Inactivation of the phenylpyruvate tautomerase activity of macrophage migration inhibitory factor by 2-oxo-4-phenyl-3-butynoate☆
Graphical abstract
Introduction
Macrophage migration inhibitory factor (MIF)1 was first identified in the 1960s as a protein involved in delayed-type hypersensitivity responses [1], [2], [3]. Since its initial discovery, MIF has been implicated in several inflammatory and autoimmune diseases, including arthritis [4], [5], [6], [7], glomerulonephritis [8], and Gram-positive and Gram-negative sepsis [9], [10]. The pro-inflammatory properties of MIF are coupled with an ability to counter the effect of corticosteroids [9], [11]. Thus, MIF inhibitors are of considerable interest as potential anti-inflammatory agents. MIF also exhibits a phenylpyruvate tautomerase (PPT) activity, catalyzing the ketonization of the enol isomers of phenylpyruvate (1, Scheme 1) [12] and p-hydroxyphenylpyruvate (2) to the respective keto isomers (3 and 4) [13]. Despite intense study, cellular receptors for MIF and the relevance of the enzymatic activity to the biological activities remain unknown.
Three-dimensional X-ray crystallographic studies of human and murine MIF demonstrate that MIF exists as a homotrimer and is structurally related to the bacterial 4-oxalocrotonate tautomerase (4-OT) and 5-carboxymethyl-2-hydroxymuconate isomerase (CHMI) [14], [15] despite low (<20%) sequence identity with these enzymes. Thus, MIF is a member of the tautomerase superfamily, a group of structurally homologous proteins characterized by a common β–α–β fold and a catalytic amino-terminal proline [16]. Pro-1 has been implicated as a critical residue in the PPT activity [13], [17].
The acetylene compounds, 2-oxo-3-pentynoate (2-OP, 5, Scheme 2) and 2-oxo-3-butynoate (2-OB, 6), are active-site-directed, irreversible inhibitors of 4-OT [18], [19]. Both compounds form a covalent bond with the amino-terminal proline, which functions as the general base in the 4-OT-catalyzed reaction. More significantly, crystallographic analysis of 4-OT inactivated by 2-OP provided much of the information about the positioning of active site residues and resulted in a proposed mechanism for the 4-OT-catalyzed reaction. The structural and functional similarities between 4-OT and MIF, which also uses Pro-1 as the catalytic base in the PPT-catalyzed reaction, suggested that the acetylene analogue of 5, 2-oxo-4-phenyl-3-butynoate (2-OPB, 7, Scheme 2) might be an irreversible inhibitor of MIF and a comparable diagnostic probe for exploring the enzymatic reactions as well as the biological activities. Hence, 2-OPB was synthesized and evaluated as a potential irreversible inhibitor of MIF. In addition, a structure of MIF crystals soaked with 7 has been determined to 1.45 Å resolution. The results are reported herein.
Section snippets
Materials and methods
Materials. All reagents, buffers, and solvents were obtained from Sigma–Aldrich Chemical Co. (St Louis, MO) with the following exceptions. The syntheses of tert-butyl glyoxylate (10) and (E)-2-fluoro-p-hydroxycinnamate (13) have been described [18], [20]. Recombinant murine MIF was purified as described previously [15]. Isopropyl-β-d-thiogalactoside (IPTG) and thin-walled PCR tubes were obtained from Ambion, Inc. (Austin, TX). Pre-packed PD-10 Sephadex G-25 columns were purchased from
Results
Synthesis of 2-oxo-4-phenyl-3-butynoate (7). The synthesis of 7 is outlined in Scheme 3 and is analogous to the procedure used for the synthesis of 2-OP [19]. Accordingly, the magnesium bromide salt of phenylacetylene was generated in situ from the commercially available compounds, methylmagnesium bromide and phenylacetylene (8). Subsequent condensation of 9 with the tert-butyl ester of glyoxylate (10) produced 11. Oxidation of 11 by activated MnO2 yielded 12 as the major product after flash
Discussion
Analogs of 2-oxo acids (such as 2-ketoglutarate and pyruvate) are potential mechanistic probes of a wide variety of enzymes [45], [46]. It is therefore not surprising that several such compounds have been synthesized and tested including the acetylene derivatives, 2-OP (5, Scheme 2), 2-OB (6), and 2-OPB (7) [18], [19], [45], [46]. Chiu and Jordan first reported a synthesis for 2-OPB in which a lithium derivative of 8 was coupled with an oxalic ester [45]. However, we chose to adapt our
Acknowledgments
The mass spectrometry described in this paper was carried out in the Analytical Instrumentation Facility Core housed in the College of Pharmacy at the University of Texas at Austin and supported by Center Grant ES07784.
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This research was supported by the National Institutes of Health Grant GM-65324 (C.P.W. and M.L.H.) and the Robert A. Welch Foundation (F-1219 to M.L.H.). SCW is a Fellow of the American Foundation for Pharmaceutical Education.