164 research outputs found
A high-throughput mass spectrometric assay for discovery of human lipoxygenase inhibitors and allosteric effectors.
Lipoxygenases (LOXs) regulate inflammation through the production of a variety of molecules whose specific downstream effects are not entirely understood due to the complexity of the inflammation pathway. The generation of these biomolecules can potentially be inhibited and/or allosterically regulated by small synthetic molecules. The current work describes the first mass spectrometric high-throughput method for identifying small molecule LOX inhibitors and LOX allosteric effectors that change the substrate preference of human lipoxygenase enzymes. Using a volatile buffer and an acid-labile detergent, enzymatic products can be directly detected using high-performance liquid chromatography-mass spectrometry (HPLC-MS) without the need for organic extraction. The method also reduces the required enzyme concentration compared with traditional ultraviolet (UV) absorbance methods by approximately 30-fold, allowing accurate binding affinity measurements for inhibitors with nanomolar affinity. The procedure was validated using known LOX inhibitors and the allosteric effector 13(S)-hydroxy-9Z,11E-octadecadienoic acid (13-HODE)
ATP allosterically activates the human 5-lipoxygenase molecular mechanism of arachidonic acid and 5(S)-hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid.
5-Lipoxygenase (5-LOX) reacts with arachidonic acid (AA) to first generate 5(S)-hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid [5(S)-HpETE] and then an epoxide from 5(S)-HpETE to form leukotriene A4, from a single polyunsaturated fatty acid. This work investigates the kinetic mechanism of these two processes and the role of ATP in their activation. Specifically, it was determined that epoxidation of 5(S)-HpETE (dehydration of the hydroperoxide) has a rate of substrate capture (Vmax/Km) significantly lower than that of AA hydroperoxidation (oxidation of AA to form the hydroperoxide); however, hyperbolic kinetic parameters for ATP activation indicate a similar activation for AA and 5(S)-HpETE. Solvent isotope effect results for both hydroperoxidation and epoxidation indicate that a specific step in its molecular mechanism is changed, possibly because of a lowering of the dependence of the rate-limiting step on hydrogen atom abstraction and an increase in the dependency on hydrogen bond rearrangement. Therefore, changes in ATP concentration in the cell could affect the production of 5-LOX products, such as leukotrienes and lipoxins, and thus have wide implications for the regulation of cellular inflammation
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
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5 S,15 S-Dihydroperoxyeicosatetraenoic Acid (5,15-diHpETE) as a Lipoxin Intermediate: Reactivity and Kinetics with Human Leukocyte 5-Lipoxygenase, Platelet 12-Lipoxygenase, and Reticulocyte 15-Lipoxygenase-1.
The reaction of 5 S,15 S-dihydroperoxyeicosatetraenoic acid (5,15-diHpETE) with human 5-lipoxygenase (LOX), human platelet 12-LOX, and human reticulocyte 15-LOX-1 was investigated to determine the reactivity and relative rates of producing lipoxins (LXs). 5-LOX does not react with 5,15-diHpETE, although it can produce LXA4 when 15-HpETE is the substrate. In contrast, both 12-LOX and 15-LOX-1 react with 5,15-diHpETE, forming specifically LXB4. For 12-LOX and 5,15-diHpETE, the kinetic parameters are kcat = 0.17 s-1 and kcat/ KM = 0.011 μM-1 s-1 [106- and 1600-fold lower than those for 12-LOX oxygenation of arachidonic acid (AA), respectively]. On the other hand, for 15-LOX-1 the equivalent parameters are kcat = 4.6 s-1 and kcat/ KM = 0.21 μM-1 s-1 (3-fold higher and similar to those for 12-HpETE formation by 15-LOX-1 from AA, respectively). This contrasts with the complete lack of reaction of 15-LOX-2 with 5,15-diHpETE [Green, A. R., et al. (2016) Biochemistry 55, 2832-2840]. Our data indicate that 12-LOX is markedly inferior to 15-LOX-1 in catalyzing the production of LXB4 from 5,15-diHpETE. Platelet aggregation was inhibited by the addition of 5,15-diHpETE, with an IC50 of 1.3 μM; however, LXB4 did not significantly inhibit collagen-mediated platelet activation up to 10 μM. In summary, LXB4 is the primary product of 12-LOX and 15-LOX-1 catalysis, if 5,15-diHpETE is the substrate, with 15-LOX-1 being 20-fold more efficient than 12-LOX. LXA4 is the primary product with 5-LOX but only if 15-HpETE is the substrate. Approximately equal proportions of LXA4 and LXB4 are produced by 12-LOX but only if LTA4 is the substrate, as described previously [Sheppard, K. A., et al. (1992) Biochim. Biophys. Acta 1133, 223-234]
Discovery of platelet-type 12-human lipoxygenase selective inhibitors by high-throughput screening of structurally diverse libraries.
Human lipoxygenases (hLO) have been implicated in a variety of diseases and cancers and each hLO isozyme appears to have distinct roles in cellular biology. This fact emphasizes the need for discovering selective hLO inhibitors for both understanding the role of specific lipoxygenases in the cell and developing pharmaceutical therapeutics. To this end, we have modified a known lipoxygenase assay for high-throughput (HTP) screening of both the National Cancer Institute (NCI) and the UC Santa Cruz marine extract library (UCSC-MEL) in search of platelet-type 12-hLO (12-hLO) selective inhibitors. The HTP screen led to the characterization of five novel 12-hLO inhibitors from the NCI repository. One is the potent but non-selective michellamine B, a natural product, anti-viral agent. The other four compounds were selective inhibitors against 12-hLO, with three being synthetic compounds and one being alpha-mangostin, a natural product, caspase-3 pathway inhibitor. In addition, a selective inhibitor was isolated from the UCSC-MEL (neodysidenin), which has a unique chemical scaffold for a hLO inhibitor. Due to the unique structure of neodysidenin, steady-state inhibition kinetics were performed and its mode of inhibition against 12-hLO was determined to be competitive (K(i)=17microM) and selective over reticulocyte 15-hLO-1 (K(i) 15-hLO-1/12-hLO\u3e30)
Kinetic and Structural Investigations of Novel Inhibitors of Human Epithelial 15-Lipoxygenase-2
Human epithelial 15-lipoxygenase-2 (h15-LOX-2, ALOX15B) is expressed in many tissues and has been implicated in atherosclerosis, cystic fibrosis and ferroptosis. However, there are few reported potent/selective inhibitors that are active ex vivo. In the current work, we report newly discovered molecules that are more potent and structurally distinct from our previous inhibitors, MLS000545091 and MLS000536924 (Jameson et al, PLoS One, 2014, 9, e104094), in that they contain a central imidazole ring, which is substituted at the 1-position with a phenyl moiety and with a benzylthio moiety at the 2-position. The initial three molecules were mixed-type, non-reductive inhibitors, with IC(50) values of 0.34 ± 0.05 μM for MLS000327069, 0.53 ± 0.04 μM for MLS000327186 and 0.87 ± 0.06 μM for MLS000327206 and greater than 50-fold selectivity versus h5-LOX, h12-LOX, h15-LOX-1, COX-1 and COX-2. A small set of focused analogs was synthesized to demonstrate the validity of the hits. In addition, a binding model was developed for the three imidazole inhibitors based on computational docking and a co-structure of h15-LOX-2 with MLS000536924. Hydrogen/deuterium exchange (HDX) results indicate a similar binding mode between MLS000536924 and MLS000327069, however, the latter restricts protein motion of helix-α2 more, consistent with its greater potency. Given these results, we designed, docked, and synthesized novel inhibitors of the imidazole scaffold and confirmed our binding mode hypothesis. Importantly, four of the five inhibitors mentioned above are active in an h15-LOX-2/HEK293 cell assay and thus they could be important tool compounds in gaining a better understanding of h15-LOX-2’s role in human biology. As such, a suite of similar pharmacophores that target h15-LOX-2 both in vitro and ex vivo are presented in the hope of developing them as therapeutic agents
Robust estimation of bacterial cell count from optical density
Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data
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Inhibition Studies of Soybean and Human 15-Lipoxygenases with Long-Chain Alkenyl Sulfate Substrates â€
Lipoxygenases are currently potential targets for therapies against asthma, artherosceloris, and cancer. Recently, inhibition studies on both soybean (SLO) and human lipoxygenase (15-HLO) revealed the presence of an allosteric site that binds both substrate, linoleic acid, and inhibitors; oleic acid (OA) and oleyl sulfate (OS). OS (K(D) approximately 0.6 microM) is a approximately 30-fold more potent inhibitor than OA (K(D) approximately 20 microM) due to the increased ionic strength of the sulfate moiety. To further investigate the role of the sulfate moiety on lipoxygenase function, SLO and 15-HLO were assayed against several fatty sulfate substrates (linoleyl sulfate (LS), cis-11,14-eicosadienoyl sulfate, and arachidonyl sulfate). The results demonstrate that SLO catalyzes all three fatty sulfate substrates and is not inhibited, indicating a binding selectivity of LS for the catalytic site and OS for the allosteric site. The 15-HLO, however, manifests parabolic inhibition kinetics with increasing substrate concentration, and it is irreversibly inhibited by these fatty sulfate substrates at high concentrations. The inhibition can be stopped, however, by the addition of detergent to the fatty sulfate mixture prior to the addition of 15-HLO. These results, combined with the modeling of the kinetic data, indicate that the inhibition of 15-HLO is due to a substrate aggregate. These substrate aggregates, however, do not inhibit SLO and could present a novel mode of inhibition for 15-HLO
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Fatty Acid Allosteric Regulation of C-H Activation in Plant and Animal Lipoxygenases
Lipoxygenases (LOXs) catalyze the (per) oxidation of fatty acids that serve as important mediators for cell signaling and inflammation. These reactions are initiated by a C-H activation step that is allosterically regulated in plant and animal enzymes. LOXs from higher eukaryotes are equipped with an N-terminal PLAT (Polycystin-1, Lipoxygenase, Alpha-Toxin) domain that has been implicated to bind to small molecule allosteric effectors, which in turn modulate substrate specificity and the rate-limiting steps of catalysis. Herein, the kinetic and structural evidence that describes the allosteric regulation of plant and animal lipoxygenase chemistry by fatty acids and their derivatives are summarized
Fatty Acid Allosteric Regulation of C-H Activation in Plant and Animal Lipoxygenases
Lipoxygenases (LOXs) catalyze the (per) oxidation of fatty acids that serve as important mediators for cell signaling and inflammation. These reactions are initiated by a C-H activation step that is allosterically regulated in plant and animal enzymes. LOXs from higher eukaryotes are equipped with an N-terminal PLAT (Polycystin-1, Lipoxygenase, Alpha-Toxin) domain that has been implicated to bind to small molecule allosteric effectors, which in turn modulate substrate specificity and the rate-limiting steps of catalysis. Herein, the kinetic and structural evidence that describes the allosteric regulation of plant and animal lipoxygenase chemistry by fatty acids and their derivatives are summarized
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