Michael acceptor containing drugs are a novel class of 5-lipoxygenase inhibitor targeting the surface cysteines C416 and C418.
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.
Institute of Pharmaceutical Chemistry, Goethe-University, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany.
Isabelle V
IV
Maucher
Institute of Pharmaceutical Chemistry, Goethe-University, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany.
Michael
M
Rühl
Institute of Pharmaceutical Chemistry, Goethe-University, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany.
Simon B M
SB
Kretschmer
Institute of Pharmaceutical Chemistry, Goethe-University, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany.
Bettina
B
Hofmann
Institute of Pharmaceutical Chemistry, Goethe-University, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany.
Benjamin
B
Kühn
Institute of Pharmaceutical Chemistry, Goethe-University, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany.
Jasmin
J
Fettel
Institute of Pharmaceutical Chemistry, Goethe-University, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany; Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Project Group for Translational Medicine and Pharmacology TMP, 60596 Frankfurt, Germany.
Anja
A
Vogel
Institute of Pharmaceutical Chemistry, Goethe-University, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany.
Karsten T
KT
Flügel
Institute of Organic Chemistry and Chemical Biology, Goethe-University, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany.
Georg
G
Manolikakes
Institute of Pharmaceutical Chemistry, Goethe-University, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany.
Nadine
N
Hellmuth
Institute of Pharmaceutical Chemistry, Goethe-University, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany.
Ann-Kathrin
AK
Häfner
Institute of Pharmaceutical Chemistry, Goethe-University, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany.
Vahid
V
Golghalyani
Institute of Pharmaceutical Chemistry, Goethe-University, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany.
Ann-Katrin
AK
Ball
Aarhus University, Department of Biomedicine & Center for Study and Prevention of Neurodegenerative Inflammation (NEURODIN), Bartholins Allé 6, 8000 Aarhus C, Denmark; Biomedical Research Laboratories, Medicine Faculty, Catholic University of Maule, Talca, Chile.
Matthias
M
Piesche
Aarhus University, Department of Biomedicine & Center for Study and Prevention of Neurodegenerative Inflammation (NEURODIN), Bartholins Allé 6, 8000 Aarhus C, Denmark.
Carmela
C
Matrone
Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Project Group for Translational Medicine and Pharmacology TMP, 60596 Frankfurt, Germany; Pharmazentrum Frankfurt/ZAFES, Institute of Clinical Pharmacology, Goethe-University, 60590 Frankfurt, Germany.
Gerd
G
Geisslinger
Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Project Group for Translational Medicine and Pharmacology TMP, 60596 Frankfurt, Germany.
Michael J
MJ
Parnham
Institute of Pharmaceutical Chemistry, Goethe-University, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany.
Michael
M
Karas
Institute of Pharmaceutical Chemistry, Goethe-University, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany.
Dieter
D
Steinhilber
Institute of Pharmaceutical Chemistry, Goethe-University, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany. Electronic address: roos@pharmchem.uni-frankfurt.de.
Jessica
J
Roos
Aarhus University, Department of Biomedicine & Center for Study and Prevention of Neurodegenerative Inflammation (NEURODIN), Bartholins Allé 6, 8000 Aarhus C, Denmark; Department of Anesthesia, Intensive Care Medicine and Pain Therapy, University Hospital Frankfurt, Frankfurt am Main, Germany. Electronic address: maier@biomed.au.dk.
Thorsten J
TJ
Maier
D003545:Cysteine / Q000187:drug effects*; D006801:Humans; D020128:Inhibitory Concentration 50; D016859:Lipoxygenase Inhibitors / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D016859:Lipoxygenase Inhibitors; D011994:Recombinant Proteins; D003545:Cysteine
Cysteine; Eicosanoids; Electrophilic molecules; Inflammation; Leukotrienes; Thymoquinone