Unlocking the potential: unveiling tyrphostins with Michael-reactive cyanoacrylate motif as promising inhibitors of human 5-lipoxygenase

Abstract

Human 5-lipoxygenase (5-LO) is the key enzyme in the biosynthesis of leukotrienes, mediators of the innate immune system that also play an important role in inflammatory diseases and cancer. In this study, we present compounds, containing a Michael-reactive cyanoacrylate moiety as potent inhibitors of 5-LO. Representatives of the tyrosine kinase inhibitor family called tyrphostins, structurally related to known 5-LO inhibitors, were screened for their 5-LO inhibitory properties using recombinant human 5-LO, intact human PMNL (polymorphonuclear leukocytes), and PMNL homogenates. Their mode of action was characterized by the addition of glutathione, using a fourfold cysteine 5-LO mutant and mass spectrometry analysis. SAR studies revealed several members of the tyrphostin family containing a Michael-reactive cyanoacrylate to efficiently inhibit 5-LO. We identified degrasyn (IC₅₀ 0.11 µM), tyrphostin A9 (IC₅₀ 0.8 µM), AG879 (IC₅₀ 78 nM), and AG556 (IC₅₀ 64 nM) as potent 5-LO inhibitors. Mass spectrometry analysis revealed that degrasyn and AG556 covalently bound to up to four cysteines, including C416 and/or C418 which surround the substrate entry site. Furthermore, the 5-LO inhibitory effect of degrasyn was remarkably impaired by the addition of glutathione or by the mutation of cysteines to serines at the surface of 5-LO. We successfully identified several tyrphostins as potent inhibitors of human 5-LO. Degrasyn and AG556 were able to covalently bind to 5-LO via their cyanoacrylate moiety. This provides a promising mechanism for targeting 5-LO by Michael acceptors, leading to new therapeutic opportunities in the field of inflammation and cancer.

Keywords: 5-Lipoxygenase; Inflammation; Leukotrienes; Michael acceptors; Tyrphostin.

Fig. 1

A Model of human 5-LO structure based on PDB 3O8Y [17]. 5-LO consists of a C2-like domain (dark blue) and a catalytic domain (grey) which contains the active site with the non-heme iron (dark red) and the iron coordinating amino acids (red). The mutated cysteines C159, C416, and C418 (yellow) are located near the putative entrance to the active site (K409, blue). B, C IC₅₀ values of selected 5-LO inhibitors in B cell-free assays using r5-LO_WT with or without the addition of 1 mM GSH (WT + GSH) or r5-LO_4C, or C with sonicated human PMNL with or without the addition of 1 mM GSH. In brief, 3 µg of protein (B) or 7.5 × 10⁶ sonicated cells/mL (C) were preincubated with an inhibitor in PBS/EDTA/ATP with or without 1 mM GSH for 15 min on ice. The reaction was started by the addition of 2 mM CaCl₂ and 10 (B) or 20 µM (C) AA for 10 min at 37 °C. Error bars depict standard deviation of IC₅₀. Asterisks indicate significant changes towards r5-LO_WT with the same inhibitor treatment. *(P < 0.05), **(P < 0.01), ***(P < 0.001); ****(P < 0.0001), n = 3

Fig. 2

Inhibition of 5-LO single cysteine mutants (C159S, C300S, C416S, or C418S) by degrasyn, tyrphostin A9, AG879, AG556, and CAPE normalized on DMSO control using 3 µg of recombinant 5-LO protein with the respective mutation. The inhibitor concentrations used correspond to the respective IC₅₀ value. Inhibitors were preincubated for 15 min in PBS/EDTA/ATP on ice before starting the reaction by the addition of 2 mM CaCl₂ and 10 µM AA for 10 min at 37 °C. Error bars depict standard deviation. Asterisks indicate significant changes towards r5-LO_WT with the same inhibitor treatment. *(P < 0.05), **(P < 0.01), ***(P < 0.001); ****(P < 0.0001), n = 3

Fig. 3

Mass spectra of r5-LO_WT or r5-LO_4C incubated with inhibitors (blue or red line). 20 µM r5-LO_WT (left) or r5-LO_4C (right) was incubated with 100 µM degrasyn (A), AG556 (B), tyrphostin A9 (C), AG879 (D), or CAPE (E) at RT for at least 1 h before analysis. Measurement was performed using an Agilent 1260 Infinity HPLC with an Agilent 6230 TOF LC/MS detector. Obtained data were processed using Mass Hunter BioConfirm (B.08.00), and resulted counts were normalized to the highest peak of the spectrum

Fig. 4

Multiplex assay of U2OS. A Normalized healthy cell count after 0 h, 12 h, 24 h, and 48 h after the addition of 1 µM (left panel) or 10 µM (right panel) inhibitor. Staurosporine (10 µM) was used as a control. Live cell count was performed in biological duplicates, normalized to vehicle control (DMSO), and depicted as mean ± SD. B Cell count ratio of tubulin effect (grey), increased mitochondrial mass (blue), and permeabilized cellular membrane (yellow) after 48 h. Error bars show SD of biological duplicates. C Confocal images of the stained U2OS cells with fluorescent (left panel) and merged brightfield images (right panel) taken after treatment with 10 µM degrasyn for 48 h in comparison to cells exposed to 0.1% DMSO. (Staining: blue: DNA, green: microtubule, red: mitochondria content, magenta: apoptosis marker annexin V)