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

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.

Synthesis and structure-activity relationship studies of 4-((2-hydroxy-3-methoxybenzyl)amino)benzenesulfonamide derivatives as potent and selective inhibitors of 12-lipoxygenase.

Human lipoxygenases (LOXs) are a family of iron-containing enzymes which catalyze the oxidation of polyunsaturated fatty acids to provide the corresponding bioactive hydroxyeicosatetraenoic acid (HETE) metabolites. These eicosanoid signaling molecules are involved in a number of physiologic responses such as platelet aggregation, inflammation, and cell proliferation. Our group has taken a particular interest in platelet-type 12-(S)-LOX (12-LOX) because of its demonstrated role in skin diseases, diabetes, platelet hemostasis, thrombosis, and cancer. Herein, we report the identification and medicinal chemistry optimization of a 4-((2-hydroxy-3-methoxybenzyl)amino)benzenesulfonamide-based scaffold. Top compounds, exemplified by 35 and 36, display nM potency against 12-LOX, excellent selectivity over related lipoxygenases and cyclooxygenases, and possess favorable ADME properties. In addition, both compounds inhibit PAR-4 induced aggregation and calcium mobilization in human platelets and reduce 12-HETE in ß-cells.

Structure and mechanism of styrene monooxygenase reductase: new insight into the FAD-transfer reaction.

The two-component flavoprotein styrene monooxygenase (SMO) from Pseudomonas putida S12 catalyzes the NADH- and FAD-dependent epoxidation of styrene to styrene oxide. In this study, we investigate the mechanism of flavin reduction and transfer from the reductase (SMOB) to the epoxidase (NSMOA) component and report our findings in light of the 2.2 Å crystal structure of SMOB. Upon rapidly mixing with NADH, SMOB forms an NADH → FADox charge-transfer intermediate and catalyzes a hydride-transfer reaction from NADH to FAD, with a rate constant of 49.1 ± 1.4 s(-1), in a step that is coupled to the rapid dissociation of NAD(+). Electrochemical and equilibrium-binding studies indicate that NSMOA binds FADhq ∼13-times more tightly than SMOB, which supports a vectoral transfer of FADhq from the reductase to the epoxidase. After binding to NSMOA, FADhq rapidly reacts with molecular oxygen to form a stable C(4a)-hydroperoxide intermediate. The half-life of apoSMOB generated in the FAD-transfer reaction is increased ∼21-fold, supporting a protein-protein interaction between apoSMOB and the peroxide intermediate of NSMOA. The mechanisms of FAD dissociation and transport from SMOB to NSMOA were probed by monitoring the competitive reduction of cytochrome c in the presence and absence of pyridine nucleotides. On the basis of these studies, we propose a model in which reduced FAD binds to SMOB in equilibrium between an unreactive, sequestered state (S state) and more reactive, transfer state (T state). The dissociation of NAD(+) after the hydride-transfer reaction transiently populates the T state, promoting the transfer of FADhq to NSMOA. The binding of pyridine nucleotides to SMOB-FADhq shifts the FADhq-binding equilibrium from the T state to the S state. Additionally, the 2.2 Å crystal structure of SMOB-FADox reported in this work is discussed in light of the pyridine nucleotide-gated flavin-transfer and electron-transfer reactions.

Nature of the reaction intermediates in the flavin adenine dinucleotide-dependent epoxidation mechanism of styrene monooxygenase.

Styrene monooxygenase (SMO) is a two-component flavoenzyme composed of an NADH-specific flavin reductase (SMOB) and FAD-specific styrene epoxidase (NSMOA). NSMOA binds tightly to reduced FAD and catalyzes the stereospecific addition of one atom of molecular oxygen to the vinyl side chain of styrene in the enantioselective synthesis of S-styrene oxide. In this mechanism, molecular oxygen first reacts with NSMOA(FAD(red)) to yield an FAD C(4a)-peroxide intermediate. This species is nonfluorescent and has an absorbance maximum of 382 nm. Styrene then reacts with the peroxide intermediate with a second-order rate constant of (2.6 ± 0.1) × 10(6) M(-1) s(-1) to yield a fluorescent intermediate with an absorbance maximum of 368 nm. We compute an activation free energy of 8.7 kcal/mol for the oxygenation step, in good agreement with that expected for a peroxide-catalyzed epoxidation, and acid-quenched samples recovered at defined time points in the single-turnover reaction indicate that styrene oxide synthesis is coincident with the formation phase of the fluorescent intermediate. These findings support FAD C(4a)-peroxide being the oxygen atom donor and the identity of the fluorescent intermediate as an FAD C(4a)-hydroxide product of the styrene epoxidation. Overall, four pH-dependent rate constants corresponding to peroxyflavin formation (pK(a) = 7.2), styrene epoxidation (pK(a) = 7.7), styrene oxide dissociation (pK(a) = 8.3), and hydroxyflavin dehydration (pK(a) = 7.6) are needed to fit the single-turnover kinetics.

Structure and ligand binding properties of the epoxidase component of styrene monooxygenase .

Styrene monooxygenase (SMO) is a two-component flavoprotein monooxygenase that transforms styrene to styrene oxide in the first step of the styrene catabolic and detoxification pathway of Pseudomonas putida S12. The crystal structure of the N-terminally histidine-tagged epoxidase component of this system, NSMOA, determined to 2.3 A resolution, indicates the enzyme exists as a homodimer in which each monomer forms two distinct domains. The overall architecture is most similar to that of p-hydroxybenzoate hydroxylase (PHBH), although there are some significant differences in secondary structure. Structural comparisons suggest that a large cavity open to the surface forms the FAD binding site. At the base of this pocket is another cavity that likely represents the styrene binding site. Flavin binding and redox equilibria are tightly coupled such that reduced FAD binds apo NSMOA approximately 8000 times more tightly than the oxidized coenzyme. Equilibrium fluorescence and isothermal titration calorimetry data using benzene as a substrate analogue indicate that the oxidized flavin and substrate analogue binding equilibria of NSMOA are linked such that the binding affinity of each is increased by 60-fold when the enzyme is saturated with the other. A much weaker approximately 2-fold positive cooperative interaction is observed for the linked binding equilibria of benzene and reduced FAD. The low affinity of the substrate analogue for the reduced FAD complex of NSMOA is consistent with a preferred reaction order in which flavin reduction and reaction with oxygen precede the binding of styrene, identifying the apoenzyme structure as the key catalytic resting state of NSMOA poised to bind reduced FAD and initiate the oxygen reaction.

Mechanism of flavin transfer and oxygen activation by the two-component flavoenzyme styrene monooxygenase.

Styrene monooxygenase (SMO) from Pseudomonas putida S12 is a two-component flavoenzyme composed of the NADH-specific flavin reductase, SMOB, and FAD-specific styrene epoxidase, SMOA. Here, we report the cloning, and expression of native and histidine-tagged versions of SMOA and SMOB and studies of the flavin transfer and styrene oxygenation reactions. In the reductive half-reaction, SMOB catalyzes the two-electron reduction of FAD with a turnover number of 3200 s(-1). Single turnover studies of the reaction of reduced SMOA with substrates indicate the formation of a stable oxygen intermediate with the absorbance characteristics of a flavin hydroperoxide. Based on the results of numerical simulations of the steady-state mechanism of SMO, we find that the observed coupling of NADH and styrene oxidation can be best explained by a model, which includes both the direct transfer and passive diffusion of reduced FAD from SMOB to SMOA.