WO2011066864A1 - Method and compositions for inhibition of macrophage migration inhibitory factor (mif) - Google Patents

Abstract

The present invention relates to methods for inhibition of tautomerase activity of macrophage migration inhibitory factor (MIF). The invention further relates to substances for irreversibly covalently binding to MIF for inhibition of tautomerase activity.

Description

Method and Compositions for inhibition of macrophage migration inhibitory factor (MIF)

The present invention relates to methods and compositions for inhibition of tautomerase activity of macrophage migration in^¬ hibitory factor (MIF) . The invention further relates to substances for irreversibly binding to MIF for inhibition of tautomerase activity.

Macrophage migration inhibitory factor (MIF) is a homotrimeric multifunctional proinflammatory cytokine that has been impli^¬ cated in the pathogenesis of several inflammatory and autoimmune diseases. MIF is expressed in a variety of cells and has been implicated in a wide range of cellular activities, including transcriptional regulation of inflammatory gene products, regu^¬ lation of glucocorticoid activity, cell cycle control by promot^¬ ing cell proliferation and survival through activation of the ERK1/2 MAPKs, the inhibition of the p53 and retinoblastoma/E2F tumor suppressor pathways and the activation the phosphoinosi- tide-3-kinase (PI3K) /Akt survival pathway. Several lines of evi^¬ dence support a central role for MIF in the pathogenesis of in^¬ flammatory and autoimmune diseases including arthritis, glomeru^¬ lonephritis, diabetes, colitis, sepsis, acute respiratory dis^¬ tress syndrome and cancer.

Therefore, targeting MIF activity has emerged as a therapeutic approach for treating these inflammatory and autoimmune diseases .

In addition to its reported biological activities, MIF is known to catalyze the tautomerization of phenylpyruvate (or hydroxyl- phenylpyruvate) and D-dopachrome methyl ester. Initial attempts to modulate the activity of MIF and elucidate its role in disease focused on the administration of neutralizing anti-MIF antibodies in several models of inflammatory dis^¬ eases (Leung, J.C., et al . , Anti-macrophage migration inhibitory factor reduces transforming growth factor-beta 1 expression in experimental IgA nephropathy. Nephrol Dial Transplant, 2004. 19(8): p. 1976-85. Huang, X.R., et al . , Macrophage migration in^¬ hibitory factor is an important mediator in the pathogenesis of gastric inflammation in rats. Gastroenterology, 2001. 121(3): p. 619-30. Kitaichi, N., et al . , Inhibition of experimental autoimmune uveoretinitis with anti-macrophage migration inhibitory factor antibodies. Curr Eye Res, 2000. 20(2): p. 109-14.).

Still, neutralization of MIF with anti-MIF antibodies might not be sufficient or specific enough for therapeutic or diagnostic purposes .

In the course of the present invention the following abbrevia^¬ tions are used:

MIF, macrophage migration inhibitory factor

ITC, Isothiocyanate

BITC, Benzyl isothiocyanate;

AITC, allyl isothiocyamate ;

EITC, ethyl isothiocyanate;

MITC, methallyl isothiocyanate ;

2-2PITC, Piperidinoethyl isothiocyanate;

CPITC, Cyclopropyl isothiocyanate;

PEITC, Phenylethyl isothiocyanate;

CD, circular dichroism;

SEC, size-exclusion chromatography;

AUC, analytical ultracentrifugation;

MALDI, matrix-assisted laser desorption ionization; HSQC, heteronuclear single quantum coherence;

Wt, wild-type.

All further terminology follows common standardized language as used and known in the art unless otherwise specified.

It is thus an object of the present invention to overcome the above mentioned drawbacks of the prior art, i.e. to provide a method for inhibition of tautomerase activity of macrophage mi^¬ gration inhibitory factor (MIF) that is not limited by the above-mentioned drawbacks and which especially allows for bioas- says for ratios of active/inactive rather than total MIF; thus providing for better diagnostic biomarkers for inflammatory and autoimmune diseases. Further objects of the invention will be^¬ come apparent to a person of routine skill in the art in view of the following detailed description of the invention.

This object and yet further objects are achieved inter alia by a composition for inhibition of tautomerase activity of macrophage inhibition factor (MIF) . Said composition comprising at least one compound selected from the group of

R-N=C=S (Formula I) mula II)

(Formula III)

(Formula IV) 

wherein R and R' are independently selected from the group con^¬ sisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl, substituted acylalkyl, heterocycle, substituted heterocycle, substi^¬ tuted cycloalkyl, hydrogen and/or halogen and the molar mass of the compound does not exceed 500 g/mol.

The carbon moieties comprise from 1 to 20 Carbon moieties.

Also provided are pharmaceutically-acceptable derivatives, in^¬ cluding salts, esters, enol ethers, enol esters, solvates, hy^¬ drates and prodrugs of the compounds described herein. Further provided are pharmaceutical compositions containing the com^¬ pounds provided herein and a pharmaceutically acceptable car^¬ rier .

In the context of the present invention "reacting with" is to be understood as any process that affects the enzymatic activity of MIF. Such can be achieved by either binding, covalently or non- covalently, or modifying, either structurally or chemically by association and/or dissociation MIF with a molecule according to the present invention.

One aspect of the present invention achieves MIF inhibition by reacting the N-terminal Prol of MIF with an isothiocyanate group of another molecule. Preferably this other molecule is a small aliphatic or aromatic molecule. A small molecule according to the present invention is a molecule whose molar mass is equal or less than 500 g/mol. Preferably the molar mass is between 1 g/mol and 400 g/mol. Inhibition in the present invention is defined as a decrease of biological activity of a particular enzyme. A successful inhibi^¬ tion in the context of the present invention takes place when a given biological process is inhibited by half.

It has surprisingly been found that different classes of small molecules can selectively and covalently modify the active form of MIF by reacting with the N-terminal proline.

Isothiocyanates comprise compounds of the general formula R- N=C=S (Formula I) . Wherein R can be a substituent chosen from the group of aliphatic or aromatic compounds, for example R can be selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl, substituted acylalkyl, heterocycle, substituted heterocycle, substituted cycloalkyl, hydrogen, halo^¬ gen, etc.

In a preferred embodiment of the present invention, the method includes the step of reacting the macrophage migration inhibi^¬ tory factor with Benzyl isothiocyanate (BITC) .

(Formula I .1 )

It has surprisingly been found that BITC binds selectively to MIF. A further unexpected result was that BITC was found only modify catalytically active forms of MIF.

In a further preferred embodiment, the method includes the step of reacting the macrophage migration inhibitory factor with one of the group of allyl isothiocyamate (AITC) , ethyl isothiocy- anate (EITC) , methallyl Isothiocyanate (MITC) , 2-Piperidinoethyl isothiocyanate (2PITC) , cyclopropyl isothiocyanate (CPITC) or Phenylethyl isothiocyanate (PEITC) .

In a further aspect of the present invention, a method of inhib^¬ iting tautomerase activity of macrophage inhibition factor (MIF) is shown by reacting the N-terminal Prol of MIF with a compound chosen from the group of: ormula II)

(Formula III)

S O

^{H H} (Formula IV)

(Formula V) wherein R and R' can be independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl, substituted acylalkyl, heterocycle, substituted heterocycle, sub^¬ stituted cycloalkyl, hydrogen, halogen, etc.

In a preferred embodiment of this aspect of the present inven^¬ tion, a method of inhibiting tautomerase activity of macrophage inhibition factor (MIF) is shown by reacting the N-terminal Prol of MIF with a compound chosen from the group of: N-(2,4 dinitro- phenoxy) thiophene-2 -carboxamide ; N- (2-chlorophenyl) - ' - [ (5- nitro-3 hienyl ) carbonyl ] thiourea, N'-[ (3-acetyl-l,3-thiazolan-2- yl) carbonyl] -1, 3-benzodioxole-5-carbohydrazide, 7, 10-dioxo-4, 5- dihydro-7H, 10H-pyrano[3,2-c]pyrrolo[3,2,l-ij]quinolin-8-yl 4- fluorobenzoate ; 1 , 4-dioxo-l , 4-dihydronaphthalen-2-yl 4- methylbenzoate, 2-oxo-l-phenyl-l , 2-dihydroquinolin-4-yl benzo- ate, 2-chloro-3-oxocyclohex-l-enyl 3- (trifluoromethyl) benzoate, 3- (4-methoxyphenyl) -3-oxoprop-l-enyl 4-chlorobenzoate, 3-cyano- 4 , 6-dimethyl-2-pyridyl thiophene-2-carboxylate, 2- (2 , 3-dihydro- 1, 3-benzothiazol-2-yliden) -3- (2-furyl) -3-oxopropanenitrile, 5- oxo-2, 3-dihydro-lH, 5H-pyrido [3,2, 1-ij ] quinolin-7-yl benzoate and/or 5, 5-dimethyl-3-oxocyclohex-l-enyl 4-chlorobenzoate.

Formula IX: N-(2,4 dinitrophenoxy) thiophene-2-carboxamide

Formula X: N- (2-chlorophenyl) - '-[ (5-nitro-3

thienyl) carbonyl] thiourea

Formula XI: N '-[ (3-acetyl-l , 3-thiazolan-2-yl) carbonyl ] -1 , 3- benzodioxole-5-carbohydrazide

Formula XII: 7, 10-dioxo-4, 5-dihydro-7H, lOH-pyrano [3, 2- c]pyrrolo[3,2,l-ij]quinolin-8-yl 4-fluorobenzoate

Formula XII.2: 1 , 4-dioxo-l , 4-dihydronaphthalen-2-yl 4 methylben- zoate

Formula XIII: 2-oxo-l-phenyl-l , 2-dihydroquinolin-4-yl benzoate

Formula XIV: 2-chloro-3-oxocyclohex-l-enyl 3- ( trifluoromethyl) benzoate

Formula XV: 3- (4-methoxyphenyl) -3-oxoprop-l-enyl 4- chlorobenzoate

Formula XVI: 3-cyano-4 , 6-dimethyl-2-pyridyl thiophene-2- carboxylate

Formula XVII: 2- (2 , 3-dihydro-l , 3-benzothiazol-2-yliden) -3- (2- furyl) -3-oxopropanenitrile

Formula XVIII : 5-oxo-2, 3-dihydro-lH, 5H-pyrido [3,2, 1-ij ] quinolin- 7-yl benzoate

Formula XIX: 5, 5-dimethyl-3-oxocyclohex-l-enyl 4-chlorobenzoate

A further aspect of the present invention is a method of inhib^¬ iting tautomerase activity of macrophage inhibitory factor (MIF) with a compound capable of forming a selenodisulfide bridge. Preferrably the selenodisulfide bridge is formed with a Cysteine of MIF, more preferably with Cys81 of MIF. In a preferred embodiment the selendisulfide bridge is formed by reacting a compound selected from the group consisting of the

Formula XVII, preferably by reacting a compound of the Formula XIII with the MIF.

(Formula XVII)

R can be independently selected from the group consisting of hy^¬ drogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, ary- lalkyl, substituted arylalkyl, acylalkyl, substituted acylalkyl, heterocycle, substituted heterocycle, substituted cycloalkyl, hydrogen, halogen, etc.

A further aspect of the present invention is a method of inhib^¬ iting tautomerase activity of macrophage inhibitory factor (MIF) by reacting MIF with a compound selected from the group consist^¬ ing of:

(Formula VII) R is independently chosen from the group of hydrogen, halogen, preferably chlorine, oxygen, hydroxide, nitrogen, amide, amino, carboxyl or a branched or linear carbon group (from Ci to Ce, preferably from Ci to C5, even more preferably from Ci to C3) . In a preferred embodiment, R is either chlorine or hydrogen. X is chosen from the group of carbon, sulphur, phosphor, selenium, oxygen or nitrogen. Preferably X is either carbon or sulphur.

Preferably the step is reacting MIF with a compound selected from the group of hexachlorophenes.

In a further preferred embodiment MIF is reacted with:

(Formula XXI)

In a further preferred embodiment MIF is reacted with:

I

(Formula XXII)

In a further preferred embodiment MIF is reacted with:

(Formula XXIII) In a further aspect of the present invention relates to a method of inhibiting tautomerase activity of macrophage inhibitory fac^¬ tor (MIF) by reacting MIF with a compound chosen from the group of: Nl- [3- (trifluoromethyl) phenyl] -3- (2-chloroanilino) -2-cyano- 3-thioxopropanamide, N ' ' , ' ' ' -di (4-hydroxybenzylidene) carbonic di-hydrazide, 6- {morpholino [4- (trifluoromethyl) phenyl] methyl } - 1,3- benzodioxol-5-ol .

Formula VIII.1: Nl- [3- (trifluoromethyl) phenyl] -3- (2- chloroanilino) -2-cyano-3-thioxopropanamide

Formula VIII .3 : 6- {morpholino [4- (trifluoromethyl) phenyl] methyl } - 1,3- benzodioxol-5-ol

Another aspect of the present invention is a substance for the treatment of a macrophage migration inhibitory factor (MIF) related disease chosen from the group of Formula I to Formula XXIII .

The substance may be used in a pharmaceutical composition either as sole active ingredient or in combination with other sub^¬ stances. Further additives may be added.

Suitable routes of administration may, for example, include topical, cutaneous, oral, rectal, transmucosal , or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intrana^¬ sal, or intraocular injections, and optionally in a depot or sustained release formulation.

Depending on the route of administration additives can be dimethyl sulfoxide, menthol, lauryl alcohol, lauric acid, arachi- donic acid and Cio -C20 polyhydroxy acids, thymol, physiologically compatible buffers, such as Hank's solution, Ringer's solution, or physiological saline buffer, sugars, including lactose, su^¬ crose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypro- pylmethylcellulose, sodium carboxymethylcellulose, polyvinylpyr^¬ rolidone (PVP) , gum arabic, talc, polyvinyl pyrrolidone, car- bopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Further the substance can be embedded in a pharmaceutically ac^¬ ceptable carrier.

Furthermore, one may administer a compound of the present inven^¬ tion in a targeted drug delivery system, for example in a lipo^¬ some. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.

Pharmaceutically compatible salts may be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc.; or bases. Examples of pharmaceutically acceptable salts, carriers or excipients are well known to those skilled in the art. Such salts include, but are not limited to, sodium, potassium, lithium, calcium, magnesium, iron, zinc, hydrochloride, hydrobromide, hydroiodide, ace^¬ tate, citrate, tartrate and malate salts, and the like.

In a preferred embodiment the substance or pharmaceutical compo^¬ sition comprising the substance is used for the treatment of an inflammatory disease.

In another preferred embodiment the substance or pharmaceutical composition comprising the substance is used for the treatment of an autoimmune disease.

In yet another preferred embodiment the substance or pharmaceu^¬ tical composition comprising the substance is used for the treatment of a disease from the group consisting of sepsis, rheumatoid arthritis or multiple sclerosis. Another aspect of the present invention is a method for treat^¬ ment or prophylaxis of a macrophage migration inhibitory factor related disease, wherein a pharmaceutically effective dosage of a substance according to the invention is administered to a mam^¬ mal .

A pharmaceutical effective dosage according to the present in^¬ vention means a dosage composition where the active ingredients are contained in an effective amount to achieve their intended purpose. More specifically, a therapeutically effective amount means an amount effective to prevent or inhibit development or progression of a disease related to MIF. Determination of the effective amounts is well within the capability of those skilled in the art.

The therapeutically effective dose can, for example, be esti^¬ mated initially from tautomerase inhibition assays and cell cul^¬ ture assays. Such information can then be used to more accu^¬ rately determine useful doses in humans.

Toxicity and therapeutic efficacy of such compounds can be de^¬ termined by standard pharmaceutical, pharmacological, and toxi- cological procedures in cell cultures or experimental animals, e.g., for determining the LD₅ o (the dose lethal to 50% of the population) and the ED₅ o (the dose therapeutically effective in 50% of the population) . The dose ratio between toxic and thera^¬ peutic effects is the therapeutic index and it can be expressed as the ratio between LD₅ o and ED₅ o . The data obtained from cell culture assays or animal studies can be used in formulating a range of dosage for use in humans. A further aspect of the present invention is a method for deter^¬ mining the concentration of macrophage migration inhibitory factor. A chemical group or chemical compound that is known to co- valently bind specifically to catalytically active MIF is at^¬ tached to a first support structure. Subsequently said first support structure is contacted with a sample containing MIF for a sufficiently long time and under suitable conditions to allow for covalent binding of the chemical group or chemical compound to MIF. The remainder of the sample is then removed from the first support structure and moved to a second support structure. Finally, the concentration of catalytically active MIF is deter^¬ mined by analyzing the support structure. The concentration of catalytically inactive MIF can be determined by analysing the second support structure. The overall concentration of MIF can be determined from the analysis of both the first and the second support structure.

With this step of immobilizing a chemical group or chemical com^¬ pound, for example, a surface of a multi-well plate can be func- tionalized. A microtiter plate can be used as first support sur^¬ face for example, usually but not necessarily such a microtiter plate is made of polystyrene. The chemical group or chemical compound can be attached either non-specifically (via adsorption to the surface) or specifically (via capture by an antibody) .

The contacting with a sample containing MIF and the removing of the remainder of the sample can be performed by robots, espe^¬ cially robots adapted for aspirating and dispensing of the liq^¬ uids. Robots for handling processes and microtiter plates are known in the art and readily available through commercial chan^¬ nels. Incubation times can be varied upon discretion of the person skilled in the art depending on temperature. In practice, 2h at room temperature have proven to be effective, for example.

Eventually, a wash step may be included. Washing of the surface can be performed, for example, by saline buffer with a low per^¬ centage of detergent to remove any unbound material. PBST phos^¬ phate buffer saline with 0.05% Tween-20 has proven to be effec^¬ tive in practice.

Analysis of the concentration of active and/or inactive MIF on the respective support structure can be performed by conven- tionial photospectroscopy . Alternatively radiological, electro^¬ chemical or fluoroscopical detection can be used. A substrate which is converted by an enzyme to elicit a chromogenic or fluorogenic or electrochemical signal can be further added. Al^¬ ternatively, magnetic beads where a magnetic label is conjugated to one element can be used. The presence of magnetic beads is then detected by a magnetic reader (magnetometer) which measures the magnetic field change induced by the beads. The signal meas^¬ ured by the magnetometer is proportional to the analyte quantity in the initial sample.

For example a spectrophotometer, spectrofluorometer, or other optical/electrochemical device can be used. Determination of concentration can be achieved by referencing with a blind probe for example.

A conventional plate reader adapted for detection of the respec^¬ tive signal can be used, for example adapted or adaptable for absorbance, fluorescence intensity, luminescence, time-resolved fluorescence, and fluorescence polarization. In a preferred embodiment an isothiocyanate group or a compund comprising an isothiocyanate group is attached to the first sup^¬ port structure.

In a further preferred embodiment a chemical compound chosen from the group of: N-(2,4 dinitrophenoxy) thiophene-2- carboxamide ; N- (2-chlorophenyl) -N'-[(5-nitro-3

hienyl ) carbonyl ] thiourea, N'-[ (3-acetyl-l,3-thiazolan-2- yl) carbonyl] -1, 3-benzodioxole-5-carbohydrazide, 7, 10-dioxo-4, 5- dihydro-7H, 10H-pyrano[3,2-c]pyrrolo[3,2,l-ij]quinolin-8-yl 4- fluorobenzoate ; 1 , 4-dioxo-l , 4-dihydronaphthalen-2-yl 4- methylbenzoate, 2-oxo-l-phenyl-l , 2-dihydroquinolin-4-yl benzo- ate, 2-chloro-3-oxocyclohex-l-enyl 3- (trifluoromethyl) benzoate, 3- (4-methoxyphenyl) -3-oxoprop-l-enyl 4-chlorobenzoate, 3-cyano- 4 , 6-dimethyl-2-pyridyl thiophene-2-carboxylate, 2- (2 , 3-dihydro- 1, 3-benzothiazol-2-yliden) -3- (2-furyl) -3-oxopropanenitrile, 5- oxo-2, 3-dihydro-lH, 5H-pyrido [3,2, 1-ij ] quinolin-7-yl benzoate and/or 5, 5-dimethyl-3-oxocyclohex-l-enyl 4-chlorobenzoate is attached to the first support structure.

In a further preferred embodiment the determining of analyte concentration either in the first support structure, or the sec^¬ ond support structure or both involves an ELISA (Enzyme-linked immunosorbent assay) or ELISA like assay.

A further aspect of the present invention is a method of diag^¬ nosing a macrophage migration inhibitory factor (MIF) related disease wherein a method as previously described is carried out.

DETAILED DESCRIPTION OF THE INVENTION The invention will now be described in even more detailed by means of specific examples and methods; however, without the in^¬ vention being limited thereto.

Fig.l: the chart shows MIF tautomerase activity inhibition by isothiocyanate .

Fig.2: the chart shows, that only active forms of MIF undergo covalent modification by BITC.

Fig.3: the chart shows the sedimentation rate of MIF in the presence or absence of BITC.

Fig.4A to 4E: a chart showing ITC based inhibition of MIF tautomerase activity. 4A: BITC, 4B: MITC, 4C: AITC, 4E: PITC, 4D: EITC.

Fig. 5: a chart of BITC effect on MIF-mediated glucocorticoid overriding activity (5a) and Akt Phoshporylation (5b) .

Fig. 6: schematic drawing of MIF Bioassay

Fig. 7: Overriding of glucocorticoid-mediated inhibition of cy^¬ tokine production

Fig. 8: IC50 determination of ebselene Fig. 9: ebselene induced MIF aggregation

Fig. 10: NMR analysis of structural effects of ebselene, hexa- chlorophenes and compounds of the Formula VIII.1, VIII.3 and VIII .2 Fig. 11: Cysteine role in MIF-ebselen interaction

Fig. 12: Structural basis for the inhibition of MIF activity

EXAMPLE 1:

BITC modification of catalytically active forms of MIF

Disruption of the conformation of the C-terminus via truncation or insertion of secondary structure-disrupting elements (proline residues) within the C-terminal region (residues 104-115) abol^¬ ished the protein activity without alering its oligomerization state. These probes were used for determining whether BITC se^¬ lectively modifies enzymatically active MIF. Six trimeric MIF variants (3 enzymatically active: Wt, R73A and N110C and 3 inac^¬ tive: R73AA5, R73AA10 and P107) were tested by incubation with BITC. MALDI-TOF analysis revealed total modification of the en^¬ zymatically active forms of MIF (Wt, R73A and N110C) , whereas no modification was observed for inactive MIF proteins (R73AA5, R73AA10 and P107) . Fig.2 shows that only enzymatically active forms of the MIF trimer undergo covalent modification by BITC and its active analogues. Six trimeric variants were tested by incubation with 10 μΜ BITC for 60 min and the samples analyzed by MALDI-TOF/TOF.

Fig. 3 further shows that covalent modification of MIF by BITC does not affect its oligomerization state. Sedimentation veloc^¬ ity profiles of MIF in the presence or absence of BITC. MIF (10 μΜ) in PBS was

preincubated with 6 μΜ BITC for 1 h at RT . Sedimentation coefficient distributions were obtained by analysis of the sedimenta^¬ tion profiles using the C (s) distribution as a variant of Lamm equation solutions. Calculations were performed using Sedfit software (www.sedfit.com). BITC inhibits MIF-mediated glucocorticoid overriding activity and enhancement of Akt phosphorylation

Among the important in vivo immunoregulatory functions of MIF is its ability to counter-regulate the immunosuppressive effect of glucocorticoids on proinflammatory cytokine production. Human macrophages were pretreated with dexamethasone (100 nM) with or without recombinant MIF (100 ng/mL) and BITC (10 μΜ) prior to stimulation with LPS. Whereas dexamethasone inhibited TNF pro^¬ duction by LPS-stimulated monocytes, preincubation with MIF overcame dexamethasone inhibition of TNF production. BITC de^¬ creased TNF production even more potently than dexamethasone

(Fig. 5a) . Similar results were obtained using the RAW 264.7 mouse macrophage cell line.

For Fig. 5a, human macrophages were preincubated for 1 h with (+) or without (-) dexamethasone (Dex 100 nM) , MIF (100 ng/mL) and BITC (10 μΜ) before stimulation with LPS (100 ng/mL) . Cell- free supernatants were collected after 4 h to quantify the con^¬ centrations of TNF.

Data are means ± SD of triplicate samples from one experiment and are representative of four independent experiments. BITC in^¬ hibits MIF-induced activation of Akt phosphorylation. HeLa cells were incubated for 2 h with 50 ng/mL MIF in the presence or the absence of 1-10 μΜ BITC and LY294002 (a selective inhibitor of the PI3K/Akt pathway) .

In Fig. 5b Akt phosphorylation was quantified using the Alpha screen SureFire phosphokinase KIT as described in Materials and Methods. Data are expressed as Alpha screen counts per mg of to^¬ tal protein means ± SD of triplicate samples from one experiment and representative of 2 independent experiments.

Inhibition of tautomerase activity Several isothiocyanate based compounds inhibit MIF tautomerase activity in a concentration dependent manner (Fig.l) . IC₅₀S concentration response curve (mean +/- SD; n=8) obtained by moni^¬ toring initial velocity of HPP Keto form tautomirization to HPP enol form by MIF. MIF 100 nM was preincubated with different concentrations of ITC analogues BITC, AITC, EITC, MITC and 2PITC for 5 min, followed by the addition of 2 mM Hydroxyphenylpury- vate, then absorbance was monitored at 300 nm for 5 min. Initial velocity was calculated and normalized to percentage of activ^¬ ity.

The ITC based inhibition of tautomerase activity interferes with MIF binding to its receptor CD74, as shown in Figures 4 A to 4 E for the various ITC; Fig. 4a: Benzyl isothiocyanate (BITC) ,

Fig.4c: allyl isothiocyamate (AITC), Fig. 4e: ethyl isothiocy^¬ anate (EITC), Fig. 4b: methallyl Isothiocyanate (MITC), Fig. 4d: Piperidinoethyl isothiocyanate (PITC) . Dose-dependent inhibition by the isothiocyante derivative BITC, AITC, EITC,

MITC, and PITC. Test compounds were pre-incubated with

of 0.2 μΜ biotinylated MIF for 2h at room temperature in the dark prior to addition to 96 well plates pre-coated with recombinant human sCD74. Data points are the mean of duplicate determina^¬ tions of bound MIF.

EXAMPLE 2

Ebselene modification of catalytically active forms of MIF

To determine if ebselen inhibition of MIF is mediated by cova- lent modification of specific cystein residues; mass spectrome^¬ try was carried out on MIF samples preincubated with increasing concentrations of Ebselen (0-10 μΜ) , for 1 hour at RT . MALDI-TOF analysis revealed only a minor peak with a m/ z = 12616, which correspond to the monomoric MIF with one bound molecule of ebse^¬ len (274 Da) . It was observed that Ebselen induces the aggrega^¬ tion of MIF. To quantify the degree of Ebselen-induced aggrega^¬ tion of MIF, the samples were centrifuged at high speed 13000 g and the supernatant and pellets were collected and analysed by SDS gels.

Ebselene induces trimer to monomer dissociation.

Wt huMIF in the presence or absence of Ebselen (4 μΜ) , BITC (6 μΜ) and HCLP at (6 μΜ) was analysed by sedimentation velocity analytical ultracentrifugation; Human wt MIF preincubated either with DMSO, as a control, HCLP or BITC sediment predominantly as a single species with a sedimentation coefficient and molecular masses corresponding to that of the trimer. In the presence of ebselen, an additional sedimenting species could be observed with average s value of 1.7 S (±0.1S), in addition to 3.15 S species (trimer) .

Fig. 8 shows IC50 determination of Ebselen using Ddopachrome methyl ester as a substrate. (B) Sedimentation velocity profiles of wt human MIF in presence or absence of Ebselen. Ten micromo- lar MIF in PBS was preincubated with 3 μΜ ebselen for 1 hour at RT . Sedimentation coefficient distributions were obtained by analysis of the sedimentation profiles using the C (s) distribu^¬ tion as a variant of Lamm equation solutions. Calculations were performed using Sedfit software. Fig.9 shows that Ebselen induces MIF aggregation via interaction with cysteine. (A) Wt human MIF (10 μΜ) dialyzed against lx PBS or (D) N110C incubated with different ebselen concentrations (0, 1, 10, 100, and 1000 μΜ) for 120 min and filtered with a 100-KDa filter at 13000g for 5 min; both the filtrate and the solubi- lized retentate in 1% SDS were analyzed in a 15% SDS gel.

Alkyalted wt MIF or cysteine mutants do not show any aggregation in presence of ebselen. (B) The three cysteines of native MIF were mostly alkylated using 10 mM maleimide. (C) Ten micromolar wt MIF, C56S, or C80S were alkylated with 10 mM maleimide. After being desalted, both the alkylated and the non-alkylated pro^¬ teins were incubated with 100 μΜ ebselen for lh at RT and cen- trifuged for 15 min at 13000g, and the supernatant obtained from both alkylated and non-alkylated proteins was analyzed in a 15% SDS gel by WB using the anti-MIF antibody Zymed at 1:20000. (E) Comparison of aggregation propensity of WT, C80S, C56S and N110C in function of ebselen concentration. (F) Cysteine alkylation and/or trimer stabilization blocks ebselen-induced dissociation and aggregation of MIF.

Example 3

MIF Bioassay

Fig. 6 shows a schematic procedure for a bioassay using ITC functionalized well plate according to the present invention. MIF levels are elevated in the serum, plasma, and/or tissue of patients suffering from several inflammatory, autoimmune, cancerous, and infectious diseases. In some cases of rheumatoid ar^¬ thritis, cancer, and sepsis, the levels of MIF correlate with disease progression and severity. Administration of anti-MIF antibodies reduces the frequency of disease and demonstrates therapeutic benefits in animal models of inflammatory and auto^¬ immune diseases, including sepsis, rheumatoid arthritis, Type 1 diabetes, glomerulonephritis, tumor angiogenesis , and multiple sclerosis .

Patient's cells are lysed using conventional methods and the re^¬ sulting lysate is transferred to the ITC functionalized plate.

Whereas the inactive MIF shows no affinity to ITC, active MIF covalently binds to the reactive group. The catalyticall active MIF will be covalently immobilized to the functionalized sur^¬ faces of functionalized Plate. The unbound material is then transferred to another plate.

Indirect ELISA and Sandwich Elisa are then performed on the plate containing the active, respectively the inactive MIF.

Ratios [Active ]/[ Inactive ] and/or [Active ]/[ Total ] MIF are an advantageous diagnostic biomarker for inflammatory and autoimmune MIF-associated diseases.

Example 4

MIF glucocorticoid overriding activity

Among the important in vivo immunoregulatory functions of MIF is its ability to counterregulate the immunosuppressive effect of glucocorticoids on proinflammatory cytokine production. MIF tautomerase inhibitors block the immunoregulatory function of MIF and ability to abrogate TNF production upon stimulation of Raw 264.7 macrophages was tested. MIF pretreated with ebselen, HCLP (including its three analogues) at concentrations of 10 μΜ was incubated with dexamethasone for lh of prestimulation with Lipopolysaccharide LPS. Ebselen, HCLP, and its identified ana^¬ logues, MDPI894 and Bithionol, significantly inhibited the ca^¬ pacity of MIF to override the immunosuppressive effect of dexa^¬ methasone on LPS (Fig.7) stimulated TNF production by murine Raw 264.7 macrophages. TNF secretion was significantly suppressed in the presence of most tautomerase inhibitors.

Fig. 7 shows overriding of glucocorticoid-mediated inhibition of cytokine production. RAW 264.7 macrophages were preincubated for 1 h with ( + ) or without (-) dexamethasone (100 nM) , MIF (100 ng/mL) and MIF inhibitors (A) Ebselen, HCLP and its analogs or (B) compounds of the formula IX, X, VIII.1, XI, XII, XII.2, VII.2, VIII.3 at 10 μΜ before stimulation with 100 ng/mL of LPS. Cell-free supernatants were collected after 4 h to quantify the concentrations of TNF. (A) Data are means ± SD of triplicate samples from one experiment and are representative of four inde^¬ pendent experiments. Data are expressed in % inhibition and are 5 replicates of two representative experiments.

Example 5

Elucidating the structural basis for the inhibition of MIF enzymatic activity using MR - Ebselen, Hexachlorophenes and Formulas VIII.1, VIII.2 and VIII.3

In order to determine the structural basis for the inhibition of MIF enzymatic activity, NMR titrations were performed with com^¬ pounds of the formula VIII.1, VIII.2 and VIII.3, Ebselen and HCLP (hexachlorophene) .

Ebselen

Equimolar mixture of MIF with ebselen results in severe reso^¬ nance broadening throughout the whole sequence, indicative of conformational exchange (Figure 10) . However, more drastic ef^¬ fects are seen for the residues 3-5, 38-39, 49-51, 61-62, 64, 99, 101, 106-108, 112. Most of these residues are in the sub- unit-subunit interface. b-Sheet core of the monomeric subunit that consists of 4 b-strands, as well as the 3 b-strands coming from the other 2 subunits are highly affected by ebselen (Figure 10) . The drop in signal intensity could be due to a combination of both conformational exchange and aggregation of the dissoci^¬ ated monomeric subunits into larger moieties that are beyond the detection limit of liquid-state NMR. Among the three cysteines, only C59 displays a minor chemical shift, such an effect is ob^¬ served for neither C56 nor C80 (Figure 11) .

Fig. 10 shows the structural basis for the inhibition of MIF en^¬ zymatic activity by NMR. (A, E) The 2D 1H-15N HSQC reference spectrum of hMIF was recorded in the presence of 1% DMSO. The addition of the coumpounds to hMIF at equimolar concentrations (red) resulted in chemical shift changes. (B, F) Averaged chemi^¬ cal shift deviation in NMR spectra upon addition of Hits. (C, G) Changes of chemical shifts upon addition of the compounds were mapped onto the 3D structure of hMIF (PDB ID: 1GD0) . The inter^¬ action surface differed among all three compounds. Figures C and D are related to each other by 90°.

Fig. 11 shows the Cysteine's role in MIF-ebselen interaction. No chemical shifts are observed for C56 and C80, wheras C59 shows a minor chemical shift. (B,D) The only cysteine residue that is surface accessible is C59.

Fig. 12 shows the structural basis for the inhibition of the enzymatic activity of MIF. (A) The 2D 1H-15N HSQC reference spectrum of hMIF was recorded in the presence of 1% DMSO. Addi^¬ tion of all compounds to hMIF at equimolar concentrations re^¬ sulted in NMR chemical shift changes and signal broadening. In case of Formula VIII.2 and VIII.3, a set of new NMR resonances appeared at equimolar ratio. Titration data of HCLP are given for comparison. (B) Averaged 1H, 15N chemical shift deviation observed for backbone resonances of hMIF in 2D 1H-15N HSQC NMR spectra upon addition of Formulas XIII.1 to XIII.3 and HCLP at equimolar ratio (shown in (A) ) . (C) Changes in chemical shifts upon addition of the compounds were mapped onto the 3D structure of hMIF (PDB ID: 1GD0) .

Hexachlorophenes

Titration of MIF with HCLP results in both resonance broadening and chemical shift changes (Figure 10 C) , indicative of an in^¬ termediate exchange regime in NMR time scale. The residues that are affected by HCLP are 3-5, 35-38, 64, 67, 95, 107-110, 112 and 114 (Figure 6F and G) . Similar to BITC and HPP, these resi^¬ dues cluster around PI. Among these, 164 and Y95 are found in the active site.

Compounds of the Formula VIII.1 and VIII.2:

Backbone amide signals of wt hMIF were monitored for both For^¬ mula VIII.1 and VIII.2 titrations. Equimolar mixture of hMIF and Formula VIII.1 resulted in chemical shift deviation averaged on 1H and 15N dimensions in residue 2-3, 35-39, 49-51, 63-66, 94- 96, 107-110, 112-113 (Figure 12) . Many of these residues are lo^¬ cated in the subunit-subunit interface and enzymatic active site (Figure 12 ) .

Addition of Formula VIII.2 to hMIF also induced chemical shift deviation, but with two sequential changes; milder interaction and then conformation change of hMIF. With up to 0.5 molar ratio of Formula VIII.2, resonances from residues 2-3, 35-37, 50, 58, 60, 61, 65-66, 75, 81, 93, 95-96, 98, 107-108, 110-111 shifted (Figure 12), which correspond to subunit-subunit interface and enzymatic active site (Figure 12) . Upon addition of VIII.2 more than 0.75 molar ratio to hMIF, several peaks in HSQC disap^¬ peared, indicating intermediate exchange of conformations, and reappeared with increasing molar ratio of VIII.2, which implies higher affinity of VIII.2 to hMIF. MATERIALS AND METHODS

Chemicals

Benzyl isothiocyanate (BITC) , allyl isothiocyamate (AITC) , ethyl isothiocyanate (EITC) , methallyl Isothiocyanate (MITC) , 2- Piperidinoethyl isothiocyanate (2PITC) , cyclopropyl isothiocy^¬ anate (CPITC) and Phenylethyl isothiocyanate (PEITC) were pur^¬ chased from Sigma or Fluka of the highest purity available.

Expression and purification of human MIF

MIF protein was expressed by heat shock transformation of the BL21DE3 E. coli strain (Stratagene) with bacterial expression vector pETllb containing the human MIF gene under control of the T7 promoter. Four h post-induction, the cells were harvested, resuspended in lysis buffer (50 mM TRIS, 50 mM KC1, 5 mM MgAc, 0.1 % Nazide) , sonicated at 200 Hz pulse repetition frequency for 20 min using a VibraCellTM sonicator, and harvested by cen- trifugation at 13000g for 25 min. The clarified cell lysate was filtered, injected onto a MonoQ anion exchange column (HiPrep TM 16/10 Q FF GE Healthcare), and eluted with a linear NaCl

gradient in the elution buffer (25 mM Tris HC1 pH 7.4, 150 mM NaCl) . The flow-through fractions containing MIF were pooled and loaded onto a Superdex 75 16/60 (HiLoadTM 16/60, SuperdexTM 75, Pharmacia Biotech) gel filtration column. Fractions correspond^¬ ing to MIF were pooled together, dialyzed against PBS, and fil^¬ tered. The identity and purity of the protein was confirmed by MALDITOF mass spectrometry, silver staining, and western blotting using the rabbit anti-MIF antibody from

Zymed (Ivitrogen) at 1:20000. When required, the proteins were concentrated using a concentrator with a MWCO of 5 kDa and stored at 4 °C until use. Uniformly 15N-labeled protein samples were prepared for NMR experiments by culturing the bacteria in M9 minimal medium containing 15N ammonium chloride (1 g/L) as the only nitrogen source. Analytical ultracentrifugation (AUC)

Analytical ultracentrifugation experiments were performed using purified and dialyzed MIF samples (10-20 μΜ) preincubated with BITC for 1 h at an MIF concentration corresponding to 100% inhibition. Radial UV scans were recorded on a Beckman Optima XL-A at a wavelength of 277 nm. Sedimentation velocity experiments were carried out at 20 °C using 380-400 μΐ of protein solution. Data were recorded at rotor speeds of 50000 rpm in continuous mode, with a step size of 0.003 cm. The experimentally deter^¬ mined partial specific volume of 0.765 mg/ml was used to calcu^¬ late the molecular weights of MIF. The sedimentation velocity profiles were analyzed as a c(s) distribution of the Lamm equa^¬ tion using SEDFIT. Molar mass distributions c (M) were obtained by transforming the corresponding c(s) using SEDFIT.

Analytical size exclusion chromatography and light scattering

Size exclusion chromatography experiments were performed on pu^¬ rified MIF incubated in the presence and absence of BITC or its analogues using an analytical Superdex 75 10/30 column at room temperature. The column was equilibrated with 10 mM Tris buffer

(pH 7.6), and MIF samples (12 μΜ in PBS IX) in volumes of 0.5 mL were injected onto the column and eluted at a flow rate of 0.4 mL/min in a Agilant HPLC system attached to both UV, refractive index and .DAWN HELLIOS multiangle light scattering detector

(Wyatt Technology Corp, Santa Barbara CA) . Absolute MWs were de^¬ termined using ASTRA version 5.3 from Wyatt Technologies.

MALDI-TOF-MS MW measurements to examine possible protein modifications

Matrix Assisted LASER Desorption lonization-Time of Flight-Mass Spectrometry (MALDI-TOF-MS) was performed to examine any HITS- induced protein modification. The matrix solution was prepared by dissolving 14 mg/ml of sinapinic acid (SA) solution in 0.1% TFA/ACN (1/1) . A thin matrix layer was generated on the mirror- polished target using a gel loading tip. One microliter of sam^¬ ple (15 μΜ MIF incubated with 10 μΜ HITS for lh) was mixed with 1 μΐ of SA matrix solution, and 0.8 μΐ of this mixture was de^¬ posited on top of the thin layer and allowed to air dry. The samples were analyzed with a 4700 MALDI-TOF/TOF instrument (Ap^¬ plied Biosystems) .

Protein digestion analysis to identify the modified residue

One microliter of MIF sample previously incubated for 1 h with BITC 10μΜ, was diluted in 5 μΐ of 100 mM BA and digested over^¬ night with trypsin (Promega) . The digestion was stopped with 1 μΐ of 10% TFA and stored at 4 °C until further use. The samples were analyzed by MALDI-MS on a 4700 MALDI-TOF/TOF instrument

(Applied Biosystems) or an Axima CFR plus instrument (Shimadzu) without further purification. One microliter of sample was mixed with 1 μΐ of DHB (20 mg/ml in 1%PA/ACN (1/1)), and 0.8 μΐ of this mixture was deposited on the target and allowed to air dry. MALDI matrices and calibrants were obtained from Sigma/Fluka

( Schnelldorf, Germany), TFA was from Pierce, and SA and 2,5- dihydroxybenzoic acid (DHB) were from Fluka. Cytochrome C and apomyoglobin were from Sigma Nuclear Magnetic Resonance (NMR) Spectroscopy. NMR spectra were acquired at 27 °C on a Bruker Avance 600 MHz NMR spectrometer using a triple-resonance cryo- probe equipped with z-axis selfshielded gradient coils. Measure^¬ ments were performed using 100-200 μΜ samples. All samples were prepared in 20 mM Na2HP04, 0.5 mM EDTA, 0.02% NaN3 (pH 7.0), and 10% D20. Two-dimensional 1H-15N heteronuclear single quantum co^¬ herence (HSQC) spectra were recorded using 256 χ 1024 complex data points in the Fl and F2 dimensions, with a relaxation delay of 1.0 s. Sixty-four scans per increment were recorded for each spectrum. Spectral widths were 2200 and 8950 Hz in the 15N and 1H dimensions, respectively. Specifically, 2D 1H-15N HSQC spec^¬ tra were recorded to follow chemical shift changes upon addition of inhibitory compounds and to map the interaction surface of MIF for each compound. For this, a reference spectrum of MIF alone was recorded in the presence of 1% DMSO. For each titra^¬ tion step of MIF with inhibitory compounds, a new sample was prepared in which MIF was mixed with the corresponding compound dissolved in DMSO, with the DMSO concentration maintained at 1%. Spectra were processed with Topspin (Bruker) and NMRPipe (36) . Visualization and manipulation were performed using the public domain graphics program Sparky 3 [Goddard TD, Kneller DG (2004) SPARKY 3. University of California, San Francisco]. Resonance assignments were previously published for the same buffer system at 30°C (37) . Both intensity and chemical shift differences were analyzed with respect to the reference spectrum.

Changes were mapped on the crystal structure using the PDB file 1GDO (1.5 A resolution) and PyMOL software.

MIF receptor binding assay

Recombinant, soluble huMIF receptor ectodomain (sCD7473-232) was cloned and purified as described by and used to coat 96 well plates by incubation at 4°C overnight. Plates were washed 4 times with TTBS (pH 7.4) and blocked with superblock buffer

(PIERCE) for 1 h at room temperature. Biotinylated huMIF (Roche Molecular Biochemical) was prepared and added together in trip^¬ licate wells with increasing concentrations of purified native huMIF, heat-denatured huMIF, or test MIF proteins. Incubation was at room temperature for 2 h. After washing wells 4 times with TTBS (pH 7.4), streptavidin-conj ugated alkaline phosphatase

(R&D) was added, incubation continued for an additional h, and the wells washed and bound complexes detected by adding p- nitrophenyl phosphate (Sigma) . Absorbance was read at 405 nm us^¬ ing a DYNATECH, MR5000 plate reader, and values plotted as per- cent OD405 relative to wells containing biotinylated-MIF alone. The plots shown are representative of at least three assays, and each plotted point depicts a SEM < 10 %.

Glucocorticoid overriding assay

PBMCs from healthy donors (recruited by the Blood Center,

Lausanne, Switzerland) were purified by Ficoll-Hypaque density gradient (GE Healthcare) . To obtain macrophages, adherent PBMCs (106 cells per well in 48-well plates) were cultured for 6 days in RPMI 1640 with Glutamax. Media were supplemented with 10% heat-inactivated FCS (Seromed) and antibiotics. MIF was incu^¬ bated with the inhibitors for 15 min at room temperature. Pri^¬ mary macrophages were pre-incubated for 1 h with 100 nM dexa- methasone, dexamethasone plus MIF (100 ng/mL) or dexamethasone plus MIF and BITC (10 μΜ) before the addition of 100 ng/ml Sal^¬ monella minnesota Ultra Pure LPS (List Biologicals Laborato^¬ ries) . TNF concentrations in cell-culture supernatants collected after 4 h were measured by bioassay.

Phospho Akt activation by MIF

Confluent HeLa cells were incubated for 2 h with recombinant hu^¬ man MIF (50 ng/mL) in the presence of BITC, LY294002 (10 and 1 μΜ) or DMSO. Phosphorylation of Akt (Ser 473) was measured using Alpha screen SureFire phosphokinase KIT, an immunosandwich assay based on alpha screen technology in two incubation steps, ac^¬ cording to the manufacturer's protocol. First, cell lysate was incubated for 2 h with an anti-Akt antibody captured to protein A conjugated acceptor beads. Then, the biotinylated anti pAkt antibody was added with streptavidin coated Alpha donor beads. The two beads are brought in close proximity only in the pres^¬ ence of phosphorylated Akt. Up on illumination at 680 nm, alpha donor beads photosensitizer, converts the ambient oxygen to an excited form, which will enable the excitation of the alpha ac^¬ ceptor beads. Data are reported as alpha screen counts.

Claims

Claims

1. Composition for the inhibition of macrophage migration inhibitory factor, in particular of tautomerase activity of macrophage migration inhibitory factor, wherein said composition comprises at least one compound selected from the group consisting of

R-N=C=S (Formula I)

(Formula II)

(Formula III)

(Formula IV)

(Formula V)

(Formula VI)

(Formula VII)

(Formula VIII.1)

mula VIII.2)

wherein R and R' are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl, substituted acylalkyl, heterocycle, substituted heterocycle, substituted cycloalkyl, hydrogen and/or halogen and the molar mass of the compound does not exceed 500 g/mol .

Composition according to claim 1, wherein the compound is capable of covalently binding to a Proline, in particular the N-terminal Prol, of MIF.

Composition according to claim 2, wherein the compound is capable of selectively binding to active MIF.

Composition according to claim 1, wherein the compound is capable of forming a selenodisulfide bridge with a Cysteine of MIF, in particular with Cys81 of MIF.

Composition according to claim 3, wherein the compound is selected from a group of compounds consisting of the general formula I: R-N=C=S and wherein the compound is an isothiocy- anate, preferably selected from the group consisting of al- lyl isothiocyamate (AITC) , ethyl isothiocyanate (EITC) , methallyl isothiocyanate (MITC) , 2-Piperidinoethyl isothio^¬ cyanate (2PITC) , cyclopropyl isothiocyanate (CPITC) , benzyl isothiocyanate (BITC) and Phenylethyl isothiocyanate

(PEITC) .

Composition according to claim 5, wherein the compound is benzyl isothiocyanate (BITC) .

Composition according to claim 3, wherein the compound is selected from the group of compounds consisting of

(Formula II)

(Formula III)

(Formula IV)

(Formula V) and wherein R and R' are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, al- kenyl, substituted alkenyl, cycloalkyl, substituted cycloal- kyl, aryl, substituted aryl, arylalkyl, substituted arylal- kyl, acylalkyl, substituted acylalkyl, heterocycle, substi^¬ tuted heterocycle, substituted cycloalkyl, hydrogen and/or halogen and the molar mass of the compound does not exceed 500 g/mol.

8. Composition according to claim 7, wherein the compound is selected from the group consisting of

(Formula IX)

(Formula XIII)

(Formula XVIII)

(Formula XIX)

9. Com osition according to claim 2, wherein the compound is

(Formula XX)

Composition according to claim 1, wherein the compound the eneral formula

(Formula VII)

and R is independently selected from the group consisting of hydrogen, halogen, preferably chlorine, oxygen, hydroxide, nitrogen, amide, amino, carboxyl, or a linear or branched carbon group (Ci to Ce) , preferably R is either chlorine or hydroxyle or hydrogen; and wherein and X is chosen from the group of carbon, sulphur, phosphor, selenium, oxygen or nitrogen; preferably X is either carbon or sulphur.

11. Composition according to claim 10, wherein the compound is selected from the group consisting of:

(Formula XXI)

(Formula XXII)

(Formula XIII)

A method of inhibiting macrophage migration inhibitory factor (MIF) , in particular of inhibiting tautomerase activity of macrophage migration inhibitory factor (MIF) , comprising the step of reacting MIF with a composition according to any one of claims 1 to 11.

The method according to claim 12, wherein the reacting comprises a covalent binding.

The method according to claim 13, wherein the step of react ing the N-terminal Prol of MIF is a step of reacting the N- terminal Prol with an isothiocyanate group of a molecule se lected from compounds of the general formula R-N=C=S, wherein R is selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl, substituted acylalkyl, heterocycle, substituted heterocycle, substituted cycloal^¬ kyl, and halogen.

15. The method according to claims 14, wherein the step of re^¬ acting is a step of reacting the N-terminal Prol of MIF with Benzyl isothiocyanate (BITC) .

16. The method according to claim 14, wherein the step of react^¬ ing is a step of reacting the N-terminal Prol of MIF with an isothiocyanate selected from the group consisting of allyl isothiocyamate (AITC) , ethyl isothiocyanate (EITC) , methai- lyl Isothiocyanate (MITC) , 2-Piperidinoethyl isothiocyanate (2PITC) , cyclopropyl isothiocyanate (CPITC) and Phenylethyl isothiocyanate (PEITC) .

17. The method according to claim 12, wherein the step of reacting is a step of reacting the N-terminal Prol of MIF with a compound selected from the group consisting of N-(2,4 dini- trophenoxy) thiophene-2-carboxamide, N- (2-chlorophenyl) -N ' -

[ (5-nitro-3 hienyl ) carbonyl ] thiourea, N ' - [ ( 3-acetyl-l , 3- thiazolan-2-yl) carbonyl] -1, 3-benzodioxole-5-carbohydrazide, 7, 10-dioxo-4, 5-dihydro-7H, 10H-pyrano[3,2-c]pyrrolo[3,2,l- ij ] quinolin-8-yl 4-fluorobenzoate, 1 , 4-dioxo-l , 4- dihydronaphthalen-2-yl 4-methylbenzoate, 2-oxo-l-phenyl-l , 2- dihydroquinolin-4-yl benzoate, 2-chloro-3-oxocyclohex-l-enyl 3- (trifluoromethyl) benzoate, 3- (4-methoxyphenyl) -3-oxoprop- 1-enyl 4-chlorobenzoate, 3-cyano-4 , 6-dimethyl-2-pyridyl thio-phene-2-carboxylate, 2- (2, 3-dihydro-l, 3-benzothiazol-2- yliden) -3- (2-furyl) -3-oxopropanenitrile, 5-oxo-2, 3-dihydro- 1H, 5H-pyrido [3, 2, 1-ij ] quinolin-7-yl benzoate and 5,5- dimethyl-3-oxocyclohex-l-enyl 4-chlorobenzoate .

18. The method according to claim 12, wherein the step of react^¬ ing the MIF is a step of reacting with a compound chosen from the group Nl- [3- (trifluoromethyl) phenyl] -3- (2- chloroanilino) -2-cyano-3-thioxopropanamide, N ' ',Ν' ' ' -di (4- hydroxybenzylidene) carbonic di-hydrazide, or 6- {morpholino [4- (trifluoromethyl) phenyl] methyl } -1, 3- benzodi- oxol-5-ol .

19. A method of inhibiting macrophage migration inhibitory factor (MIF) , in particular of inhibiting tautomerase activety of macrophage migration inhibitory factor (MIF) , comprising the step of reacting the MIF with a compound of the general formula :

and wherein R is chosen from the group consisting of hydrogen, halogen, preferably chlorine, oxygen, hydroxide, nitro^¬ gen, amide, amino, carboxyl, or a linear or branched carbon group (Ci to Ce) , preferably R is either chlorine or hydro^¬ gen; and wherein and X is chosen from the group of carbon, sulphur, phosphor, selenium, oxygen or nitrogen; preferably X is either carbon or sulphur.

The method of claim 19, wherein the step of reacting the N terminal Prol of MIF is a step of reacting MIF with a com- pound selected from the group consisting of substituted or non-substituted hexachlorophenes .

21. A composition according to any one of claims 1-11, for the treatment of an inflammatory disease.

22. A composition according to any one of claims 1-11, for the treatment of an autoimmune disease.

23. A composition according to any one of claims 1-11, for the treatment of a disease chosen from the group consisting of sepsis, rheumatoid arthritis, multiple sclerosis.

24. Use of at least one composition of any one of claims 1-12 in a pharmaceutically effective dosage for manufacturing a me^¬ dicament for the treatment or prophylaxis of a macrophage migration inhibitory factor (MIF) related disease in a mammal .

25. A method for determining the concentration of macrophage migration inhibitory factor (MIF), comprising the steps of: i) Providing a first support structure to which is attached a chemical group or chemical compound that is known to covalently bind specifically to catalytically active MIF;

ii) Contacting the first support structure with a sam^¬ ple containing MIF for a sufficiently long time and under suitable conditions to allow for cova- lent binding of the chemical group or chemical compound to MIF;

iii) Removing the remainder of the sample from the

first support structure and transfer of this re^¬ mainder to a second support structure; iv) Determining the concentration of catalytically active MIF from analysis of the first support struc^¬ ture ;

and/or

determining the concentration of catalytically inactive MIF from analysis of the second support structure ;

and/or

determining the overall concentration of MIF from analysis of both the first and the second support structure .

26. The method of claim 25, wherein the first support structure and/or the second support structure are multi-well plates.

27. The method of claim 25, wherein the chemical group referred to in step i) is an isothiocyanate group.

28. The method of claim 25, wherein the chemical compound re^¬ ferred to in step i) is a composition according to any one of claims 1 - 12.

29. The method of claim 25, wherein determination of MIF concentration referred to in step iv) involves an ELISA assay.

30. A method of diagnosing a macrophage migration inhibitory

factor (MIF) related disease, comprising the step of carrying out a method for determining the concentration of MIF according to claim 25.