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 the current paper, we present a novel method to accurately measure 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 over 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), only affects the ratio of 15-hLO-1 (over 5-fold). In addition, the reduced products, 13-(S)-hydroxyoctadecadienoic acid (13-HODE) and 12-(S)-hydroxyeicosatetraenoic acid (12-HETE), also affect substrate specificity, indicating that iron oxidation is not responsible for the change in the [k cat /K m ] AA /[k cat /K m ] LA ratio. These results, coupled with the dependence of the 15-hLO-1, k cat /K m kinetic isotope effect ( D k cat /K m ) on the presence of 12-HPETE and 12-HETE, indicate that the allosteric site, previously identified in 15-hLO-1 [Mogul, R., Johansen, E., Holman, T. R. (1999) Biochemistry 39, 4801-4807], is responsible for the change in substrate specificity. The ability of LO products to regulate substrate specificity may have relevancy to cancer progression and warrants further investigation into the role of this product-feedback loop in the cell.Human lipoxygenases (hLO) are a family of structurally related enzymes that catalyze the hydroperoxidation of polyunsaturated fatty acids using molecular oxygen (Scheme 1) (1). There are three main isozymes of pharmacological interest: 5-hLO, 12-hLO and 15-hLO, which are named according to their positional specificity on arachidonic acid (AA), producing their respective hydroperoxyeicosatetraenoic acid (HPETE) products. Each of these lipoxygenase isozymes plays a distinct biological role in human disease; 5-hLO is implicated in asthma (2) and cancer (3,4), 12-hLO is implicated in psoriasis (5) and cancer (4,6,7) and 15-hLO is implicated in atherosclerosis (8) and cancer (4,9).Defining the exact role of LO in human disease is complicated by the incomplete understanding of three fundamental biochemical properties of hLO: substrate specificity, activation specificity and allosteric regulation. With regards to substrate specificity, enzymes are typically highly specific and react with only one particular substrate, such as 5-hLO and platelet 12-hLO, which only react with AA. However, reticulocyte 15-hLO-1 and epithelial 15-hLO-2 react with both LA and AA, albeit with different efficiencies. 15-hLO-1 reacts preferentially $ This work was supported by the National Institutes of Health (GM56062 and S10-RR20939 (MS equipment grant)) *To whom the correspondence should be addressed: tholman@chemistry.ucsc.edu. % Current address, KaloBios, 3427 Hillview Ave., Sui...

Abstract. 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 indicates that baicalein is not selective against 12-hLO versus human reticulocyte 15-LO-1 (15-hLO-1) (15/12 = 1.3), in vitro. However, in the presence of detergents baicalein is slightly more selective (15/12 = 7), which may imply greater selectivity in a cell based assay but has yet to be proven. The mechanism of baicalein inhibition of 15-hLO is reductive, which computer docking suggests is through direct binding of the catecholic moiety of baicalein to the iron. A structurally related flavonoid, apigenin, is not reductive, however, computer docking suggests a hydrogen bond with Thr591, may account for its inhibitor potency.

There are a variety of lipoxygenases in the human body (hLO), each having a distinct role in cellular biology. Human reticulocyte 15-Lipoxygenase-1 (15-hLO-1), which catalyzes the dioxygenation of 1,4-cis,cis-pentadiene-containing polyunsaturated fatty acids, is implicated in a number of diseases including cancer, atherosclerosis, and neurodegenerative conditions. Despite the potential therapeutic relevance of this target, few inhibitors have been reported that are both potent and selective. To this end, we have employed a quantitative high-throughput (qHTS) screen against ~74,000 small molecules in search of reticulocyte 15-hLO-1 selective inhibitors. This screen led to the discovery of a novel chemotype for 15-hLO-1 inhibition, which displays nM potency and is >7,500-fold selective against the related isozymes, 5-hLO, platelet 12-hLO, epithelial 15-hLO-2, ovine cyclooxygenase-1 and human cyclooxygenase-2. In addition, kinetic experiments were performed which indicate that this class of inhibitor is tight binding, reversible, and appears not to reduce the active-site ferric ion.

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A key challenge facing drug discovery today is variability of the drug target between species, such as with 12/15-lipoxygenase (12/15-LOX), which contributes to ischemic brain injury, but its human and rodent isozymes have different inhibitor specificities. In the current work, we have utilized a quantitative high-throughput (qHTS) screen to identify compound 1 (ML351), a novel chemotype for 12/15-LOX inhibition that has nanomolar potency (IC50 = 200 nM) against human 12/15-LOX and is protective against oxidative glutamate toxicity in mouse neuronal HT22 cells. In addition, it exhibited greater than 250-fold selectivity versus related LOX isozymes, was a mixed inhibitor, and did not reduce the active-site ferric ion. Lastly, 1 significantly reduced infarct size following permanent focal ischemia in a mouse model of ischemic stroke. As such, this represents the first report of a selective inhibitor of human 12/15-LOX with demonstrated in vivo activity in proof-of-concept mouse models of stroke.

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 Dkcat/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. For AA, there are multiple rate determining steps (RDS) at both high and low temperature, with both diffusion and hydrogen bonding rearrangements contributing at high temperature, but only hydrogen bonding rearrangements contributing at low temperature. The observed kinetic dependency on the hydrogen bonding rearrangement is eliminated upon addition of the allosteric effector, 13-(S)-hydroxyoctadecadienoic acid (13-HODE), however, no allosteric effects were seen on diffusion or hydrogen atom abstraction. The (kcat/KM)AA/(kcat/KM)LA ratio was observed to have a pH dependence, which was fit with a titration curve (pKa = 7.7), suggesting the protonation of a histidine residue, which could hydrogen bond with the carboxylate of 13-HODE. Assuming this interaction, 13-HODE was docked to the solvent exposed histidines of a 15-hLO-2 homology model and found to bind well with H627, suggesting a potential location for the allosteric site. Utilizing d31-LA as an inhibitor, it was demonstrated that the binding of d31-LA to the allosteric site changes the conformation of 15-hLO-2 such that the affinity for substrate increases. This result suggests that allosteric binding locks the enzyme into a catalytically competent state, which facilitates binding of LA and decreases the (kcat/KM)AA/(kcat/KM)LA ratio. Finally, the magnitude of the 13-HODE KD for 15-hLO-2 is over 200-fold lower than that of 13-HODE for 15-hLO-1, changing the substrate specificity of 15-hLO-2 to 1.9, which would alter the LO product distribution by increasing the production of the pro-tumorigenic 13-HODE, possibly representing a pro-tumorigenic feedback loop for 13-HODE and 15-hLO-2.

Human lipoxygenase (hLO) isozymes have been implicated in a number of disease states and have attracted much attention with respect to their inhibition. One class of inhibitors, the flavonoids, have been shown to be potent lipoxygenase inhibitors but their study has been restricted to those compounds found in nature, which have limited structural variability. We have therefore carried out a comprehensive study to determine the structural requirements for flavonoid potency and selectivity against platelet 12-hLO, reticulocyte 15-hLO-1, and prostate epithelial 15-hLO-2. We conclude from this study that catechols are essential for high potency, that isoflavones and isoflavonones tend to select against 12-hLO, that isoflavons tend to select against 15-hLO-1, but few flavonoids target 15-hLO-2.

The lipid-metabolizing enzyme 12/15-lipoxygenase (12/15-LOX) mediates cell death resulting from oxidative stress in both neurons and oligodendrocytes. Specifically, it may contribute to the pathophysiology of stroke and Alzheimer's and Parkinson's diseases. We report here that two of three specific 12/15-LOX inhibitors, derived from a virtual screen by computer modeling and validated by inhibition of recombinant human 15-LOX in vitro, are able to rescue both neuronal as well as oligodendroglial cells from cell death induced by oxidative stress. Thus, in a fairly streamlined process, an initial virtual screen of 50,000 compounds in a library of drug-like molecules has led to the identification of two novel drug candidates for targeting LOX. Future studies of these novel neuroprotective inhibitors of 12/15-LOX may provide new therapeutic opportunities to combat stroke and other neurodegenerative diseases.