A new class of isothiocyanate-based irreversible inhibitors of macrophage migration inhibitory factor.
Macrophage migration inhibitory factor (MIF) is a homotrimeric multifunctional proinflammatory cytokine that has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. Current therapeutic strategies for targeting MIF focus on developing inhibitors of its tautomerase activity or modulating its biological activities using anti-MIF neutralizing antibodies. Herein we report a new class of isothiocyanate (ITC)-based irreversible inhibitors of MIF. Modification by benzyl isothiocyanate (BITC) and related analogues occurred at the N-terminal catalytic proline residue without any effect on the oligomerization state of MIF. Different alkyl and arylalkyl ITCs modified MIF with nearly the same efficiency as BITC. To elucidate the mechanism of action, we performed detailed biochemical, biophysical, and structural studies to determine the effect of BITC and its analogues on the conformational state, quaternary structure, catalytic activity, receptor binding, and biological activity of MIF. Light scattering, analytical ultracentrifugation, and NMR studies on unmodified and ITC-modified MIF demonstrated that modification of Pro1 alters the tertiary, but not the secondary or quaternary, structure of the trimer without affecting its thermodynamic stability. BITC induced drastic effects on the tertiary structure of MIF, in particular residues that cluster around Pro1 and constitute the tautomerase active site. These changes in tertiary structure and the loss of catalytic activity translated into a reduction in MIF receptor binding activity, MIF-mediated glucocorticoid overriding, and MIF-induced Akt phosphorylation. Together, these findings highlight the role of tertiary structure in modulating the biochemical and biological activities of MIF and present new opportunities for modulating MIF biological activities in vivo.
Ouertatani-Sakouhi
Hajer
H
Laboratory of Molecular Neurobiology and Functional Neuroproteomics, Brain Mind Institute and Institute of Biotechnology and Bioengineering, Ecole Polytechnique Federale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland.
El-Turk
Farah
F
Fauvet
Bruno
B
Roger
Thierry
T
Le Roy
Didier
D
Karpinar
Damla Pinar
DP
Leng
Lin
L
Bucala
Richard
R
Zweckstetter
Markus
M
Calandra
Thierry
T
Lashuel
Hilal A
HA
D001327:Autoimmune Diseases / Q000503:physiopathology; D002850:Chromatography, Gel; D016207:Cytokines / Q000737:chemistry; D005938:Glucocorticoids / Q000494:pharmacology; D006801:Humans; D007249:Inflammation / Q000503:physiopathology; D019746:Intramolecular Oxidoreductases / Q000037:antagonists & inhibitors; D017879:Isothiocyanates / Q000494:pharmacology*; D008263:Macrophage Migration-Inhibitory Factors / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008958:Models, Molecular; D010766:Phosphorylation; D011487:Protein Conformation; D012542:Scattering, Radiation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D016207:Cytokines; D005938:Glucocorticoids; D017879:Isothiocyanates; D008263:Macrophage Migration-Inhibitory Factors; D019746:Intramolecular Oxidoreductases; C515598:MIF protein, human