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. Supplementary Information The online version contains supplementary material available at 10.1186/s10020-024-00803-0.


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Page 2 Full wwPDB X-ray Structure Validation Report (*For Manuscript Review*) 8SPN 1 Overall quality at a glance i ○ The following experimental techniques were used to determine the structure: X-RAY DIFFRACTION The reported resolution of this entry is 1.73 Å.
Percentile scores (ranging between 0-100) for global validation metrics of the entry are shown in the following graphic.The table shows the number of entries on which the scores are based.The table below summarises the geometric issues observed across the polymeric chains and their fit to the electron density.The red, orange, yellow and green segments of the lower bar indicate the fraction of residues that contain outliers for >=3, 2, 1 and 0 types of geometric quality criteria respectively.A grey segment represents the fraction of residues that are not modelled.

Metric
The numeric value for each fraction is indicated below the corresponding segment, with a dot representing fractions <=5% The upper red bar (where present) indicates the fraction of residues that have poor fit to the electron density.The numeric value is given above the bar.The following

Entry composition i ○
There are 6 unique types of molecules in this entry.The entry contains 3046 atoms, of which 0 are hydrogens and 0 are deuteriums.
In the tables below, the ZeroOcc column contains the number of atoms modelled with zero occupancy, the AltConf column contains the number of residues with at least one atom in alternate conformation and the Trace column contains the number of residues modelled with at most 2 atoms.
• Molecule 1 is a protein called Macrophage migration inhibitory factor.3 Residue-property plots i ○ These plots are drawn for all protein, RNA, DNA and oligosaccharide chains in the entry.The first graphic for a chain summarises the proportions of the various outlier classes displayed in the second graphic.The second graphic shows the sequence view annotated by issues in geometry and electron density.Residues are color-coded according to the number of geometric quality criteria for which they contain at least one outlier: green = 0, yellow = 1, orange = 2 and red = 3 or more.A red dot above a residue indicates a poor fit to the electron density (RSRZ > 2).Stretches of 2 or more consecutive residues without any outlier are shown as a green connector.Residues present in the sample, but not in the model, are shown in grey.

Mol Chain Residues Atoms ZeroOcc AltConf Trace
• Molecule 1: Macrophage migration inhibitory factor Chain A: • Molecule 1: Macrophage migration inhibitory factor Chain B: • Molecule 1: Macrophage migration inhibitory factor Chain C: There are no bond angle outliers.
There are no chirality outliers.
There are no planarity outliers.

Too-close contacts i ○
In the following table, the Non-H and H(model) columns list the number of non-hydrogen atoms and hydrogen atoms in the chain respectively.The H(added) column lists the number of hydrogen atoms added and optimized by MolProbity.The Clashes column lists the number of clashes within the asymmetric unit, whereas Symm-Clashes lists symmetry-related clashes.The all-atom clashscore is defined as the number of clashes found per 1000 atoms (including hydrogen atoms).The all-atom clashscore for this structure is 2.

Mol Chain Non-H H(model) H(added) Clashes Symm-Clashes
All (12) close contacts within the same asymmetric unit are listed below, sorted by their clash magnitude.
Atom There are no symmetry-related clashes.

Protein backbone i ○
In the following table, the Percentiles column shows the percent Ramachandran outliers of the chain as a percentile score with respect to all X-ray entries followed by that with respect to entries of similar resolution.
The Analysed column shows the number of residues for which the backbone conformation was analysed, and the total number of residues.There are no Ramachandran outliers to report.

Protein sidechains i ○
In the following table, the Percentiles column shows the percent sidechain outliers of the chain as a percentile score with respect to all X-ray entries followed by that with respect to entries of similar resolution.
The Analysed column shows the number of residues for which the sidechain conformation was analysed, and the total number of residues.There are no protein residues with a non-rotameric sidechain to report.

Mol
Sometimes sidechains can be flipped to improve hydrogen bonding and reduce clashes.All (2) such sidechains are listed below:

RNA i ○
There are no RNA molecules in this entry.

Non-standard residues in protein, DNA, RNA chains i ○
There are no non-standard protein/DNA/RNA residues in this entry.

Carbohydrates i ○
There are no monosaccharides in this entry.

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Page 12 Full wwPDB X-ray Structure Validation Report (*For Manuscript Review*) 8SPN

Ligand geometry i ○
5 ligands are modelled in this entry.
In the following The following is a two-dimensional graphical depiction of Mogul quality analysis of bond lengths, bond angles, torsion angles, and ring geometry for all instances of the Ligand of Interest.In addition, ligands with molecular weight > 250 and outliers as shown on the validation Tables will also be included.For torsion angles, if less then 5% of the Mogul distribution of torsion angles is within 10 degrees of the torsion angle in question, then that torsion angle is considered an outlier.
Any bond that is central to one or more torsion angles identified as an outlier by Mogul will be highlighted in the graph.For rings, the root-mean-square deviation (RMSD) between the ring in question and similar rings identified by Mogul is calculated over all ring torsion angles.If the average RMSD is greater than 60 degrees and the minimal RMSD between the ring in question and any Mogul-identified rings is also greater than 60 degrees, then that ring is considered an outlier.The outliers are highlighted in purple.The color gray indicates Mogul did not find sufficient equivalents in the CSD to analyse the geometry.

Other polymers i ○
There are no such residues in this entry.

Polymer linkage issues i ○
There are no chain breaks in this entry.

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Page 15 Full wwPDB X-ray Structure Validation Report (*For Manuscript Review*) 8SPN 6 Fit of model and data i ○ 6.1 Protein, DNA and RNA chains i ○ In the following table, the column labelled '#RSRZ> 2' contains the number (and percentage) of RSRZ outliers, followed by percent RSRZ outliers for the chain as percentile scores relative to all X-ray entries and entries of similar resolution.The OWAB column contains the minimum, median, 95 th percentile and maximum values of the occupancy-weighted average B-factor per residue.The column labelled 'Q< 0.9' lists the number of (and percentage) of residues with an average occupancy less than 0.9.There are no non-standard protein/DNA/RNA residues in this entry.

Carbohydrates i ○
There are no monosaccharides in this entry.

Ligands i ○
In the following table, the Atoms column lists the number of modelled atoms in the group and the number defined in the chemical component dictionary.The B-factors column lists the minimum, median, 95 th percentile and maximum values of B factors of atoms in the group.The column labelled 'Q< 0.9' lists the number of atoms with occupancy less than 0.9.The following is a graphical depiction of the model fit to experimental electron density of all instances of the Ligand of Interest.In addition, ligands with molecular weight > 250 and outliers as shown on the geometry validation Tables will also be included.Each fit is shown from different orientation to approximate a three-dimensional view.
Electron density around 7TN C 201: 2mF o -DF c (at 0.7 rmsd) in gray mF o -DF c (at 3 rmsd) in purple (negative) and green (positive)

Other polymers i ○
There are no such residues in this entry.
table lists non-polymeric compounds, carbohydrate monomers and non-standard residues in protein, DNA, RNA chains that are outliers for geometric or electron-density-fit criteria:

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• Molecule 5 is Iguratimod (three-letter code: 7TN) (formula: C 17 H 14 N 2 O 6 S) (labeled as "Ligand of Interest" by depositor).F

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e wBond lengths and bond angles in the following residue types are not validated in this section: SO4, 7TN, GOL, IPAThe Z score for a bond length (or angle) is the number of standard deviations the observed value is removed from the expected value.A bond length (or angle) with |Z| > 5 is considered an outlier worth inspection.RMSZ is the root-mean-square of all Z scores of the bond lengths (or angles).
table, the Counts columns list the number of bonds (or angles) for which Mogul statistics could be retrieved, the number of bonds (or angles) that are observed in the model and the number of bonds (or angles) that are defined in the Chemical Component Dictionary.The Link column lists molecule types, if any, to which the group is linked.The Z score for a bond length (or angle) is the number of standard deviations the observed value is removed from the expected value.A bond length (or angle) with |Z| > 2 is considered an outlier worth inspection.RMSZ is the root-mean-square of all Z scores of the bond lengths (or angles).In the following table, the Chirals column lists the number of chiral outliers, the number of chiral centers analysed, the number of these observed in the model and the number defined in the Chemical Component Dictionary.Similar counts are reported in the Torsion and Rings columns.'-' means no outliers of that kind were identified.

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Continued on next page... Continued from previous page... Mol Type Chain Res Atoms RSCC RSR B-factors(Å 2 ) Q<0.9 Mol Type Chain Res Atoms RSCC RSR B-factors(Å 2 ) Q<0.9