D-dopachrome tautomerase (D-DT or MIF-2): doubling the MIF cytokine family

D-dopachrome tautomerase (D-DT) is a newly described cytokine and a member of the macrophage migration inhibitory factor (MIF) protein superfamily. MIF is a broadly expressed pro-inflammatory cytokine that regulates both the innate and the adaptive immune response. MIF activates the MAP kinase cascade, modulates cell migration, and counter-acts the immunosuppressive effects of glucocorticoids. For many cell types, MIF also acts as an important survival or anti-apoptotic factor. Circulating MIF levels are elevated in the serum in different infectious and autoimmune diseases, and neutralization of the MIF protein via antibodies or small molecule antagonists improves the outcome in numerous animal models of human disease. Recently, a detailed investigation of the biological role of the closely homologous protein D-DT, which is encoded by a gene adjacent to MIF, revealed an overlapping functional spectrum with MIF. The D-DT protein also is present in most tissues and circulates in serum at similar concentrations as MIF. D-DT binds the MIF cell surface receptor complex, CD74/CD44, with high affinity and induces similar cell signaling and effector functions. Furthermore, an analysis of the signaling properties of the two proteins showed that they work cooperatively, and that neutralization of D-DT in vivo significantly decreases inflammation. In this review, we highlight the similarities and differences between MIF and D-DT, which we propose to designate "MIF-2", and discuss the implication of D-DT/MIF-2 expression for MIF-based therapies.     

A structurally preserved allosteric site in the MIF superfamily affects enzymatic activity and CD74 activation in D-dopachrome tautomerase

The macrophage migration inhibitory factor (MIF) family of cytokines contains multiple ligand-binding sites and mediates immunomodulatory processes through an undefined mechanism(s). Previously, we reported a dynamic relay connecting the MIF catalytic site to an allosteric site at its solvent channel. Despite structural and functional similarity, the MIF homolog D-dopachrome tautomerase (also called MIF-2) has low sequence identity (35%), prompting the question of whether this dynamic regulatory network is conserved. Here, we establish the structural basis of an allosteric site in MIF-2, showing with solution NMR that dynamic communication is preserved in MIF-2 despite differences in the primary sequence. X-ray crystallography and NMR detail the structural consequences of perturbing residues in this pathway, which include conformational changes surrounding the allosteric site, despite global preservation of the MIF-2 fold. Molecular simulations reveal MIF-2 to contain a comparable hydrogen bond network to that of MIF, which was previously hypothesized to influence catalytic activity by modulating the strength of allosteric coupling. Disruption of the allosteric relay by mutagenesis also attenuates MIF-2 enzymatic activity in vitro and the activation of the cluster of differentiation 74 receptor in vivo, highlighting a conserved point of control for nonoverlapping functions in the MIF superfamily.     

2,5-Pyridinedicarboxylic acid is a bioactive and highly selective inhibitor of D-dopachrome tautomerase

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Inhibition of macrophage migration inhibitory factor (MIF) tautomerase activity by dopachrome analogs

A macrophage migration inhibitory factor (MIF) dopachrome tautomerization assay was employed for identification of MIF inhibitors. One group of dopachrome analogs was identified which inhibits MIF tautomerase activity. In particular, the analogs with a leaving group at beta position displayed activity at a concentration of tenfold less than that of the MIF-substrate. These findings could lead to a better understanding of MIF biological activities and the development of agents for the treatment of MIF related diseases.     

Crystal structure of human D-dopachrome tautomerase, a homologue of macrophage migration inhibitory factor, at 1.54 A resolution

D-Dopachrome tautomerase shares a low homologous amino acid sequence (33% homology) with the macrophage migration inhibitory factor (MIF) and possesses similar tautomerase activity as well. MIF is a cytokine involved in inflammatory reactions and immune responses. Whereas recent studies have identified MIF as a pituitary hormone and immunoregulator, much less is known about the structural basis of these physiological functions and the real significance of tautomerase activity. Therefore, interest in the structure-function relationship between D-dopachrome tautomerase and MIF has increased, especially with regard to inflammation and immune responses. We have determined the X-ray crystal structure of human D-dopachrome tautomerase at 1.54 A resolution. D-Dopachrome tautomerase folds to form a homotrimer that has extensive contact between subunits by intersubunit beta-sheets. Its overall topology and trimeric formations are similar to those of human MIF. The N-terminal proline is located at the bottom of a positively charged pocket in which the conformations of Lys32 and Ser63 are highly conserved. These positively charged properties are also seen in the active site pocket of human MIF, bacterial 5-(carboxymethyl)-2-hydroxymuconate isomerase (CHMI), and 4-oxalocrotonate tautomerase (4-OT). A detailed comparison of these structures revealed significant differences in the environment around the potential active site, the intersubunit contacts, and charge distribution on the molecular surface. It can be concluded that these features are related to the physiological role and tautomerase activity of MIF and D-dopachrome tautomerase. The present structural study could be helpful for designing effective inhibitors that modulate immunoregulatory and hormone-like effects.     

An integrative in silico methodology for the identification of modulators of macrophage migration inhibitory factor (MIF) tautomerase activity

Macrophage migration inhibitory factor (MIF) is a major proinflammatory cytokine that has been increasingly implicated in the pathogenesis of several inflammatory, autoimmune, infectious and oncogenic diseases. Accumulating evidence suggests that the tautomerase activity of MIF plays a role in modulating some of its intra- and extra-cellular activities. Therefore, the identification and development of small-molecule inhibitors targeting the catalytic activity of MIF has emerged as an attractive and viable therapeutic strategy to attenuate its function in health and disease. Herein we report a novel virtual screening protocol for the discovery of new inhibitors of MIF’s tautomerase activity. Our protocol takes into account the flexibility and dynamics of the catalytic site by coupling molecular dynamics (MD) simulations aimed at modeling the protein’s flexibility in solution to (i) docking with FlexX, or (ii) docking with FlexX and pharmacophoric filtering with Unity. In addition, we applied in parallel a standalone docking using the new version of Surflex software. The three approaches were used to screen the ChemBridge chemical library and the inhibitory activity of the top-ranked 333 compound obtained from each approach (1000 compound in total) was assessed in vitro using the tautomerase assay. This biochemical validation process resulted in the identification of 12 novel MIF inhibitors corresponding to a 1.2% hit rate. Six of these hits came from Surflex docking; two from FlexX docking with MD simulations and four hits were identified with MDS and pharmacophore filtering with minimal overlap between the hits from each approach. Six hits were identified with IC₅₀ values lower than 10 μM (three hits with IC₅₀ lower than 1 μM); four were shown to be suicide inhibitors and act via covalent modification of the N-terminal catalytic residues Pro1. One additional inhibitor, N-phenyl-N-1,3,4-thiadiazol-2-yl-thiourea, (IC₅₀ = 300 nM) was obtained from FlexX docking combined to pharmacophoric filtering on one of the eight MD structures. These results demonstrate the power of integrative in silico approaches in the discovery of new modulator of MIF’s tautomerase activity. The chemical diversity and mode of action of these compounds suggest that they could be used as molecular probes to elucidate the functions and biology of MIF and as lead candidates in drug developments of anti-MIF drugs.     

Fragment screening of inhibitors for MIF tautomerase reveals a cryptic surface binding site

In the course of a fragment screening campaign by in silico docking followed by X-ray crystallography, a novel binding site for migration inhibitory factor (MIF) inhibitors was demonstrated. The site is formed by rotation of the side-chain of Tyr-36 to reveal a surface binding site in MIF that is hydrophobic and surrounded by aromatic side-chain residues. The crystal structures of two small inhibitors that bind to this site and of a quinolinone inhibitor, that spans the canonical deep pocket near Pro-1 and the new surface binding site, have been solved. These results suggest new opportunities for structure-based design of MIF inhibitors.     

Conserved worldwide linkage disequilibrium in the human factor XI gene

We have identified, in four diverse human populations, five common single-nucleotide polymorphisms (SNPs) in the coding region of the gene for the blood coagulation protease factor XI. Each SNP has an allele frequency >5% in at least one population. Three of the SNPs (C472T, A844G, and T1234C), spread out over approximately 10 kb of genomic DNA, are in marked linkage disequilibrium (LD) with one another (P < 10(-4)). Interestingly, haplotypes associated with the linked SNPs are conserved across all populations studied, despite significantly different allele frequencies between populations. The presence of such common, widely dispersed haplotypes could complicate the interpretation of LD studies and emphasizes the need for a better understanding of general patterns of LD to facilitate identification of genes for common disorders.     

Discovery of covalent inhibitors for MIF tautomerase via cocrystal structures with phantom hits from virtual screening

Biochemical and X-ray crystallographic studies confirmed that hydroxyquinoline derivatives identified by virtual screening were actually covalent inhibitors of the MIF tautomerase. Adducts were formed by N-alkylation of the Pro-1 at the catalytic site with a loss of an amino group of the inhibitor.     

Critical modifications of the ISO-1 scaffold improve its potent inhibition of macrophage migration inhibitory factor (MIF) tautomerase activity

Based on the scaffold of (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1), an inhibitor of the proinflammatory cytokine MIF, two critical modifications and chiral resolution have significantly improved the potency of the inhibition. Compound (R)-17 is 20-fold more potent than ISO-1 and inhibits MIF tautomerase activity with an IC(50) of 550 nM.     

An approach to incorporate linkage disequilibrium structure into genomic association analysis

In this study, we propose to use the principal component analysis (PCA) and regression model to incorporate linkage disequilibrium (LD) in genomic association data analysis. To accommodate LD in genomic data and reduce multiple testing, we suggest performing PCA and extracting the PCA score to capture the variation of genomic data, after which regression analysis is used to assess the association of the disease with the principal component score. An empirical analysis result shows that both genotype-basod correlation matrix and haplotype-based LD matrix can produce similar results for PCA. Principal component score seems to be more powerful in detecting genetic association because the principal component score is quantitatively measured and may be able to capture the effect of multiple loci.     

The human factor H-related gene 2 (FHR2): structure and linkage to the coagulation factor XIIIb gene

The human factor H-related gene 2 (FHR2) encodes a serum protein structurally and immunologically related to complement factor H. We describe the isolation and genomic organization of the human FHR2 gene from a yeast artificial chromosome library. The FHR2 gene is organized in five exosn and span about 7 kilobases (kb) of human genomic DNA. A comparison with the corresponding cDNA sequence (clone DDESK59) shows that the analyzed FHR2 gene has a deleted region within exon 4. A new splice acceptor site created in the truncated exon indicates that the analyzed gene could be translated to a truncated protein. Further, we demonstrate that the genes for FHR2 and subunit of coagulation factor XIII are located in the same 165 kb YAC DNA. Thus, the three structurally related genes FXIIIb, FHR2, and factor H are linked on human chromosome 1 in the regulators of complement activiation (RCA) gene cluster. The physical linkage of the FHR2 and the factor H genes provides additional evidence for a close relatedness of complement factor H and the factor H-related proteins. The linkage and the almost exclusive organization in short consensus repeat-containing domains indicates a close evolutionary relationship of the FXIIIb, FHR2, and factor H genes.The nucleotide sequence data reported in this paper have been submitted to the EMBL and GenBank nucleotide sequence databases and have been assigned the accession number X86564 (exon 1), X86565 (exon 2), X86566 (exon 3 and 4), and X86567 (exon 5)