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. 2024 Jun 3;300(7):107443.
doi: 10.1016/j.jbc.2024.107443. Online ahead of print.

A small-molecule allele-selective transcriptional inhibitor of the MIF immune susceptibility locus

Jia Li  1 , Lin Leng  1 , Georgios Pantouris  2 , Ramu Manjula  2 , Marta Piecychna  1 , Laura Abriola  3 , Buqu Hu  1 , Elias Lolis  2 , Michelle E Armstrong  4 , Seamas C Donnelly  4 , Richard Bucala  5
Affiliations

A small-molecule allele-selective transcriptional inhibitor of the MIF immune susceptibility locus

Jia Li et al. J Biol Chem. .

Abstract

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 (namely, 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.

Keywords: ICBP90; MIF; MIF allele; MIF polymorphism; autoimmunity; inflammation; macrophage migration inhibitory factor; microsatellite; precision medicine; transcription inhibitor.

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Conflict of interest statement

Conflict of interests The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1
Figure 1
Development and validation of an interaction assay between MIF promoter microsatellite and ICBP90.A, the human MIF gene (rs5844572) showing its three exons, the −794 CATT5–8 promoter microsatellite, and the ICBP90 transcription factor. The numerals refer to nucleotides upstream from the transcription start site. B, the upper panel shows the gel electrophoretic analysis of FPLC fractions collected by imidazole gradient elution of recombinant histidine-tagged ICBP90413-618 (100% imidazole = 500 mM). The lower panel shows the purity of the two ICBP90413-618 fractions (predicted MW 24.3 kDa) selected for high-throughput screening together with the E. coli lysate protein expression starting material. C, verification of recombinant ICBP90413-618 immunoreactivity by Western blot detection with anti-histidine and anti-ICBP90 antibodies. D, melting curve analysis of the −794 CATT8 microsatellite DNA (nucleotides −865 to −752) assessed by the fluorescence of the dsDNA binding dye SYBR green (λmax 520), showing dose-dependent duplex stabilization by ICBP90413-618 (0.2, 0.1 nmol) but not by the control proteins CD7473-232 (sCD74) or human serum albumin (HSA) (both at 0.2 nmol). The melting temperature of a corresponding −794 CATT0MIF promoter DNA (nucleotides −833 to −752) was not affected by ICBP90413-618 addition (control). The addition of anti-ICBP90 antibody but not control IgG also prevented melting curve stabilization (not shown). E, assessment of the stability of the ICBP90 - MIF promoter solution interaction by capture ELISA. Recombinant ICBP90413-618 was pre-incubated with increasing concentrations of a 5′ biotin-labelled MIF promoter CATT8 oligonucleotide (0.125–2.0 pmol), with or without excess 20 pmol unlabeled MIF promoter CATT8 oligonucleotide, followed by addition to immobilized (plate-bound) streptavidin, incubation, washing, and detection with horseradish-peroxidase labeled anti-ICBP90. Values shown are mean ± SD and representative of two independent studies (∗p < 0.01 by Student’s t test). F, electromobility shift assay (EMSA) showing retardation of the electrophoretic migration of a 5′ biotin-MIF promoter CATT8 oligonucleotide by ICBP90413-618 in the presence of a MIF promoter oligonucleotide lacking the CATT8 microsatellite (5′CATT0 oligo) and reduction by the presence of excess MIF promoter CATT8 oligonucleotide (5′CATT8 oligo). All data shown are representative of at least two independent determinations.
Figure 2
Figure 2
Development of a high-throughput AlphaScreen for candidate inhibitors of the MIF promoter microsatellite-ICBP90 interaction.A, AlphaScreen analyses of the dose-dependent solution interaction of the 5′-biotin MIF promoter CATT8 oligonucleotide (5′Biotin-CATT8, 6.8–5000 nM) with ICBP90413-618 at the tested concentrations of 6.8, 21.0, and 62.0 nM. Fluorescence intensity is expressed in counts per second (cps). B, dose-dependent inhibition of anti-ICPB90 IgG or an isotypic control IgG antibody on AlphaScreen solution interaction of ICBP90413-618 (62 nM) with 5′biotin MIF promoter CATT8 oligonucleotide (10 nM). C, electrophoretic mobility shift assay (EMSA) of five representative compound hits identified by AlphaScreen (Compound 1: NSC 3852, 2: NSC 5159, 3: NSC 106995 (CMFT), 4: NSC 114341, and 5: NSC 11107). The lanes show retention of ICBP90413-618/5′Biotin-CATT8 molecular complexes, dependence on ICBP90413-618, and inhibition by compound 3; Lane 1: 5′Biotin MIF promoter CATT8 only; Lane 2: 5′Biotin MIF promoter CATT8 plus ICBP90413-618; Lanes 3 to 7: 5′Biotin MIF promoter CATT8 + ICBP90413-618 plus test Compounds 1 to 5; Lanes 8 to 12: 5′Biotin MIF promoter CATT8 plus test compounds 1 to 5 without ICBP90413-618. D, dose-dependent inhibition of ICBP90413-618/5′Biotin-CATT8 solution interaction by CMFT (NSC106995) and 3 structural congeners identified in the screened libraries (NSC 1569419, 382951, 382953). E, dose-dependent inhibition by CMFT of ICBP90413-618/5′Biotin-CATT8 complexation assessed by EMSA. Lane 1: 5′Biotin MIF promoter CATT8 only; Lane 2: 5′Biotin MIF promoter CATT8 plus ICBP90413-618; Lane 3: 5′Biotin MIF promoter CATT8 plus ICBP90413-618 plus excess 5′ MIF promoter CATT8 oligonucleotide. Lanes 4 to 11: 5′Biotin MIF promoter CATT8 plus ICBP90413-618 plus increasing concentrations of CMFT (0, 7.8, 15.6, 31.2, 62.5, 125, 250, and 500 μM). Graphed data points are duplicate or triplicate determinations with mean ± SD (Student’s t test, two-tailed). EMSA determinations are representative of at least two independent experiments.
Figure 3
Figure 3
Structural modeling of CMFT bound to ICBP90 and interaction with DNA.A, X-ray co-crystal structure of the ICBP90 SRA domain in its DNA-bound form showing flexible loops (green), β-strands (yellow), and α-helices (red). The duplex DNA is shown as a surface representation with backbone atoms and bases (blue) together with the interdigitating NKR (asparagine, lysine, and arginine) motif (magenta) (15). B, an energy-minimized structure of the SRA domain (aqua) docked with CMFT (orange), showing hydrogen bonding interactions (yellow dashes) with D469, G448, A463, G465, Y466, and K540. The hydrophibic and ionic interactions are shown in grey and orange dashes, respectively (C). Superimposed structures of the SRA domain bound to DNA double helix (orange) from the 3CLZ.pdb, modeled CMFT bound SRA (yellow) and methylated cytosine (green).
Figure 4
Figure 4
Impact of CMFT on cellular MIF RNA and protein expression.A, human THP-1 monocytes transfected with MIF promoter-luciferase reporter plasmids bearing 0, 5, 6, 7, and 8 CATT repeats were stimulated with lipopolysaccharide (LPS, 100 ng/ml), treated with CMFT (3 μM) or vehicle control (0.4% DMSO) for 6 h and luciferase activity assessed by Dual-Luciferase assay. B, quantitative PCR analysis of MIF mRNA of bone marrow-derived macrophages (BMDMs) isolated from humanized MIFCATT5 and MIFCATT7 mice, stimulated in vitro with LPS (100 ng/ml), and treated with CMFT (2.5 μM, 6 h) or vehicle control (0.4% DMSO). C, ELISA analysis of human MIF in supernatants (triplicate measurements) of cultured LPS-stimulated (100 ng/ml) BMDMs from MIFCATT5 and MIFCATT7 mice 6 h after treatment with CMFT (2.5 μM) or vehicle control (0.4% DMSO). Data are the mean+SD and representative of two replicated experiments (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 by Student’s t test, two-tailed).
Figure 5
Figure 5
Transcriptomic analysis of the impact of CMFT on the inflammatory activation of high-genotypic, MIF expressing MIFCATT7macrophages. Stimulation of triplicate BMDM cultures were as in Figure 4B, with CMFT (+) or vehicle (−) addition for 6 h followed by RNAseq analysis (Agilent Bioanalyzer). Expression heatmaps of responsive genes for 2.0-fold differential expression with an FDR<0.05 in gene expression sets for (A): inflammation (Inflammatory Signaling, Toll-like Receptors, and Cytokines/Chemokine) and (B): metabolism (Homeostasis/Glycolysis).
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