ALOX15 protein, human

The mathematical model of the AA metabolic network in PMNs. Based on KEGG and a survey of the literature, a group of ODEs were devised to develop the mathematical model of the AA metabolic network in human PMNs (see details in Protocol S1 and Dataset S1). Michaelis–Menten equations are used to describe the catalysis in the network:
where [S] is the concentration of substrate, [E_t] is the total concentration of enzyme, K_cat is turnover number, and K_m is the Michaelis–Menten constant.
If competitive reversible inhibitors are involved in the catalysis, the equation is:
where [I] is the concentration of inhibitor and Ki is the inhibition constant, which is defined as:

If the inhibitors are irreversible, we assume the enzymes would decay according to the following equation:
where K is a constant.
When activators are involved in the catalysis, we use the following equation:
where [A] is the concentration of activator and KI is a constant.
PGE2 can upregulate 15-LOX through transcription in this network. Based on the experimental data, we describe its effect with the following equation:
where [g] is the concentration of PGE2 and K is a constant.
The ode15s routine of Matlab 6.5 (Mathworks, http://www.mathworks.com) was used to solve the ODEs. LTB4 and ω-LTB4 metabolic curves under different exogenous AA concentrations were calculated. These calculated curves were fit to the experimental data by empirically modulating parameters that had no direct values from published experiments, while the other parameters remained fixed to their experimental values. The parameter set that fit the experimental data well was chosen for further studies.
Simulating the therapeutic effects of the dual functional inhibitor and the mixture. The inhibition behavior on different enzymes is assumed to be independent and can be calculated by the following equations (only competitive reversible inhibitors are studied here):
where [I] is the concentration of inhibitor, and Ki is the dissociation constant. We use these equations with the consideration that the binding affinity of drugs is usually strong and the necessary concentration of inhibitors is in the same magnitude with the concentration of the enzyme. Then the enzyme-inhibitor complex cannot be neglected, and the above equations are required.
To evaluate the efficacy of inhibitors, inhibition intensity on the production of inflammatory mediators is defined as:
where [PGs]₁ is the concentration of PGs after taking drugs, [PGs]₀ is the concentration of PGs before treatment, [LTs]₁ is the concentration of LTs after taking drugs, and [LTs]₀ is the concentration of LTs before treatment.
We use the same value of total inhibition ability and total concentration of the dual functional inhibitor and the mixture to ensure the equality in the comparison. The total inhibition ability is defined as the product of inhibit constant to COX-2 and 5-LOX (Ki_COX-2 × Ki_5-LOX) and is fixed to 1 × 10⁻¹⁴ in all simulations. ER is defined as:
where A is the activity of enzyme, and C is the concentration. The ER value in the current model is 0.02.

All target thiouracil analogs 3–9 were further tested for 15-LOX inhibitory activity using Cayman’s Lipoxygenase Inhibitor Screening Assay Kit (Catalog No. 760700, Cayman Chemical, USA.
90 µL of 15-LOX was pipetted into a 96-well plate quickly. The test chemical was then dissolved in DMSO in 10 µL portions at concentrations of 2.5 µM, 5.0 µM, and 10 µM and added to each well. Arachidonic acid, a 110 µL substrate, was added to start the reaction, and the plate was shaken for at least five minutes. In order to interrupt enzyme catalysis and advance the reaction, 100 µL of chromogen was added to each well and made in accordance with the manufacturer’s instructions. In blank wells, 100 µL of assay buffer (0.1 M Tris-HCl, pH 7.4) was utilized. The positive control and 100% beginning activity were Quercetin and DMSO, respectively. The solution’s absorbance was determined at λ 490–500 nm. The percentage inhibition was calculated according to the following equation:
where (IA) is the 100% initial activity and (A_{inhibitor sample)} is the absorbance of the test sample. A dose-response curve was plotted between % inhibition and the drug concentration. The non-linear dose-response curve was used for calculating drug concentration showing 50% enzyme inhibition.

The 15-LOX inhibitory activity of Ca-EE was determined according to the method described by Yasin et al. with a slight modification [66 (link)]. In addition, 15-LOXs promote the reaction between linoleic acid and oxygen, producing 13-hydroperoxyoctadecadienoic acid that can increase the absorbance at 234 nm. To prepare the mixture, 200 μL of sample (Ca-EE or quercetin) and 400 μL of soybean lipoxygenase solution (167 U/mL) were reacted in 3.2 mL of 100 mM sodium phosphate buffer (pH 7.4). The mixture was reacted at 25 °C for 10 min. Quercetin was used for positive control and prepared by omitting the sample from the mixture and adding only DMSO. The reaction was started by putting 200 μL of 2.5 mM sodium linoleic acid. The absorbance was measured at 234 nm every 30 s for 3 min using a UV/Vis spectrophotometer (BioTek Instruments Inc., Winooski, VT, USA). Lipoxygenase inhibitory activity was calculated by following equation:
where ∆A₁/∆t is the enzymatic activity in the control group, and ∆A₂∆t is that in the sample group.