Michael acceptor containing drugs are a novel class of 5-lipoxygenase inhibitor targeting the surface cysteines C416 and C418

Biochem Pharmacol. 2017 Feb 1;125:55-74. doi: 10.1016/j.bcp.2016.11.004. Epub 2016 Nov 4.

Abstract

Recently, we published that nitro-fatty acids (NFA) are potent electrophilic molecules which inhibit 5-lipoxygenase (5-LO) by interacting catalytically with cysteine residues next to a substrate entry channel. The electrophilicity is derived from an intramolecular Michael acceptor moiety consisting of an electron-withdrawing group in close proximity to a double bond. The potential of the Michael acceptor moiety to interact with functionally relevant cysteines of proteins potentially renders them effective and sustained enzyme activity modulators. We screened a large library of naturally derived and synthetic electrophilic compounds to investigate whether other types of Michael acceptor containing drugs suppress 5-LO enzyme activity. The activity was measured by assessing the effect on the 5-LO product formation of intact human polymorphonuclear leukocytes. We demonstrated that a number of structurally different compounds were suppressive in the activity assays and showed that Michael acceptors of the quinone and nitro-alkene group produced the strongest inhibition of 5-LO product formation. Reactivity with the catalytically relevant cysteines 416 and 418 was confirmed using mutated recombinant 5-LO and mass spectrometric analysis (MALDI-MS). In the present study, we show for the first time that a number of well-recognized naturally occurring or synthetic anti-inflammatory compounds carrying a Michael acceptor, such as thymoquinone (TQ), the paracetamol metabolite NAPQI, the 5-LO inhibitor AA-861, and bardoxolone methyl (also known as RTA 402 or CDDO-methyl ester) are direct covalent 5-LO enzyme inhibitors that target the catalytically relevant cysteines 416 and 418.

Keywords: Cysteine; Eicosanoids; Electrophilic molecules; Inflammation; Leukotrienes; Thymoquinone.

MeSH terms

  • Cysteine / drug effects*
  • Humans
  • Inhibitory Concentration 50
  • Lipoxygenase Inhibitors / pharmacology*
  • Recombinant Proteins / metabolism
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Substances

  • Lipoxygenase Inhibitors
  • Recombinant Proteins
  • Cysteine