Inhibition of MIF bioactivity by rational design of pharmacological inhibitors of MIF tautomerase activity.
The pro-inflammatory mediator macrophage migration inhibitory factor (MIF) is produced by immune and endocrine cells and inhibits the antiinflammatory activities of glucocorticoids. MIF also catalyzes the tautomerization of the non-naturally occurring D-isomer of dopachrome, phenylpyruvate, and certain catecholamines, suggesting that MIF might exert its biological effects via enzymatic action on a substrate. However, no physiologically relevant substrate for MIF has been identified. Site-directed mutagenesis studies have not consistently supported a requirement for an intact, functional catalytic site as a prerequisite for MIF bioactivity. We hypothesized that the catalytically active site, but not the enzymatic activity per se, nevertheless plays a critical role in MIF pro-inflammatory activity. Accordingly, we designed small druglike molecules that bind at the catalytically active tautomerase site of MIF and tested the complex for MIF bioactivity. We describe herein the rational design and synthesis of a class of imine conjugates produced by coupling amino acids to a range of benzaldehyde derivatives that inhibit MIF tautomerase and biological activities. We found that aromatic amino acid Schiff bases were better inhibitors of MIF enzymatic and bioactivities compared to the aliphatic ones. For instance, the IC(50) inhibition of MIF tautomerase activity by aromatic amino acid Schiff base methyl esters was achieved at a concentration between 1.65 and 50 microM, suggesting a critical role for the additional binding of the aromatic residues within the vicinity of the active site. The most potent inhibitor of MIF tautomerase activity was 2-[(4-hydroxybenzylidene)amino]-3-(1H-indol-3-yl)propionic acid methyl ester (8), with an IC(50) of 1.65 microM. We found that compound 8 binding to MIF active site resulted in the inhibition of MIF bioactivity in three established bioassays: ERK-1/2 MAP kinase activation, p53-dependent apoptosis, and proliferation of serum-starved cells. Compound 8 inhibited MIF interaction with its as yet unidentified cognate cell surface receptor as shown by flow cytometry, concluding a critical role for the tautomerase active site in receptor binding. Thus the inhibitory effect of compound 8 on MIF bioactivities strongly correlated with the inhibition of MIF tautomerase activity, a connection not made previously through use of small-molecule MIF inhibitors. The inhibitory activity of amino acid-benzaldehyde Schiff base-type MIF antagonists is the first step toward a meaningful structure/function analysis of inhibitors of MIF cellular bioactivities.
Dios
Angeles
A
The Picower Institute for Medical Research, Manhasset, NY 11030, USA.
Mitchell
Robert A
RA
Aljabari
Bayan
B
Lubetsky
Jodi
J
O'Connor
KerryAnn
K
Liao
Hong
H
Senter
Peter D
PD
Manogue
Kirk R
KR
Lolis
Elias
E
Metz
Christine
C
Bucala
Richard
R
Callaway
David J E
DJ
Al-Abed
Yousef
Y
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D020134:Catalytic Domain; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D007097:Imines / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D007211:Indoles / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D007535:Isomerases / Q000037:antagonists & inhibitors*; D008263:Macrophage Migration-Inhibitory Factors / Q000037:antagonists & inhibitors*; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008934:Mitogens / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D008958:Models, Molecular; D010766:Phosphorylation; D011485:Protein Binding; D012545:Schiff Bases / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D013237:Stereoisomerism; D013329:Structure-Activity Relationship
C458001:2-((4-hydroxybenzylidene)amino)-3-(1H-indol-3-yl)propionic acid methyl ester; D004791:Enzyme Inhibitors; D007097:Imines; D007211:Indoles; D008263:Macrophage Migration-Inhibitory Factors; D008934:Mitogens; D012545:Schiff Bases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D007535:Isomerases