A Small Molecule Allele-selective Transcriptional Inhibitor of the MIF Immune Susceptibility Locus.

Functional variants of the gene for the cytokine macrophage migration inhibitory factor (MIF) are defined by a 4-nucleotide promoter microsatellite (-794 CATT5-8, rs5844572) and confer risk for autoimmune, infectious, and oncologic diseases. We describe herein the discovery of a prototypic, small molecule inhibitor of MIF transcription with selectivity for high microsatellite repeat number and correspondingly high gene expression. Utilizing a high-throughput luminescent proximity screen, we identify 1-carbomethoxy-5-formyl-4,6,8-trihydroxyphenazine (CMFT) to inhibit the functional interaction between the transcription factor ICBP90 (a.k.a. UHRF1) and the MIF -794 CATT5-8 promoter microsatellite. CMFT inhibits MIF mRNA expression in a -794 CATT5-8 length-dependent manner with an IC50 of 470 nM, and preferentially reduces ICBP90-dependent MIF mRNA and protein expression in high-genotypic versus low-genotypic MIF - expressing macrophages. RNA expression analysis also showed CMFT to downregulate MIF-dependent, inflammatory gene expression with little evidence of off-target metabolic toxicity. These findings provide proof-of-concept for advancing the pharmacogenomic development of precision-based MIF inhibitors for diverse autoimmune and inflammatory conditions.

The C-terminal Region of D-DT Regulates Molecular Recognition for Protein-Ligand Complexes.

Systematic analysis of molecular recognition is critical for understanding the biological function of macromolecules. For the immunomodulatory protein D-dopachrome tautomerase (D-DT), the mechanism of protein-ligand interactions is poorly understood. Here, 17 carefully designed protein variants and wild type (WT) D-DT were interrogated with an array of complementary techniques to elucidate the structural basis of ligand recognition. Utilization of a substrate and two selective inhibitors with distinct binding profiles offered previously unseen mechanistic insights into D-DT-ligand interactions. Our results demonstrate that the C-terminal region serves a key role in molecular recognition via regulation of the active site opening, protein-ligand interactions, and conformational flexibility of the pocket's environment. While our study is the first comprehensive analysis of molecular recognition for D-DT, the findings reported herein promote the understanding of protein functionality and enable the design of new structure-based drug discovery projects.

Iguratimod, an allosteric inhibitor of macrophage migration inhibitory factor (MIF), prevents mortality and oxidative stress in a murine model of acetaminophen overdose.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that has been implicated in multiple inflammatory and non-inflammatory diseases, including liver injury induced by acetaminophen (APAP) overdose. Multiple small molecule inhibitors of MIF have been described, including the clinically available anti-rheumatic drug T-614 (iguratimod); however, this drug's mode of inhibition has not been fully investigated. METHODS: We conducted in vitro testing including kinetic analysis and protein crystallography to elucidate the interactions between MIF and T-614. We also performed in vivo experiments testing the efficacy of T-614 in a murine model of acetaminophen toxicity. We analyzed survival in lethal APAP overdose with and without T-614 and using two different dosing schedules of T-614. We also examined MIF and MIF inhibition effects on hepatic hydrogen peroxide (H2O2) as a surrogate of oxidative stress in non-lethal APAP overdose. RESULTS: Kinetic analysis was consistent with a non-competitive type of inhibition and an inhibition constant (Ki) value of 16 µM. Crystallographic analysis revealed that T-614 binds outside of the tautomerase active site of the MIF trimer, with only the mesyl group of the molecule entering the active site pocket. T-614 improved survival in lethal APAP overdose when given prophylactically, but this protection was not observed when the drug was administered late (6 h after APAP). T-614 also decreased hepatic hydrogen peroxide concentrations during non-lethal APAP overdose in a MIF-dependent fashion. CONCLUSIONS: T-614 is an allosteric inhibitor of MIF that prevented death and decreased hepatic hydrogen peroxide concentrations when given prophylactically in a murine model of acetaminophen overdose. Further studies are needed to elucidate the mechanistic role of MIF in APAP toxicity.

Analysis of CD74 Occurrence in Oncogenic Fusion Proteins.

CD74 is a type II cell surface receptor found to be highly expressed in several hematological and solid cancers, due to its ability to activate pathways associated with tumor cell survival and proliferation. Over the past 16 years, CD74 has emerged as a commonly detected fusion partner in multiple oncogenic fusion proteins. Studies have found CD74 fusion proteins in a range of cancers, including lung adenocarcinoma, inflammatory breast cancer, and pediatric acute lymphoblastic leukemia. To date, there are five known CD74 fusion proteins, CD74-ROS1, CD74-NTRK1, CD74-NRG1, CD74-NRG2α, and CD74-PDGFRB, with a total of 16 different variants, each with unique genetic signatures. Importantly, the occurrence of CD74 in the formation of fusion proteins has not been well explored despite the fact that ROS1 and NRG1 families utilize CD74 as the primary partner for the formation of oncogenic fusions. Fusion proteins known to be oncogenic drivers, including those of CD74, are typically detected and targeted after standard chemotherapeutic plans fail and the disease relapses. The analysis reported herein provides insights into the early intervention of CD74 fusions and highlights the need for improved routine assessment methods so that targeted therapies can be applied while they are most effective.

Protocol for purification and enzymatic characterization of members of the human macrophage migration inhibitory factor superfamily.

Macrophage migration inhibitory factor (MIF) and D-dopachrome tautomerase (D-DT or MIF-2) are two proteins serving a key role in the pathogenesis of multiple disorders, including cancer.1 Here, we present a protocol for the purification and enzymatic characterization of MIF and D-DT using keto-enol tautomerase activity. This approach measures enzymatic activity through the formation of an enol-borate complex. We describe steps for expressing and purifying proteins, preparing the 96-well microplate, and assay implementation including monitoring of keto-enol tautomerase activity. For complete details on the use and execution of this protocol, please refer to Parkins et al.2,3.

A novel enzymatic assay for the identification of 4-hydroxyphenylpyruvate dioxygenase modulators.

4-hydroxyphenylpyruvate dioxygenase (HPPD) is a key enzyme involved in the pathogenesis of tyrosinemia III and cancer. Herein, we describe a spectroscopy-based assay to detect HPPD dioxygenase activity in the presence or absence of small-molecule modulators. We describe steps for transformation, expression, and purification of HPPD and preparation of the assay plate. We detail initiation and completion of the enzymatic reaction followed by detection of remaining substrate in the form of enol-HPP/borate complex. This assay is applicable for high-throughput screening. For complete details on the use and execution of this protocol, please refer to Parkins et al.1.

Underrepresented Impurities in 4-Hydroxyphenylpyruvate Affect the Catalytic Activity of Multiple Enzymes.

Macrophage migration inhibitory factor (MIF) is a key immunostimulatory protein with regulatory properties in several disorders, including inflammation and cancer. All the reported inhibitors that target the biological activities of MIF have been discovered by testing against its keto/enol tautomerase activity. While the natural substrate is still unknown, model MIF substrates are used for kinetic experiments. The most extensively used model substrate is 4-hydroxyphenyl pyruvate (4-HPP), a naturally occurring intermediate of tyrosine metabolism. Here, we examine the impact of 4-HPP impurities in the precise and reproducible determination of MIF kinetic data. To provide unbiased evaluation, we utilized 4-HPP powders from five different manufacturers. Biochemical and biophysical analyses showed that the enzymatic activity of MIF is highly influenced by underrepresented impurities found in 4-HPP. Besides providing inconsistent turnover results, the 4-HPP impurities also influence the accurate calculation of ISO-1's inhibition constant, an MIF inhibitor that is broadly used for in vitro and in vivo studies. The macromolecular NMR data show that 4-HPP samples from different manufacturers result in differential chemical shift perturbations of amino acids in MIF's active site. Our MIF-based conclusions were independently evaluated and confirmed by 4-hydroxyphenylpyruvate dioxygenase (HPPD) and D-dopachrome tautomerase (D-DT); two additional enzymes that utilize 4-HPP as a substrate. Collectively, these results explain inconsistencies in previously reported inhibition values, highlight the effect of impurities on the accurate determination of kinetic parameters, and serve as a tool for designing error-free in vitro and in vivo experiments.

Ligand-induced conformational changes enable intersubunit communications in D-dopachrome tautomerase.

D-Dopachrome tautomerase (D-DT; or MIF-2) is a multifunctional protein with immunomodulatory properties and a documented pathogenic role in inflammation and cancer that is associated with activation of the cell surface receptor CD74. Alongside D-DT, macrophage migration inhibitory factor (MIF) is also known to activate CD74, promoting pathogenesis. While the role of the MIF/CD74 axis has been extensively studied in various disease models, the late discovery of the D-DT/CD74 axis has led to a poor investigation into the D-DT-induced activation mechanism of CD74. A previous study has identified 4-(3-carboxyphenyl)-2,5-pyridinedicarboxylic acid (4-CPPC) as the first selective and reversible inhibitor of D-DT and reported its potency to block the D-DT-induced activation of CD74 in a cell-based model. In this study, we employ molecular dynamics simulations and nuclear magnetic resonance experiments to study 4-CPPC-induced changes to the dynamic profile of D-DT. We found that binding of the inhibitor remarkably promotes the conformational flexibility of C-terminal without impacting the structural stability of the biological assembly. Consequently, long-range intrasubunit (>11 Å) and intersubunit (>30 Å) communications are enabled between distal regions. Communication across the three subunits is accomplished via 4-CPPC, which serves as a communication bridge after Val113 is displaced from its hydrophobic pocket. This previously unrecognized structural property of D-DT is not shared with its human homolog, MIF, which exhibits an impressive C-terminal rigidity even in the presence of an inhibitor. Considering the previously reported role of MIF's C-terminal in the activation of CD74, our results break new ground for understanding the functionality of D-DT in health and disease.

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

Macrophage migration inhibitory factor (MIF) and D-dopachrome tautomerase (D-DT) are two pleotropic cytokines, which are coexpressed in various cell types to activate the cell surface receptor CD74. Via the MIF/CD74 and D-DT/CD74 axes, the two proteins exhibit either beneficial or deleterious effect on human diseases. In this study, we report the identification of 2,5-pyridinedicarboxylic acid (a.k.a. 1) that effectively blocks the D-DT-induced activation of CD74 and demonstrates an impressive 79-fold selectivity for D-DT over MIF. Crystallographic characterization of D-DT-1 elucidates the binding features of 1 and reveals previously unrecognized differences between the MIF and D-DT active sites that explain the ligand's functional selectivity. The commercial availability, low cost, and high selectivity make 1 the ideal tool for studying the pathophysiological functionality of D-DT in disease models. At the same time, our comprehensive biochemical, computational, and crystallographic analyses serve as a guide for generating highly potent and selective D-DT inhibitors.

A Cysteine Variant at an Allosteric Site Alters MIF Dynamics and Biological Function in Homo- and Heterotrimeric Assemblies.

Macrophage migration inhibitory factor (MIF) is an inflammatory protein with various non-overlapping functions. It is not only conserved in mammals, but it is found in parasites, fish, and plants. Human MIF is a homotrimer with an enzymatic cavity between two subunits with Pro1 as a catalytic base, activates the receptors CD74, CXCR2, and CXCR4, has functional interactions in the cytosol, and is reported to be a nuclease. There is a solvent channel down its 3-fold axis with a recently identified gating residue as an allosteric site important for regulating, to different extents, the enzymatic activity and CD74 binding and signaling. In this study we explore the consequence of converting the allosteric residue Tyr99 to cysteine (Y99C) and characterize its crystallographic structure, NMR dynamics, stability, CD74 function, and enzymatic activity. In addition to the homotrimeric variant, we develop strategies for expressing and purifying a heterotrimeric variant consisting of mixed wild type and Y99C for characterization of the allosteric site to provide more insight.

MIF but not MIF-2 recruits inflammatory macrophages in an experimental polymicrobial sepsis model.

Excessive inflammation drives the progression from sepsis to septic shock. Macrophage migration inhibitory factor (MIF) is of interest because MIF promoter polymorphisms predict mortality in different infections, and anti-MIF antibody improves survival in experimental models when administered 8 hours after infectious insult. The recent description of a second MIF superfamily member, D-dopachrome tautomerase (D-DT/MIF-2), prompted closer investigation of MIF-dependent responses. We subjected Mif-/- and Mif-2-/- mice to polymicrobial sepsis and observed a survival benefit with Mif but not Mif-2 deficiency. Survival was associated with reduced numbers of small peritoneal macrophages (SPMs) that, in contrast to large peritoneal macrophages (LPMs), were recruited into the peritoneal cavity. LPMs produced higher quantities of MIF than SPMs, but SPMs expressed higher levels of inflammatory cytokines and the MIF receptors CD74 and CXCR2. Adoptive transfer of WT SPMs into Mif-/- hosts reduced the protective effect of Mif deficiency in polymicrobial sepsis. Notably, MIF-2 lacks the pseudo-(E)LR motif present in MIF that mediates CXCR2 engagement and SPM migration, supporting a specific role for MIF in the recruitment and accumulation of inflammatory SPMs.

The N-terminus of MIF regulates the dynamic profile of residues involved in CD74 activation.

Macrophage migration inhibitory factor (MIF) is an immunomodulatory protein with a pathogenic activity in various inflammatory disorders, autoimmune diseases, and cancer. The majority of MIF-triggered pathological conditions are associated with activation of the cell surface receptor CD74. In the absence of small molecule antagonists that directly target CD74, MIF variants and MIF-ligand complexes have served as modulators of CD74 activity. These molecules have been reported to have either antagonistic or agonistic effects against the receptor, although the mechanistic parameters that distinguish the two groups are largely unknown. Through molecular dynamics simulations and NMR experiments, we explored the relationship between MIF's catalytically active N-terminus and the surface residues important for the activation of CD74. We found that the two sites are connected via backbone dynamics that are propagated to the CD74 activation surface of MIF, from the ß2 and ß4 strands. Our results also provide mechanistic evidence that explain the functional characteristics of MIF variants, serving as CD74 agonists or antagonists. Such findings are of high importance for understanding the MIF-induced activation of CD74 as well as for the development of highly potent CD74 therapeutics.

The N-terminal length and side-chain composition of CXCL13 affect crystallization, structure and functional activity.

CXCL13 is the cognate chemokine agonist of CXCR5, a class A G-protein-coupled receptor (GPCR) that is essential for proper humoral immune responses. Using a `methionine scanning' mutagenesis method on the N-terminus of CXCL13, which is the chemokine signaling region, it was shown that minor length alterations and side-chain substitutions still result in CXCR5 activation. This observation indicates that the orthosteric pocket of CXCR5 can tolerate these changes without severely affecting the activity. The introduction of bulk on the ligand was well tolerated by the receptor, whereas a loss of contacts was less tolerated. Furthermore, two crystal structures of CXCL13 mutants were solved, both of which represent the first uncomplexed structures of the human protein. These structures were stabilized by unique interactions formed by the N-termini of the ligands, indicating that CXCL13 exhibits substantial N-terminal flexibility while the chemokine core domain remains largely unchanged. Additionally, it was observed that CXCL13 harbors a large degree of flexibility in the C-terminal extension of the ligand. Comparisons with other published structures of human and murine CXCL13 validate the relative rigidity of the core domain as well as the N- and C-terminal mobilities. Collectively, these mutants and their structures provide the field with additional insights into how CXCL13 interacts with CXCR5.

An allosteric site on MKP5 reveals a strategy for small-molecule inhibition.

The mitogen-activated protein kinase (MAPK) phosphatases (MKPs) have been considered "undruggable," but their position as regulators of the MAPKs makes them promising therapeutic targets. MKP5 has been suggested as a potential target for the treatment of dystrophic muscle disease. Here, we identified an inhibitor of MKP5 using a p38α MAPK-derived, phosphopeptide-based small-molecule screen. We solved the structure of MKP5 in complex with this inhibitor, which revealed a previously undescribed allosteric binding pocket. Binding of the inhibitor to this pocket collapsed the MKP5 active site and was predicted to limit MAPK binding. Treatment with the inhibitor recapitulated the phenotype of MKP5 deficiency, resulting in activation of p38 MAPK and JNK. We demonstrated that MKP5 was required for TGF-ß1 signaling in muscle and that the inhibitor blocked TGF-ß1-mediated Smad2 phosphorylation. TGF-ß1 pathway antagonism has been proposed for the treatment of dystrophic muscle disease. Thus, allosteric inhibition of MKP5 represents a therapeutic strategy against dystrophic muscle disease.

Regulation of MIF Enzymatic Activity by an Allosteric Site at the Central Solvent Channel.

In proteins with multiple functions, such as macrophage migration inhibitory factor (MIF), the study of its intramolecular dynamic network can offer a unique opportunity to understand how a single protein is able to carry out several nonoverlapping functions. A dynamic mechanism that controls the MIF-induced activation of CD74 was recently discovered. In this study, the regulation of tautomerase activity was explored. The catalytic base Pro1 is found to form dynamic communications with the same allosteric node that regulates CD74 activation. Signal transmission between the allosteric and catalytic sites take place through intramolecular aromatic interactions and a hydrogen bond network that involves residues and water molecules of the MIF solvent channel. Once thought to be a consequence of trimerization, a regulatory function for the solvent channel is now defined. These results provide mechanistic insights into the regulation of catalytic activity and the role of solvent channel water molecules in MIF catalysis.

Elucidating the role of an immunomodulatory protein in cancer: From protein expression to functional characterization.

Several fundamental discoveries made over the last two decades, in the field of cancer biology, have increased our understanding of the complex tumor micro- and macroenvironments. This has shifted the current empirical cancer therapies to more rationalized treatments targeting immunomodulatory proteins. From the point of identification, a protein target undergoes several interrogations, which are necessary to truly define its druggability. Here, we outline some basic steps that can be followed for in vitro characterization of a potential immunomodulatory protein target. We describe procedures for recombinant protein expression and purification including key annotations on protein cloning, expression systems, purification strategies and protein characterization using structural and biochemical approaches. For functional characterization, we provide detailed protocols for using flow-cytometric techniques in cell lines or primary cells to study protein expression profiles, proliferation, apoptosis and cell-cycle changes. This multilevel approach can provide valuable, in-depth understanding of any protein target with potential immunomodulatory effects.

A selective small-molecule inhibitor of macrophage migration inhibitory factor-2 (MIF-2), a MIF cytokine superfamily member, inhibits MIF-2 biological activity.

Cytokine macrophage migration inhibitory factor-2 (MIF-2 or D-dopachrome tautomerase) is a recently characterized second member of the MIF cytokine superfamily in mammalian genomes. MIF-2 shares pro-inflammatory and tumorigenic properties with the clinical target MIF (MIF-1), but the precise contribution of MIF-2 to immune physiology or pathology is unclear. Like MIF-1, MIF-2 has intrinsic keto-enol tautomerase activity and mediates biological functions by engaging the cognate, common MIF family receptor CD74. Evidence that the catalytic site of MIF family cytokines has a structural role in receptor binding has prompted exploration of tautomerase inhibitors as potential biological antagonists and therapeutic agents, although few catalytic inhibitors inhibit receptor activation. Here we describe the discovery and biochemical characterization of a selective small-molecule inhibitor of MIF-2. An in silico screen of 1.6 million compounds targeting the MIF-2 tautomerase site yielded several hits for potential catalytic inhibitors of MIF-2 and identified 4-(3-carboxyphenyl)-2,5-pyridinedicarboxylic acid (4-CPPC) as the most functionally potent compound. We found that 4-CPPC has an enzymatic IC50 of 27 µm and 17-fold selectivity for MIF-2 versus MIF-1. An in vitro binding assay for MIF-1/MIF-2 to the CD74 ectodomain (sCD74) indicated that 4-CPPC inhibits MIF-2-CD74 binding in a dose-dependent manner (0.01-10 µm) without influencing MIF-1-CD74 binding. Notably, 4-CPPC inhibited MIF-2-mediated activation of CD74 and reduced CD74-dependent signal transduction. These results open opportunities for development of more potent and pharmacologically auspicious MIF-2 inhibitors to investigate the distinct functions of this MIF family member in vivo.

Expression, purification and crystallization of the novel Xenopus tropicalis ALDH16B1, a homologue of human ALDH16A1.

ALDH16 is a novel family of the aldehyde dehydrogenase (ALDH) superfamily with unique structural characteristics that distinguish it from the other ALDH superfamily members. In addition to structural characteristics, there is an evolutionary-related grouping within the ALDH 16 genes. The ALDH16 isozymes in frog, lower animals, and bacteria possess a critical Cys residue in their active site, which is absent from ALDH16 in mammals and fish. Genomic analysis and plasma metabolomic studies have associated ALDH16A1 with the pathogenesis of gout in humans, although its actual involvement in this disease is poorly understood. Insight into the structure of ALDH16A1 is an important step in deciphering its function in gout. Herein, we report our efforts towards the structural characterization of Xenopus tropicalis ALDH16B1 (the homolog of human ALDH16A1) that was predicted to be catalytically-active. Recombinant ALDH16B1 was expressed in Sf9 cells and purified using affinity and size exclusion chromatography. Crystallization of ALDH16B1 was achieved by vapor diffusion. A data set was collected at 2.5 Šand preliminary crystallographic analysis showed that the frog ALDH16B1 crystals belong to the P 212 121 space group with unit cell parameters a = 80.48 Å, b = 89.73 Å, c = 190.92 Å, α = ß = γ = 90.00°. Structure determination is currently in progress.

Structural Plasticity in the C-Terminal Region of Macrophage Migration Inhibitory Factor-2 Is Associated with an Induced Fit Mechanism for a Selective Inhibitor.

We report the first reversible and selective small molecule inhibitor of pro-inflammatory protein macrophage migration inhibitory factor-2 (also known as MIF-2 or d-DT). 4-(3-Carboxyphenyl)-2,5-pyridinedicarboxylic acid (4-CPPC) shows competitive binding with a 13-fold selectivity for human MIF-2 versus human MIF-1. The crystal structure of MIF-2 complexed with 4-CPPC reveals an induced fit mechanism that is not observed in the numerous MIF-1/inhibitor complexes. Crystallographic analysis demonstrates the structural source of 4-CPPC binding and selectivity for MIF-2. 4-CPPC can be employed to reveal previously unrecognized functions of MIF-1 in biological systems in which both MIF-1 and MIF-2 are expressed, to improve our knowledge of the MIF family of proteins, and to provide new mechanistic insights that can be utilized for the development of potent and selective pharmacological modulators of MIF-2.

Nanosecond Dynamics Regulate the MIF-Induced Activity of CD74.

Macrophage migration inhibitory factor (MIF) activates CD74, which leads to severe disorders including inflammation, autoimmune diseases and cancer under pathological conditions. Molecular dynamics (MD) simulations up to one microsecond revealed dynamical correlation between a residue located at the opening of one end of the MIF solvent channel, previously thought to be a consequence of homotrimerization, and residues in a distal region responsible for CD74 activation. Experiments verified the allosteric regulatory site and identified a pathway to this site via the MIF ß-strands. The reported findings provide fundamental insights on a dynamic mechanism that controls the MIF-induced activation of CD74.