Pseudoperoxidase investigations of hydroperoxides and inhibitors with human lipoxygenases

AbstractUnderstanding the mode of action for lipoxygenase (LOX) inhibitors is critical to determining their efficacy in the cell. The pseudoperoxidase assay is an important tool for establishing if a LOX inhibitor is reductive in nature, however, there have been difficulties identifying the proper conditions for each of the many human LOX isozymes. In the current paper, both the 234nM decomposition (UV) and iron-xylenol orange (XO) assays are shown to be effective methods of detecting pseudoperoxidase activity for 5-LOX, 12-LOX, 15-LOX-1 and 15-LOX-2, but only if 13-(S)-HPODE is used as the hydroperoxide substrate. The AA products, 12-(S)-HPETE and 15-(S)-HPETE, are not consistent hydroperoxide substrates since they undergo a competing transformation to the di-HETE products. Utilizing the above conditions, the selective 12-LOX and 15-LOX-1 inhibitors, probes for diabetes, stroke and asthma, are characterized for their inhibitory nature. Interestingly, ascorbic acid also supports the pseudoperoxidase assay, suggesting that it may have a role in maintaining the inactive ferrous form of LOX in the cell. In addition, it is observed that nordihydroguaiaretic acid (NDGA), a known reductive LOX inhibitor, appears to generate radical species during the pseudoperoxidase assay, which are potent inhibitors against the human LOX isozymes, producing a negative pseudoperoxidase result. Therefore, inhibitors that do not support the pseudoperoxidase assay with the human LOX isozymes, should also be investigated for rapid inactivation, to clarify the negative pseudoperoxidase result

Enzymatic Studies of Isoflavonoids as Selective and Potent Inhibitors of Human Leukocyte 5-Lipo-Oxygenase.

Continuing our search to find more potent and selective 5-LOX inhibitors, we present now the enzymatic evaluation of seventeen isoflavones (IR) and nine isoflavans (HIR), and their in vitro and in cellulo potency against human leukocyte 5-LOX. Of the 26 compounds tested, 10 isoflavones and 9 isoflavans possessed micromolar potency, but only three were selective against 5-LOX (IR-2, HIR-303, and HIR-309), with IC50 values at least 10 times lower than those of 12-LOX, 15-LOX-1, and 15-LOX-2. Of these three, IR-2 (6,7-dihydroxy-4-methoxy-isoflavone, known as texasin) was the most selective 5-LOX inhibitor, with over 80-fold potency difference compared with other isozymes; Steered Molecular Dynamics (SMD) studies supported these findings. The presence of the catechol group on ring A (6,7-dihydroxy versus 7,8-dihydroxy) correlated with their biological activity, but the reduction of ring C, converting the isoflavones to isoflavans, and the substituent positions on ring B did not affect their potency against 5-LOX. Two of the most potent/selective inhibitors (HIR-303 and HIR-309) were reductive inhibitors and were potent against 5-LOX in human whole blood, indicating that isoflavans can be potent and selective inhibitors against human leukocyte 5-LOX in vitro and in cellulo

Enzymatic Studies of Isoflavonoids as Selective and Potent Inhibitors of Human Leukocyte 5-Lipo-Oxygenase.

Continuing our search to find more potent and selective 5-LOX inhibitors, we present now the enzymatic evaluation of seventeen isoflavones (IR) and nine isoflavans (HIR), and their in vitro and in cellulo potency against human leukocyte 5-LOX. Of the 26 compounds tested, 10 isoflavones and 9 isoflavans possessed micromolar potency, but only three were selective against 5-LOX (IR-2, HIR-303, and HIR-309), with IC50 values at least 10 times lower than those of 12-LOX, 15-LOX-1, and 15-LOX-2. Of these three, IR-2 (6,7-dihydroxy-4-methoxy-isoflavone, known as texasin) was the most selective 5-LOX inhibitor, with over 80-fold potency difference compared with other isozymes; Steered Molecular Dynamics (SMD) studies supported these findings. The presence of the catechol group on ring A (6,7-dihydroxy versus 7,8-dihydroxy) correlated with their biological activity, but the reduction of ring C, converting the isoflavones to isoflavans, and the substituent positions on ring B did not affect their potency against 5-LOX. Two of the most potent/selective inhibitors (HIR-303 and HIR-309) were reductive inhibitors and were potent against 5-LOX in human whole blood, indicating that isoflavans can be potent and selective inhibitors against human leukocyte 5-LOX in vitro and in cellulo

Using Enzyme Assays to Evaluate the Structure and Bioactivity of Sponge-Derived Meroterpenes

Enzyme screening of crude sponge extracts prioritized a 2005 Papua New Guinea collection of Hyrtios sp. for further study. The MeOH extract contained puupehenone and four puupehenone analogues (1, 2, 3, 5, and 7) along with a new diastereomer, 20-epi-hydroxyhaterumadienone (4), and a new analogue, 15-oxo-puupehenoic acid (6). The drimane terpene core of 4 and 6 was rapidly dereplicated, and the modified Mosher's method identified 4, while 1D and 2D NMR techniques were used to solve 6. These compounds plus noteworthy repository natural products and standards were tested against three lipoxygenase isozymes, human 5-, 12-, and 15-lipoxygenases. Significant potency and selectivity profiles were exhibited in the human 5-lipoxygenase assay by puupehenone (1) and jaspaquinol (9) and structural factors responsible for activity identified

Kinetic and Structural Investigations into the Allosteric and pH Effect on the Substrate Specificity of Human Epithelial 15-Lipoxygenase‑2

Lipoxygenases, important enzymes in inflammation, can regulate their substrate specificity by allosteric interactions with their own hydroperoxide products. In this work, addition of both 13-(<i>S</i>)-hydroxy-(9<i>Z</i>,11<i>E</i>)-octadecadienoic acid [13-(<i>S</i>)-HODE] and 13-(<i>S</i>)-hydroperoxy-(6<i>Z</i>,9<i>Z</i>,11<i>E</i>)-octadecatrienoic acid to human epithelial 15-lipoxygenase-2 (15-LOX-2) increases the <i>k</i>_cat/<i>K</i>_M substrate specificity ratio of arachidonic acid (AA) and γ-linolenic acid (GLA) by 4-fold. 13-(<i>S</i>)-HODE achieves this change by activating <i>k</i>_cat/<i>K</i>_M^AA but inhibiting <i>k</i>_cat/<i>K</i>_M^GLA, which indicates that the allosteric structural changes at the active site discriminate between the length and unsaturation differences of AA and GLA to achieve opposite kinetic effects. The substrate specificity ratio is further increased, 11-fold in total, with an increase in pH, suggesting mechanistic differences between the pH and allosteric effects. Interestingly, the loss of the PLAT domain affects substrate specificity but does not eliminate the allosteric properties of 15-LOX-2, indicating that the allosteric site is located in the catalytic domain. However, the removal of the PLAT domain does change the magnitude of the allosteric effect. These data suggest that the PLAT domain moderates the communication pathway between the allosteric and catalytic sites, thus affecting substrate specificity. These results are discussed in the context of protein dimerization and other structural changes

Determination of IC₅₀ values for ketoconazole and ketaminazole with CaCYP51 and HsCYP51.

<p>CYP51 reconstitution assays (0.5-ml total volume) containing 1 µM CaCYP51 (A) or 0.3 µM HsCYP51 (B) were performed as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065928#s2" target="_blank">Materials and Methods</a>. Ketoconazole (solid circles) and ketaminazole (hollow circles) concentrations were varied from 0 to 4 µM for CaCYP51 and up to 190 µM for HsCYP51 with the DMSO concentration kept constant at 1% (vol/vol). Mean values from two replicates are shown along with associated standard deviation bars. Relative velocities of 1.0 were equivalent to 1.04 and 2.69 nmoles 14α-demetylated lanosterol produced per minute per nmole CYP51 (min⁻¹) for CaCYP51 and HsCYP51, respectively.</p

Selectivity profile of representative analogues (µM), with errors in parentheses^a.

a<p>The UV-based manual inhibition data (3 replicates) were fit as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065928#s2" target="_blank">Materials and Methods</a> section.</p>b<p>N/D = Not determined.</p

IC₅₀ values of dual anti-fungal, anti-inflammatory inhibitors (µM), with error in parentheses.

<p>The UV-based manual inhibition data (3 replicates) were fit as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065928#s2" target="_blank">Materials and Methods</a> section. N/D = Not determined.</p

5-LOX IC₅₀ values of representative analogues (µM), with errors in brackets.

<p>The UV-based manual inhibition data (3 replicates) were fit as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065928#s2" target="_blank">Materials and Methods</a> section.</p

Buffer conditions for IC₅₀ assays, with constant substrate concentration and varying inhibitor concentration^a.

a<p>The UV-based manual inhibition data (3 replicates) were fit as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065928#s2" target="_blank">Materials and Methods</a> section.</p