Platelet 12-LOX is essential for FcγRIIa-mediated platelet activation.

Platelets are essential in maintaining hemostasis following inflammation or injury to the vasculature. Dysregulated platelet activity often results in thrombotic complications leading to myocardial infarction and stroke. Activation of the FcγRIIa receptor leads to immune-mediated thrombosis, which is often life threatening in patients undergoing heparin-induced thrombocytopenia or sepsis. Inhibiting FcγRIIa-mediated activation in platelets has been shown to limit thrombosis and is the principal target for prevention of immune-mediated platelet activation. In this study, we show for the first time that platelet 12(S)-lipoxygenase (12-LOX), a highly expressed oxylipin-producing enzyme in the human platelet, is an essential component of FcγRIIa-mediated thrombosis. Pharmacologic inhibition of 12-LOX in human platelets resulted in significant attenuation of FcγRIIa-mediated aggregation. Platelet 12-LOX was shown to be essential for FcγRIIa-induced phospholipase Cγ2 activity leading to activation of calcium mobilization, Rap1 and protein kinase C activation, and subsequent activation of the integrin αIIbß3. Additionally, platelets from transgenic mice expressing human FcγRIIa but deficient in platelet 12-LOX, failed to form normal platelet aggregates and exhibited deficiencies in Rap1 and αIIbß3 activation. These results support an essential role for 12-LOX in regulating FcγRIIa-mediated platelet function and identifies 12-LOX as a potential therapeutic target to limit immune-mediated thrombosis.

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.

Azole Antifungal Sensitivity of Sterol 14α-Demethylase (CYP51) and CYP5218 from Malassezia globosa.

Malassezia globosa cytochromes P450 CYP51 and CYP5218 are sterol 14α-demethylase (the target of azole antifungals) and a putative fatty acid metabolism protein (and a potential azole drug target), respectively. Lanosterol, eburicol and obtusifoliol bound to CYP51 with Kd values of 32, 23 and 28 µM, respectively, catalyzing sterol 14α-demethylation with respective turnover numbers of 1.7 min(-1), 5.6 min(-1) and 3.4 min(-1). CYP5218 bound a range of fatty acids with linoleic acid binding strongest (Kd 36 µM), although no metabolism could be detected in reconstitution assays or role in growth on lipids. Clotrimazole, fluconazole, itraconazole, ketoconazole, voriconazole and ketaminazole bound tightly to CYP51 (Kd ≤ 2 to 11 nM). In contrast, fluconazole did not bind to CYP5218, voriconazole and ketaminazole bound weakly (Kd ~107 and ~12 µM), whereas ketoconazole, clotrimazole and itraconazole bound strongest to CYP5218 (Kd ~1.6, 0.5 and 0.4 µM) indicating CYP5218 to be only a secondary target of azole antifungals. IC50 determinations confirmed M. globosa CYP51 was strongly inhibited by azole antifungals (0.15 to 0.35 µM). MIC100 studies showed itraconazole should be considered as an alternative to ketoconazole given the potency and safety profiles and the CYP51 assay system can be used in structure-activity studies in drug development.

Discovery of a novel dual fungal CYP51/human 5-lipoxygenase inhibitor: implications for anti-fungal therapy.

We report the discovery of a novel dual inhibitor targeting fungal sterol 14α-demethylase (CYP51 or Erg11) and human 5-lipoxygenase (5-LOX) with improved potency against 5-LOX due to its reduction of the iron center by its phenylenediamine core. A series of potent 5-LOX inhibitors containing a phenylenediamine core, were synthesized that exhibit nanomolar potency and >30-fold selectivity against the LOX paralogs, platelet-type 12-human lipoxygenase, reticulocyte 15-human lipoxygenase type-1, and epithelial 15-human lipoxygenase type-2, and >100-fold selectivity against ovine cyclooxygenase-1 and human cyclooxygnease-2. The phenylenediamine core was then translated into the structure of ketoconazole, a highly effective anti-fungal medication for seborrheic dermatitis, to generate a novel compound, ketaminazole. Ketaminazole was found to be a potent dual inhibitor against human 5-LOX (IC50 = 700 nM) and CYP51 (IC50 = 43 nM) in vitro. It was tested in whole blood and found to down-regulate LTB4 synthesis, displaying 45% inhibition at 10 µM. In addition, ketaminazole selectively inhibited yeast CYP51 relative to human CYP51 by 17-fold, which is greater selectivity than that of ketoconazole and could confer a therapeutic advantage. This novel dual anti-fungal/anti-inflammatory inhibitor could potentially have therapeutic uses against fungal infections that have an anti-inflammatory component.