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

The business case for women leaders: Meta-analysis, research critique, and path forward

Since the 1990s, a growing body of research has sought to quantify the relationship between women’s representation in leadership positions and organizational financial performance. Commonly known as the “business case” for women’s leadership, the idea is that having more women leaders is good for business. Through meta-analysis ( k = 78, n = 117,639 organizations) of the direct effects of women’s representation in leadership (as CEOs, on top management teams, and on boards of directors) on financial performance, and tests that proxy theoretical arguments for moderated relationships, we call attention to equivocal findings. Our results suggest women’s leadership may affect firm performance in general and sales performance in particular. And women’s leadership—overall and, specifically, the presence of a female CEO—is more likely to positively relate to firms’ financial performance in more gender egalitarian cultures. Yet taking our findings as a whole, we argue that commonly used methods of testing the business case for women leaders may limit our ability as scholars to understand the value that women bring to leadership positions. We do not advocate that the business case be abandoned altogether but, rather, improved and refined. We name exemplary research studies to show how different perspectives on gender, alternative conceptualizations of value, and the specification of underlying mechanisms linking leadership to performance can generate changes in both the dominant ontology and the epistemology underlying this body of research.</jats:p

Structural and Therapeutic Investigations of Human Lipoxygenase

The research in this dissertation describes the investigations of potential therapeutics as well as structural and allosteric properties of human lipoxygenases. Lipoxygenases (LOX) are a ubiquitous enzyme found in plants and mammals, of which are responsible for regulation of inflammation in humans. Uncontrolled inflammation in humans may result in various types of cancers and inflammatory diseases, for which LOX is implicated. This has prompted the Holman lab to explore a diverse range on potential therapeutic targets in hopes of discovery of novel selective LOX inhibitors, while concurrently investigating the structural and kinetic properties of the enzyme.Through use of conventional kinetic and structural studies we investigated the role of the polycystin-1 lipoxygenase alpha-toxin (PLAT) domain's role in enzyme catalysis and allosteric regulation. Previous studies had implicated the PLAT domain as being a critical aspect of the allosteric binding site. This theory was explored through extensive investigations into the resulting effects elicited by removal of the PLAT domain from human epithelial 15-lipoxygenase-2 (15-LOX-2). In chapter 2 we present our findings supporting our previous concept, that indeed the PLAT domain plays a key role in the allosteric properties of 15-LOX-2.Chapter 3 describes collaboration with the National Institutes Chemical Genomic Center, where we report the discovery of a novel dual inhibitor targeting fungal sterol 14á-demethylase (CYP51 or Erg11) and human 5-lipoxygenase (5-LOX) with improved potency against 5-LOX due to its reduction of the iron center by its phenylenediamine core. The phenylenediamine core was then translated into the structure of ketoconazole, a highly effective anti-fungal medication for seborrheic dermatitis, to generate a novel compound, ketaminazole. Ketaminazole was found to be a potent dual selective inhibitor against human 5-LOX and CYP51 in vitro.Understanding the mode of action of lipoxygenase (LOX) inhibitors is critical to determining their efficacy in the cell. The pseudoperoxidase assay is an important tool for establishing if an inhibitor is reductive in nature. In chapter 4, we evaluate the effectiveness of two distinct pseudoperoxidase methods in characterizing known inhibitor's redox properties; the "234 nm" decomposition and xylenol orange assay. In addition, we identified rapid inactivation occurring with particular inhibitors in the pseudoperoxidase assay. To account for the resulting inaccuracy attributed to this inhibitor dependent inactivation, we modified the pre-existing "234 nm" assay allowing for observation of this inactivation

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-(S)-hydroxy-(9Z,11E)-octadecadienoic acid [13-(S)-HODE] and 13-(S)-hydroperoxy-(6Z,9Z,11E)-octadecatrienoic acid to human epithelial 15-lipoxygenase-2 (15-LOX-2) increases the kcat/KM substrate specificity ratio of arachidonic acid (AA) and γ-linolenic acid (GLA) by 4-fold. 13-(S)-HODE achieves this change by activating kcat/KM(AA) but inhibiting kcat/KM(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

The business case for women leaders : meta-analysis, research critique, and path forward

Since the 1990s, a growing body of research has sought to quantify the relationship between women’s representation in leadership positions and organizational financial performance. Commonly known as the “business case” for women’s leadership, the idea is that having more women leaders is good for business. Through meta-analysis (k = 78, n = 117,639 organizations) of the direct effects of women’s representation in leadership (as CEOs, on top management teams, and on boards of directors) on financial performance, and tests that proxy theoretical arguments for moderated relationships, we call attention to equivocal findings. Our results suggest women’s leadership may affect firm performance in general and sales performance in particular. And women’s leadership—overall and, specifically, the presence of a female CEO—is more likely to positively relate to firms’ financial performance in more gender egalitarian cultures. Yet taking our findings as a whole, we argue that commonly used methods of testing the business case for women leaders may limit our ability as scholars to understand the value that women bring to leadership positions. We do not advocate that the business case be abandoned altogether but, rather, improved and refined. We name exemplary research studies to show how different perspectives on gender, alternative conceptualizations of value, and the specification of underlying mechanisms linking leadership to performance can generate changes in both the dominant ontology and the epistemology underlying this body of research.A grant (No. 80588) provided by the National Research Foundation of South Africa.https://journals.sagepub.com/home/jomhj2019Human Resource Managemen

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