesses 20-fold selectivity toward 15-LOX-2 over the other oxygenases, listed above.Finally, neither compound possesses reductive activity towards the active-site ferrous ion.

View Article: PubMed Central - PubMed

Affiliation: Chemistry and Biochemistry Department, University of California Santa Cruz, Santa Cruz, California, United States of America.

ABSTRACT
Lipoxygenase (LOX) enzymes catalyze the hydroperoxidation of arachidonic acid and other polyunsaturated fatty acids to hydroxyeicosatetraenoic acids with varying positional specificity to yield important biological signaling molecules. Human epithelial 15-lipoxygenase-2 (15-LOX-2) is a highly specific LOX isozyme that is expressed in epithelial tissue and whose activity has been correlated with suppression of tumor growth in prostate and other epithelial derived cancers. Despite the potential utility of an inhibitor to probe the specific role of 15-LOX-2 in tumor progression, no such potent/specific 15-LOX-2 inhibitors have been reported to date. This study employs high throughput screening to identify two novel, specific 15-LOX-2 inhibitors. MLS000545091 is a mixed-type inhibitor of 15-LOX-2 with a Ki of 0.9+/-0.4 µM and has a 20-fold selectivity over 5-LOX, 12-LOX, 15-LOX-1, COX-1, and COX-2. MLS000536924 is a competitive inhibitor with a Ki of 2.5+/-0.5 µM and also possesses 20-fold selectivity toward 15-LOX-2 over the other oxygenases, listed above. Finally, neither compound possesses reductive activity towards the active-site ferrous ion.

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Steady-state kinetics data for the determination of Ki for 15-LOX-2 with MLS000536924.(A) Initial enzymatic rate (µmol/min/mg) versus substrate concentration (µM) at inhibitor concentrations of 0 µM (open circles) 1 µM (open squares) 2 µM (open diamonds) and 5 µM (closed circles) fitted to the Henri–Michaelis– Menten equation to yield Vmax (µmol/min/mg) and Vmax/KM (µmol/min/mg/µM). All measurements were done in triplicate. (B) KM/Vmax replot (closed circles) (units are µM/µmol/min/mg) versus [Inhibitor] (µM), which yielded a Ki of 2.5+/−0.5 µM. 1/Vmax replot (open circles) (units are 1/µmol/min/mg) versus [Inhibitor] (µM), value did not change with increasing inhibitor concentration, indicating competitive inhibition.
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pone-0104094-g005: Steady-state kinetics data for the determination of Ki for 15-LOX-2 with MLS000536924.(A) Initial enzymatic rate (µmol/min/mg) versus substrate concentration (µM) at inhibitor concentrations of 0 µM (open circles) 1 µM (open squares) 2 µM (open diamonds) and 5 µM (closed circles) fitted to the Henri–Michaelis– Menten equation to yield Vmax (µmol/min/mg) and Vmax/KM (µmol/min/mg/µM). All measurements were done in triplicate. (B) KM/Vmax replot (closed circles) (units are µM/µmol/min/mg) versus [Inhibitor] (µM), which yielded a Ki of 2.5+/−0.5 µM. 1/Vmax replot (open circles) (units are 1/µmol/min/mg) versus [Inhibitor] (µM), value did not change with increasing inhibitor concentration, indicating competitive inhibition.

Mentions: The nature of LOX inhibition by the current compounds was further investigated using steady-state kinetics (Table 2). Analysis was performed with MLS000545091 and MLS000536924 by monitoring the formation of 15-HPETE as a function of substrate and inhibitor concentration in the presence of 0.01% Triton X-100. The microscopic rate constants, Vmax (µmol/min/mg) and Vmax/KM (µmol/min/mg/µM) were determined for MLS000545091 (Figure 4A) and MLS000536924 (Figure 5A) using the Henri–Michaelis–Menten equation. For MLS000545091, the kinetic rate constants were replotted with KM/Vmax and 1/Vmax versus inhibitor concentration (Figure 4B), yielding Ki and Ki′, respectively. The Ki was 0.9+/−0.4 µM and the Ki′ was 9.9+/−0.7 µM, indicating mixed-type inhibition. The error for Ki is relatively high, which could be due to the hydrophobic nature of the inhibitor and its insolubility. For MLS000536924, KM/Vmax was replotted (Figure 5B) to yield a Ki of 2.5+/−0.5 µM. The 1/Vmax was also replotted, but did not change with increasing inhibitor concentration, indicating competitive inhibition. In order to confirm that these compounds do not bind to the allosteric site of 15-LOX-2 appreciably [30], [31], their IC50 values were compared using both AA and LA as a substrate, but no significant difference in inhibitor binding was observed (data not shown). This data indicated that allosteric binding does not affect inhibitor potency greatly, which is to be expected given the difference in magnitude between Ki and Ki′ for MLS000545091.


A high throughput screen identifies potent and selective inhibitors to human epithelial 15-lipoxygenase-2.

Jameson JB, Kantz A, Schultz L, Kalyanaraman C, Jacobson MP, Maloney DJ, Jadhav A, Simeonov A, Holman TR - PLoS ONE (2014)

Steady-state kinetics data for the determination of Ki for 15-LOX-2 with MLS000536924.(A) Initial enzymatic rate (µmol/min/mg) versus substrate concentration (µM) at inhibitor concentrations of 0 µM (open circles) 1 µM (open squares) 2 µM (open diamonds) and 5 µM (closed circles) fitted to the Henri–Michaelis– Menten equation to yield Vmax (µmol/min/mg) and Vmax/KM (µmol/min/mg/µM). All measurements were done in triplicate. (B) KM/Vmax replot (closed circles) (units are µM/µmol/min/mg) versus [Inhibitor] (µM), which yielded a Ki of 2.5+/−0.5 µM. 1/Vmax replot (open circles) (units are 1/µmol/min/mg) versus [Inhibitor] (µM), value did not change with increasing inhibitor concentration, indicating competitive inhibition.
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Related In: Results  -  Collection

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pone-0104094-g005: Steady-state kinetics data for the determination of Ki for 15-LOX-2 with MLS000536924.(A) Initial enzymatic rate (µmol/min/mg) versus substrate concentration (µM) at inhibitor concentrations of 0 µM (open circles) 1 µM (open squares) 2 µM (open diamonds) and 5 µM (closed circles) fitted to the Henri–Michaelis– Menten equation to yield Vmax (µmol/min/mg) and Vmax/KM (µmol/min/mg/µM). All measurements were done in triplicate. (B) KM/Vmax replot (closed circles) (units are µM/µmol/min/mg) versus [Inhibitor] (µM), which yielded a Ki of 2.5+/−0.5 µM. 1/Vmax replot (open circles) (units are 1/µmol/min/mg) versus [Inhibitor] (µM), value did not change with increasing inhibitor concentration, indicating competitive inhibition.
Mentions: The nature of LOX inhibition by the current compounds was further investigated using steady-state kinetics (Table 2). Analysis was performed with MLS000545091 and MLS000536924 by monitoring the formation of 15-HPETE as a function of substrate and inhibitor concentration in the presence of 0.01% Triton X-100. The microscopic rate constants, Vmax (µmol/min/mg) and Vmax/KM (µmol/min/mg/µM) were determined for MLS000545091 (Figure 4A) and MLS000536924 (Figure 5A) using the Henri–Michaelis–Menten equation. For MLS000545091, the kinetic rate constants were replotted with KM/Vmax and 1/Vmax versus inhibitor concentration (Figure 4B), yielding Ki and Ki′, respectively. The Ki was 0.9+/−0.4 µM and the Ki′ was 9.9+/−0.7 µM, indicating mixed-type inhibition. The error for Ki is relatively high, which could be due to the hydrophobic nature of the inhibitor and its insolubility. For MLS000536924, KM/Vmax was replotted (Figure 5B) to yield a Ki of 2.5+/−0.5 µM. The 1/Vmax was also replotted, but did not change with increasing inhibitor concentration, indicating competitive inhibition. In order to confirm that these compounds do not bind to the allosteric site of 15-LOX-2 appreciably [30], [31], their IC50 values were compared using both AA and LA as a substrate, but no significant difference in inhibitor binding was observed (data not shown). This data indicated that allosteric binding does not affect inhibitor potency greatly, which is to be expected given the difference in magnitude between Ki and Ki′ for MLS000545091.

Bottom Line: MLS000545091 is a mixed-type inhibitor of 15-LOX-2 with a Ki of 0.9+/-0.4 µM and has a 20-fold selectivity over 5-LOX, 12-LOX, 15-LOX-1, COX-1, and COX-2.MLS000536924 is a competitive inhibitor with a Ki of 2.5+/-0.5 µM and also possesses 20-fold selectivity toward 15-LOX-2 over the other oxygenases, listed above.Finally, neither compound possesses reductive activity towards the active-site ferrous ion.

View Article: PubMed Central - PubMed

Affiliation: Chemistry and Biochemistry Department, University of California Santa Cruz, Santa Cruz, California, United States of America.

ABSTRACT
Lipoxygenase (LOX) enzymes catalyze the hydroperoxidation of arachidonic acid and other polyunsaturated fatty acids to hydroxyeicosatetraenoic acids with varying positional specificity to yield important biological signaling molecules. Human epithelial 15-lipoxygenase-2 (15-LOX-2) is a highly specific LOX isozyme that is expressed in epithelial tissue and whose activity has been correlated with suppression of tumor growth in prostate and other epithelial derived cancers. Despite the potential utility of an inhibitor to probe the specific role of 15-LOX-2 in tumor progression, no such potent/specific 15-LOX-2 inhibitors have been reported to date. This study employs high throughput screening to identify two novel, specific 15-LOX-2 inhibitors. MLS000545091 is a mixed-type inhibitor of 15-LOX-2 with a Ki of 0.9+/-0.4 µM and has a 20-fold selectivity over 5-LOX, 12-LOX, 15-LOX-1, COX-1, and COX-2. MLS000536924 is a competitive inhibitor with a Ki of 2.5+/-0.5 µM and also possesses 20-fold selectivity toward 15-LOX-2 over the other oxygenases, listed above. Finally, neither compound possesses reductive activity towards the active-site ferrous ion.

Show MeSH
Related in: MedlinePlus