Macrophage migration inhibitory factor (MIF) tautomerase active site [1]

Macrophage migration inhibitory factor (MIF) [2]

Macrophage migration inhibitory factor (MIF) [3]

Without recombinant MIF added, ISO-1 (0.1–20 μM; 16 hours) has a mild inhibitory effect on Cox-2 secretion [1].

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ISO-1 inhibited MIF D-dopachrome tautomerase activity in a dose-dependent manner with an IC50 of approximately 7 μM. The non-hydroxylated phenyl analog (compound 2) was 10-15 times less potent. The 4-methoxy analog (compound 3) showed no activity. Oxidation of ISO-1 to isoxazole (compound 4) eliminated activity, and reduction to compound 5 also led to inactivity [1].

ISO-1 inhibited MIF-induced arachidonic acid release in RAW 264.7 macrophages transfected with a MIF-expressing plasmid in a dose-dependent manner; compounds 2, 3, and 4 failed to inhibit at most concentrations, except compound 2 at 100 μM showed some inhibition [1].

ISO-1 dose-dependently antagonized MIF-dependent inhibition of dexamethasone in LPS-activated human monocytes, decreasing TNFα, PGE2, and Cox-2 production at concentrations from 0.1 to 20 μM. A slight inhibitory effect on cytokine secretion without added recombinant MIF was also observed, possibly due to inhibition of native MIF [1].

ISO-1 inhibited intracellular MIF tautomerase activity in RAW 267.4 macrophages in a dose-dependent manner (1-100 μM) after 30 min treatment, as measured by L-dopachrome methyl ester conversion [2].

ISO-1 dose-dependently inhibited LPS-induced TNF secretion from RAW 267.4 macrophages (1-100 μM) [2].

ISO-1 inhibited LPS-induced NF-κB activation in RAW 267.4 macrophages, with up to 70% inhibition at 100 μM as measured by electrophoretic mobility shift assay [2].

Fluorescently labeled ISO-1 (FL-ISO-1) was taken up by RAW 267.4 macrophages and localized in the cytoplasm and nuclei within 30 min [2].

ISO-1 (ip; 3.5–35 mg/kg; twice daily; 2 weeks) has anti-inflammatory properties and increases survival from lethal endotoxemia [2]. Intraperitoneal injection ISO-1 (35 mg/kg, twice daily, 3 days) significantly lowers Flk1 expression in the implants and decreases mean implant size [3].

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ISO-1 (35 mg/kg i.p., twice daily for 3 days) significantly protected against lethal endotoxemia in male Balb/C mice (LPS 5 mg/kg i.p.), improving survival in a dose-dependent manner (p < 0.001 at 35 mg/kg) compared to vehicle. The effect was comparable to anti-MIF antibody treatment [2].

ISO-1 treatment initiated 24 h after cecal ligation and puncture (CLP)-induced polymicrobial sepsis (35 mg/kg i.p., twice daily for 3 days) resulted in 77% survival compared to 38% in vehicle-treated controls (p < 0.001). Anti-MIF antibody given in the same time frame improved survival comparable to ISO-1 [2].

ISO-1 inhibited TNF release by 67% in peritoneal macrophages from LPS-treated wild-type mice but had no effect on cytokine release from MIF-deficient mice [2].

In a mouse model of endometriosis (C57BL/6-Tg(ACTB-EGFP) recipients with uterine fragments injected intraperitoneally), treatment with ISO-1 (35 mg/kg i.p., twice daily for 3 consecutive days, starting 1 week after induction) significantly reduced average endometriotic implant size compared to vehicle. Flk1 (VEGF receptor 2) mRNA expression, an indicator of vascularity, was also significantly reduced. Reproductive cyclicity was not disrupted [3].

D-Dopachrome tautomerase activity assay: L-Dopachrome methyl ester was prepared by oxidation of L-3,4-dihydroxyphenylalanine methyl ester with sodium periodate. Activity was measured at room temperature by adding dopachrome methyl ester to a cuvette containing MIF in potassium phosphate buffer (pH 6.0, 0.5 mM EDTA). The decrease in absorbance at 475 nm from 2 to 20 seconds was recorded spectrophotometrically. Inhibitors dissolved in DMSO were added to the cuvette with MIF before adding dopachrome. IC50 values were determined from dose-response curves [1].

Intracellular MIF tautomerase activity assay: RAW 267.4 macrophages were treated with various concentrations of ISO-1 for 30 min, then washed and lysed in non-denaturing buffer. The lysates were assayed for tautomerase activity using L-dopachrome methyl ester as described above, measuring absorbance decrease at 475 nm [2].

MIF activity assessment in peritoneal fluid: Peritoneal fluid was collected from mice, protein concentrations normalized, and samples mixed with assay buffer (50 mM potassium phosphate, pH 6.0, 1 mM EDTA). Dopachrome methyl ester was added and absorbance read at 475 nm using a microplate spectrophotometer. Activity was expressed as fold change relative to controls [3].

Western Blot Analysis[1]
Cell Types: RAW 264.7 macrophages
Tested Concentrations: 0.1μM, 1μM, 10μM, 20μM
Incubation Duration: 16 hrs (hours)
Experimental Results: Inhibition of Cox-2 secretion.

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Glucocorticoid override assay: Human or murine mononuclear cells were isolated from whole blood by density gradient centrifugation, and monocytes were purified by adherence. Cells (1×10^6/well) were preincubated for 1 h with dexamethasone (10^-8 M), dexamethasone plus MIF (100 ng/ml wild-type or mutant), or dexamethasone plus MIF and ISO-1 (0.1-20 μM) before adding LPS (0.5 μg/ml). After 16 h, supernatants were collected for TNFα and PGE2 determination by ELISA. Cox-2 expression was analyzed by Western blot using specific antibodies [1].

Arachidonic acid release assay: RAW 264.7 macrophages were plated with [14C]arachidonic acid overnight, then washed and transfected with MIF-expressing plasmid or empty vector in the presence or absence of vehicle or ISO-1. After 16 h, supernatants were collected and 14C-labeled arachidonic acid was quantitated by scintillation counting [1].

TNF secretion assay: RAW 267.4 macrophages were treated with ISO-1 (1-100 μM) for 30 min prior to LPS (100 ng/ml) addition. After 16 h incubation, cell culture supernatants were collected for TNF determination by ELISA [2].

NF-κB activation assay (EMSA): RAW 267.4 macrophages were treated with ISO-1 (1-100 μM) 30 min before LPS addition. Nuclear extracts were isolated after 2 h. Nuclear protein (5 μg) was incubated with 32P-labeled double-stranded oligonucleotide containing the NF-κB consensus sequence. Samples were resolved on 4% polyacrylamide gel and visualized by autoradiography. Bound NF-κB bands were quantified by densitometry [2].

Intracellular localization of fluorescent ISO-1: RAW 267.4 macrophages plated on coverslips were treated with fluorescently labeled ISO-1 (FL-ISO-1), then washed, fixed with formaldehyde, stained with DAPI, and imaged using fluorescence microscopy [2].

mRNA expression analysis: Total RNA was extracted from endometriotic implants or uterine tissues, and cDNA was synthesized. qRT-PCR was performed to quantify Mif and Flk1 mRNA levels, normalized to 18S rRNA. Western blot analysis was performed on protein extracts using anti-MIF antibody and normalized to β-actin [3].

Animal/Disease Models: C57Bl/6 MIF+/+ and MIF–/– mice [2]
Doses: 3.5-35 mg/kg
Route of Administration: intraperitoneal (ip) injection; 3.5-35 mg/kg; twice (two times) daily; 2 weeks
Experimental Results: Yes Preventing fatal sepsis.

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Animal/Disease Models: Female C57BL/6-Tg(ACTB-EGFP)1Osb/J mice [3]
Doses: 35 mg/kg
Route of Administration: intraperitoneal (ip) injection; 35 mg/kg; twice (two times) daily; 3 days
Experimental Results: In utero The average size of the endometriotic implants diminished.

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Endotoxemia model: Male Balb/C mice (8 weeks old) were injected intraperitoneally with LPS (5 mg/kg). ISO-1 (3.5-35 mg/kg) or vehicle (5% DMSO in aqueous solution) was administered intraperitoneally 30 min before and 6 h after LPS infusion, then twice daily for 3 days. Animals were monitored for survival for 2 weeks [2].

Cecal ligation and puncture (CLP) model: Mice were anesthetized, the cecum was isolated and ligated below the ileocecal valve, and punctured once with a 22-gauge needle, extruding a small amount of stool. Antibiotics (0.5 mg/kg Premaxin) were given subcutaneously immediately after CLP, and resuscitative fluid (normal saline, 20 ml/kg) was given subcutaneously. ISO-1 (35 mg/kg) or vehicle (5% DMSO) treatment was started 24 h after CLP, administered intraperitoneally twice daily for 3 days. Animal survival was monitored for 2 weeks. In parallel experiments, anti-MIF antibody (2.8 mg/kg) was given intraperitoneally once daily for 3 days starting 24 h after CLP [2].

Endometriosis mouse model: Female C57BL/6-Tg(ACTB-EGFP) mice served as recipients. Donor uteri from immature C57BL/6 mice were harvested after PMSG injection, and uterine fragments (1 mm^3) were injected into the peritoneal cavity of recipients. One week after induction, mice received ISO-1 (35 mg/kg body weight) or vehicle (5% DMSO in saline) intraperitoneally twice daily (12 h apart) for 3 consecutive days. Mice were sacrificed one week after the last injection, and endometriotic implants were counted and sized (mm^3). Estrous cycles were monitored by vaginal cytology for 14 days (4 days before treatment until sacrifice) [3].

ISO-1 and its derivatives were not toxic at the pharmacological doses used, as assessed by trypan blue exclusion assays in cell cultures [1].

ISO-1 showed no lethality in mice up to 250 mg/kg, a dose approximately 7-fold higher than the maximum dose used in in vivo efficacy studies [2].

Treatment with ISO-1 did not disrupt the reproductive estrous cycle in mice, indicating no significant reproductive toxicity under the experimental conditions [3].

[1]. The tautomerase active site of macrophage migration inhibitory factor is a potential target for discovery of novel anti-inflammatory agents. J Biol Chem. 2002 Jul 12;277(28):24976-82.

[2]. ISO-1 binding to the tautomerase active site of MIF inhibits its pro-inflammatory activity and increases survival in severe sepsis. J Biol Chem. 2005 Nov 4;280(44):36541-4.

[3]. Inhibition of macrophage migration inhibitory factor reduces endometriotic implant size in mice with experimentally induced disease. J Endometr. 2011 Sep 30;3(3):135-142.

5-Isoxazoacetic acid, 4,5-dihydro-3-(4-hydroxyphenyl)-methyl ester, is a member of the phenolic class of compounds.

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MIF is a pro-inflammatory cytokine involved in sepsis, arthritis, glomerulonephritis, and other inflammatory diseases. The tautomerase active site of MIF is a potential target for anti-inflammatory agents. ISO-1 binds to the same position in the MIF active site as p-hydroxyphenylpyruvic acid (a substrate). The crystal structure of the MIF-ISO-1 complex revealed that only the R-isomer binds to the MIF pocket, with interactions including hydrogen bonds to Lys-32, Ile-64, and Asn-97, and aromatic interactions with Tyr-95. The PAM mutant (alanine insertion between Pro-1 and Met-2) which disrupts the catalytic site is defective in glucocorticoid counter-regulatory activity [1].

ISO-1 is the first small molecule inhibitor of MIF pro-inflammatory activities with therapeutic implications. It targets the tautomerase active site, a non-essential enzymatic function. Serum MIF levels increased to 70% of maximum within 24 h post-CLP and peaked at 36 h, indicating MIF as a late mediator in sepsis. Inhibition of MIF by ISO-1 recapitulates the phenotype of MIF-deficient macrophages (hyporesponsive to endotoxin) [2].

In endometriosis, MIF expression is elevated in ectopic implants. ISO-1 reduces implant size and vascularity without affecting reproductive cyclicity, suggesting potential for treating endometriosis in humans [3].

C12H13NO4

235.239

235.084

478336-92-4

478336-92-4;

4633677

White to off-white solid powder

1.3±0.1 g/cm3

365.0±48.0 °C at 760 mmHg

114 °C

174.6±29.6 °C

0.0±0.8 mmHg at 25°C

1.582

0.89

1

5

4

17

308

0

AIXMJTYHQHQJLU-UHFFFAOYSA-N

InChI=1S/C12H13NO4/c1-16-12(15)7-10-6-11(13-17-10)8-2-4-9(14)5-3-8/h2-5,10,14H,6-7H2,1H3

4,5-dihydro-3-(4-hydroxyphenyl)-5-isoxazoleacetic acid, methyl ester

ISO-1 ISO 1 ISO1 MIF Antagonist Macrophage Migration Inhibitory Factor

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~212.55 mM)

Solubility in Formulation 1: 5 mg/mL (21.25 mM) in 5% DMSO + 95% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (10.63 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (10.63 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (10.63 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)