RESUMEN
Targeting newly identified damage pathways in the ischemic brain can help to circumvent the currently severe limitations of acute stroke therapy. Here we show that the activity of 12/15-lipoxygenase was increased in the ischemic mouse brain, and 12/15-lipoxygenase colocalized with a marker for oxidized lipids, MDA2. This colocalization was also detected in the brain of 2 human stroke patients, where it also coincided with increased apoptosis-inducing factor. A novel inhibitor of 12/15-lipoxygenase, LOXBlock-1, protected neuronal HT22 cells against oxidative stress. In a mouse model of transient focal ischemia, the inhibitor reduced infarct sizes both 24 hours and 14 days poststroke, with improved behavioral parameters. Even when treatment was delayed until at least 4 hours after onset of ischemia, LOXBlock-1 was protective. Furthermore, it reduced tissue plasminogen activator-associated hemorrhage in a clot model of ischemia/reperfusion. This study establishes inhibition of 12/15-lipoxygenase as a viable strategy for first-line stroke treatment.
Asunto(s)
Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/metabolismo , Inhibidores de la Lipooxigenasa/uso terapéutico , Accidente Cerebrovascular/tratamiento farmacológico , Accidente Cerebrovascular/enzimología , Anciano , Animales , Femenino , Humanos , Inhibidores de la Lipooxigenasa/administración & dosificación , Masculino , Ratones , Persona de Mediana Edad , Resultado del TratamientoRESUMEN
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.
Asunto(s)
Araquidonato 15-Lipooxigenasa/química , Araquidonato 15-Lipooxigenasa/metabolismo , Sitio Alostérico , Humanos , Concentración de Iones de Hidrógeno , Cinética , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/metabolismo , Inhibidores de la Lipooxigenasa/farmacología , Reticulocitos/enzimología , Accidente Cerebrovascular/tratamiento farmacológico , Animales , Ensayos Analíticos de Alto Rendimiento , Humanos , Inhibidores de la Lipooxigenasa/uso terapéutico , Ratones , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Araquidonato 12-Lipooxigenasa/metabolismo , Derivados del Benceno/síntesis química , Inhibidores de la Lipooxigenasa/síntesis química , Sulfonamidas/síntesis química , Animales , Derivados del Benceno/química , Derivados del Benceno/farmacología , Disponibilidad Biológica , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Calcio/metabolismo , Humanos , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/enzimología , Inhibidores de la Lipooxigenasa/química , Inhibidores de la Lipooxigenasa/farmacología , Ratones , Agregación Plaquetaria/efectos de los fármacos , Relación Estructura-Actividad , Sulfonamidas/química , Sulfonamidas/farmacologíaRESUMEN
We report the discovery of novel small molecule inhibitors of platelet-type 12-human lipoxygenase, which display nanomolar activity against the purified enzyme, using a quantitative high-throughput screen (qHTS) on a library of 153607 compounds. These compounds also exhibit excellent specificity, >50-fold selectivity vs the paralogues, 5-human lipoxygenase, reticulocyte 15-human lipoxygenase type-1, and epithelial 15-human lipoxygenase type-2, and >100-fold selectivity vs ovine cyclooxygenase-1 and human cyclooxygenase-2. Kinetic experiments indicate this chemotype is a noncompetitive inhibitor that does not reduce the active site iron. Moreover, chiral HPLC separation of two of the racemic lead molecules revealed a strong preference for the (-)-enantiomers (IC(50) of 0.43 ± 0.04 and 0.38 ± 0.05 µM) compared to the (+)-enantiomers (IC(50) of >25 µM for both), indicating a fine degree of selectivity in the active site due to chiral geometry. In addition, these compounds demonstrate efficacy in cellular models, which underscores their relevance to disease modification.