Probing the Electrostatic and Steric Requirements for Substrate Binding in Human Platelet-Type 12-Lipoxygenase.

Human platelet ALOX12 (hALOX12 or h12-LOX) has been implicated in a variety of human diseases. The present study investigates the active site of hALOX12 to more thoroughly understand how it positions the substrate and achieves nearly perfect regio- and stereospecificities (i.e.

A handheld platform for target protein detection and quantification using disposable nanopore strips.

Accessible point-of-care technologies that can provide immunoassay and molecular modalities could dramatically enhance diagnostics, particularly for infectious disease control in low-resource settings. Solid-state nanopores are simple and durable sensors with low-energy instrumentation requirements. While nanopore sensors have demonstrated efficacy for nucleic acid targets, selective detection and quantification of target proteins from sample background has not been demonstrated.

Human 15-LOX-1 active site mutations alter inhibitor binding and decrease potency.

Human 15-lipoxygenase-1 (h15-LOX-1 or h12/15-LOX) reacts with polyunsaturated fatty acids and produces bioactive lipid derivatives that are implicated in many important human diseases. One such disease is stroke, which is the fifth leading cause of death and the first leading cause of disability in America. The discovery of h15-LOX-1 inhibitors could potentially lead to novel therapeutics in the treatment of stroke, however, little is known about the inhibitor/active site interaction.

Biochemical and Cellular Characterization and Inhibitor Discovery of Pseudomonas aeruginosa 15-Lipoxygenase.

Pseudomonas aeruginosa is an opportunistic pathogen that can cause nosocomial and chronic infections in immunocompromised patients. P. aeruginosa secretes a lipoxygenase, LoxA, but the biological role of this enzyme is currently unknown.

Kinetic and structural investigations of the allosteric site in human epithelial 15-lipoxygenase-2.

Allosteric regulation of human lipoxygenase (hLO) activity has recently been implicated in the cellular biology of prostate cancer. In the current work, we present isotope effect, pH, and substrate inhibitor data of epithelial 15-hLO-2, which probe the allosteric effects on its mechanistic behavior. The Dk(cat)/KM for 15-hLO-2, with AA and LA as substrate, is large indicating hydrogen atom abstraction is the principle rate-determining step, involving a tunneling mechanism for both substrates.

Substrate specificity changes for human reticulocyte and epithelial 15-lipoxygenases reveal allosteric product regulation.

Human reticulocyte 15-lipoxygenase (15-hLO-1) and epithelial 15-lipoxygenase (15-hLO-2) have been implicated in a number of human diseases, with differences in their substrate specificity potentially playing a central role. In this paper, we present a novel method for accurately measuring the substrate specificity of the two 15-hLO isozymes and demonstrate that both cholate and specific LO products affect substrate specificity. The linoleic acid (LA) product, 13-hydroperoxyoctadienoic acid (13-HPODE), changes the ( k cat/ K m) (AA)/( k cat/ K m) (LA) ratio more than 5-fold for 15-hLO-1 and 3-fold for 15-hLO-2, while the arachidonic acid (AA) product, 12-( S)-hydroperoxyeicosatetraenoic acid (12-HPETE), affects only the ratio of 15-hLO-1 (more than 5-fold).

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.

Baicalein is a potent in vitro inhibitor against both reticulocyte 15-human and platelet 12-human lipoxygenases.

Lipoxygenases (LO) have been implicated in asthma, immune disorders, and various cancers and as a consequence, there is great interest in isolating selective LO isozyme inhibitors. Currently, there is much use of baicalein as a selective human platelet 12-LO (12-hLO) inhibitor, however, our current steady-state inhibition data indicate that baicalein is not selective against 12-hLO versus human reticulocyte 15-LO-1 (15-hLO-1) (15/12=1.3), in vitro.

Stereochemical determination and bioactivity assessment of (S)-(+)-curcuphenol dimers isolated from the marine sponge Didiscus aceratus and synthesized through laccase biocatalysis.

Electrospray ionization mass spectrometry-guided isolation of extracts from Didiscus aceratus led to the discovery of several new derivatives of the bioactive bisabolene-type sponge metabolite (S)-(+)-curcuphenol (1). The compounds obtained by this method included a mixture of known (2) and new (3) dihydroxylated analogs as well as a novel family of dimeric derivatives, dicurcuphenols A-E (4-8), and dicurcuphenol ether F (9). Dimers 4-9 were also subsequently obtained through a hemisynthetic method in which 1 was incubated with the enzyme laccase.

Lipoxygenase inhibition by anadanthoflavone, a new flavonoid from the aerial parts of Anadenanthera colubrina.

Chemical investigation of the aerial parts of Anadenanthera colubrina led to the isolation of a new flavonoid named anadanthoflavone ( 1), along with 11 known compounds: alnusenol, lupenone, lupeol, betulinic acid, alpha-amyrin, beta-amyrin, beta-sitosterol, stigmasterol, apigenin, 4-hydroxybenzoic acid and cinnamic acid. The isolated compounds were evaluated for their inhibitory activity on human platelet 12-lipoxygenase (12-hLO), human reticulocyte 15-lipoxygenase (15-hLO) and soybean lipoxygenase-1 (15-sLO). Compound 1 was found to be active against 12-hLO and 15-hLO with IC50 values of 13 +/- 3 microM and 17 +/- 3 microM, respectively.