Small Molecule MIF Modulation Enhances Ferroptosis by Impairing DNA Repair Mechanisms.

Ferroptosis is a form of regulated cell death that can be modulated by small molecules and has the potential for the development of therapeutics for oncology. Although excessive lipid peroxidation is the defining hallmark of ferroptosis, DNA damage may also play a significant role. In this study, a potential mechanistic role for MIF in homologous recombination (HR) DNA repair is identified. The inhibition or genetic depletion of MIF or other HR proteins, such as breast cancer type 1 susceptibility protein (BRCA1), is demonstrated to significantly enhance the sensitivity of cells to ferroptosis. The interference with HR results in the translocation of the tumor suppressor protein p53 to the mitochondria, which in turn stimulates the production of reactive oxygen species. Taken together, the findings demonstrate that MIF-directed small molecules enhance ferroptosis via a putative MIF-BRCA1-RAD51 axis in HR, which causes resistance to ferroptosis. This suggests a potential novel druggable route to enhance ferroptosis by targeted anticancer therapeutics in the future.

Design, Synthesis, and Evaluation of Inhibitors of Hedgehog Acyltransferase.

Hedgehog signaling is involved in embryonic development and cancer growth. Functional activity of secreted Hedgehog signaling proteins is dependent on N-terminal palmitoylation, making the palmitoyl transferase Hedgehog acyltransferase (HHAT), a potential drug target and a series of 4,5,6,7-tetrahydrothieno[3,2-c]pyridines have been identified as HHAT inhibitors. Based on structural data, we designed and synthesized 37 new analogues which we profiled alongside 13 previously reported analogues in enzymatic and cellular assays. Our results show that a central amide linkage, a secondary amine, and (R)-configuration at the 4-position of the core are three key factors for inhibitory potency. Several potent analogues with low- or sub-µM IC50 against purified HHAT also inhibit Sonic Hedgehog (SHH) palmitoylation in cells and suppress the SHH signaling pathway. This work identifies IMP-1575 as the most potent cell-active chemical probe for HHAT function, alongside an inactive control enantiomer, providing tool compounds for validation of HHAT as a target in cellular assays.

Allosteric Inhibitors of Macrophage Migration Inhibitory Factor (MIF) Interfere with Apoptosis-Inducing Factor (AIF) Co-Localization to Prevent Parthanatos.

Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine and essential signaling protein associated with inflammation and cancers. One of the newly described roles of MIF is binding to apoptosis-inducing factor (AIF) that "brings" cells to death in pathological conditions. The interaction between MIF and AIF and their nuclear translocation stands as a central event in parthanatos. However, classical competitive MIF tautomerase inhibitors do not interfere with MIF functions in parthanatos. In this study, we employed a pharmacophore-switch to provide allosteric MIF tautomerase inhibitors that interfere with the MIF/AIF co-localization. Synthesis and screening of a focused compound collection around the 1,2,3-triazole core enabled identification of the allosteric tautomerase MIF inhibitor 6y with low micromolar potency (IC50 = 1.7 ± 0.1 µM). This inhibitor prevented MIF/AIF nuclear translocation and protects cells from parthanatos. These findings indicate that alternative modes to target MIF hold promise to investigate MIF function in parthanatos-mediated diseases.

Cyclic diguanylate analogues: Facile synthesis, STING binding mode and anti-tumor immunity delivered by cytidinyl/cationic lipid.

Herein 2-cyanoethoxy-N,N,N',N'-tetraisopropyl-phosphorodiamidite(10, PIII, 3.5 eq.) could synergistically react with 3',5'-dihydroxyl groups in a dinucleotide(PV) at the cyclization step for the synthesis of cyclic dinucleotides (CDNs) (c-di-GMP, cGAMP etc.) and their phosphorothioated analogues. A dynamic PIII-PV coordination mechanism has been proposed for the cyclization procedure which is confirmed by the variant 31P NMR data and molecular simulation. Among the mono-phosphorothioated CDNs, two stereoisomers showed different capacity for STING activation and the reason was predicted by molecular modeling. While compound 12b1 showed most potent ability to elicit cytokines (IFNß, IL-6, Cxcl9 and Cxcl10) induction compared to another stereoisomer. Also, 12b1 significantly inhibited the tumor growth in the EO771 model with both 0.1 µg (i.t.) and 2 µg (i.v.) administration through the aid of a Mix delivery system developed by our group, and achieved a 31% long-term survival rate of tumor-bearing mice. 12b1/Mix significantly improved the percentage of CD8+ or CD4+ effector memory T (Tem, CD44highCD62Llow) cells and CD8+ central memory T (Tcm, CD44highCD62Lhigh) cells in the blood of EO771 mice, inducing the immune memory against EO771 tumor cells. Relatively lower dose regimens of 12b1(0.1 µg)/Mix displayed better tumor suppression by more potent STING pathway activation and higher levels of cytokines induction in the tumor.

Thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione Derivative Inhibits d-Dopachrome Tautomerase Activity and Suppresses the Proliferation of Non-Small Cell Lung Cancer Cells.

The homologous cytokines macrophage migration inhibitory factor (MIF) and d-dopachrome tautomerase (d-DT or MIF2) play key roles in cancers. Molecules binding to the MIF tautomerase active site interfere with its biological activity. In contrast, the lack of potent MIF2 inhibitors hinders the exploration of MIF2 as a drug target. In this work, screening of a focused compound collection enabled the identification of a MIF2 tautomerase inhibitor R110. Subsequent optimization provided inhibitor 5d with an IC50 of 1.0 µM for MIF2 tautomerase activity and a high selectivity over MIF. 5d suppressed the proliferation of non-small cell lung cancer cells in two-dimensional (2D) and three-dimensional (3D) cell cultures, which can be explained by the induction of cell cycle arrest via deactivation of the mitogen-activated protein kinase (MAPK) pathway. Thus, we discovered and characterized MIF2 inhibitors (5d) with improved antiproliferative activity in cellular models systems, which indicates the potential of targeting MIF2 in cancer treatment.

4-Iodopyrimidine Labeling Reveals Nuclear Translocation and Nuclease Activity for Both MIF and MIF2.

Macrophage migration inhibitory factor (MIF) and its homolog MIF2 (also known as D-dopachrome tautomerase or DDT) play key roles in cell growth and immune responses. MIF and MIF2 expression is dysregulated in cancers and neurodegenerative diseases. Accurate and convenient detection of MIF and MIF2 will facilitate research on their roles in cancer and other diseases. Herein, we report the development and application of a 4-iodopyrimidine based probe 8 for the selective labeling of MIF and MIF2. Probe 8 incorporates a fluorophore that allows in situ imaging of these two proteins. This enabled visualization of the translocation of MIF2 from the cytoplasm to the nucleus upon methylnitronitrosoguanidine stimulation of HeLa cells. This observation, combined with literature on nuclease activity for MIF, enabled the identification of nuclease activity for MIF2 on human genomic DNA.

HDAC/MIF dual inhibitor inhibits NSCLC cell survival and proliferation by blocking the AKT pathway.

Non-small-cell lung carcinoma (NSCLC) is one of the most common forms of lung cancer, and a leading cause of cancer death among human beings. There is an urgent demand for novel therapeutics for the treatment of NSCLC to enhance the efficacy of the currently applied Tyrosine kinase inhibitors (TKIs) therapy and to overcome therapy-resistance. Here, we report a novel small-molecule inhibitor that simultaneously targets histone deacetylase (HDAC) and macrophage migration inhibitory factor (MIF). The HDAC/MIF dual inhibitor proved to be toxic for EGFR mutated (H1650, TKI-resistant) or knock out (A549 EGFR-/-) NSCLC cell lines. Further experiments showed that HDAC inhibition inhibits cell survival and proliferation, while MIF inhibition downregulates pAKT or AKT expression level, which both interfere with cell survival. Furthermore, the combination treatment of TKI and HDAC/MIF dual inhibitor showed that the dual inhibitor enhanced TKI inhibitory efficacy, highlighting the advantages of HDAC/MIF dual inhibitor for more effective treatment of NSCLC.

Identification of a Bromodomain-like Region in 15-Lipoxygenase-1 Explains Its Nuclear Localization.

Lipoxygenase (LOX) activity provides oxidative lipid metabolites, which are involved in inflammatory disorders and tumorigenesis. Activity-based probes to detect the activity of LOX enzymes in their cellular context provide opportunities to explore LOX biology and LOX inhibition. Here, we developed Labelox B as a potent covalent LOX inhibitor for one-step activity-based labeling of proteins with LOX activity. Labelox B was used to establish an ELISA-based assay for affinity capture and antibody-based detection of specific LOX isoenzymes. Moreover, Labelox B enabled efficient activity-based labeling of endogenous LOXs in living cells. LOX proved to localize in the nucleus, which was rationalized by identification of a functional bromodomain-like consensus motif in 15-LOX-1. This indicates that 15-LOX-1 is not only involved in oxidative lipid metabolism, but also in chromatin binding, which suggests a potential role in chromatin modifications.

Proteolysis Targeting Chimera (PROTAC) for Macrophage Migration Inhibitory Factor (MIF) Has Anti-Proliferative Activity in Lung Cancer Cells.

Macrophage migration inhibitory factor (MIF) is involved in protein-protein interactions that play key roles in inflammation and cancer. Current strategies to develop small molecule modulators of MIF functions are mainly restricted to the MIF tautomerase active site. Here, we use this site to develop proteolysis targeting chimera (PROTAC) in order to eliminate MIF from its protein-protein interaction network. We report the first potent MIF-directed PROTAC, denoted MD13, which induced almost complete MIF degradation at low micromolar concentrations with a DC50 around 100 nM in A549 cells. MD13 suppresses the proliferation of A549 cells, which can be explained by deactivation of the MAPK pathway and subsequent induction of cell cycle arrest at the G2/M phase. MD13 also exhibits antiproliferative effect in a 3D tumor spheroid model. In conclusion, we describe the first MIF-directed PROTAC (MD13) as a research tool, which also demonstrates the potential of PROTACs in cancer therapy.

7-Hydroxycoumarins Are Affinity-Based Fluorescent Probes for Competitive Binding Studies of Macrophage Migration Inhibitory Factor.

Macrophage migration inhibitory factor (MIF) is a cytokine with key roles in inflammation and cancer, which qualifies it as a potential drug target. Apart from its cytokine activity, MIF also harbors enzyme activity for keto-enol tautomerization. MIF enzymatic activity has been used for identification of MIF binding molecules that also interfere with its biological activity. However, MIF tautomerase activity assays are troubled by irregularities, thus creating a need for alternative methods. In this study, we identified a 7-hydroxycoumarin fluorophore with high affinity for the MIF tautomerase active site (Ki = 18 ± 1 nM) that binds with concomitant quenching of its fluorescence. This property enabled development of a novel competition-based assay format to quantify MIF binding. We also demonstrated that the 7-hydroxycoumarin fluorophore interfered with the MIF-CD74 interaction and inhibited proliferation of A549 cells. Thus, we provide a high-affinity MIF binder as a novel tool to advance MIF-oriented research.

Structure-activity relationships for binding of 4-substituted triazole-phenols to macrophage migration inhibitory factor (MIF).

Macrophage migration inhibitory factor (MIF) is a versatile protein that plays a role in inflammation, autoimmune diseases and cancers. Development of novel inhibitors will enable further exploration of MIF as a drug target. In this study, we investigated structure-activity relationships of MIF inhibitors using a MIF tautomerase activity assay to measure binding. Importantly, we notified that transition metals such as copper (II) and zinc (II) interfere with the MIF tautomerase activity under the assay conditions applied. EDTA was added to the assay buffer to avoid interference of residual heavy metals with tautomerase activity measurements. Using these assay conditions the structure-activity relationships for MIF binding of a series of triazole-phenols was explored. The most potent inhibitors in this series provided activities in the low micromolar range. Enzyme kinetic analysis indicates competitive binding that proved reversible. Binding to the enzyme was confirmed using a microscale thermophoresis (MST) assay. Molecular modelling was used to rationalize the observed structure-activity relationships. The most potent inhibitor 2d inhibited proliferation of A549 cells in a clonogenic assay. In addition, 2d attenuated MIF induced ERK phosphorylation in A549 cells. Altogether, this study provides insights in the structure-activity relationships for MIF binding of triazole-phenols and further validates this class of compounds as MIF binding agents in cell-based studies.

Cross-kingdom mimicry of the receptor signaling and leukocyte recruitment activity of a human cytokine by its plant orthologs.

Human macrophage migration-inhibitory factor (MIF) is an evolutionarily-conserved protein that has both extracellular immune-modulating and intracellular cell-regulatory functions. MIF plays a role in various diseases, including inflammatory diseases, atherosclerosis, autoimmunity, and cancer. It serves as an inflammatory cytokine and chemokine, but also exhibits enzymatic activity. Secreted MIF binds to cell-surface immune receptors such as CD74 and CXCR4. Plants possess MIF orthologs but lack the associated receptors, suggesting functional diversification across kingdoms. Here, we characterized three MIF orthologs (termed MIF/d-dopachrome tautomerase-like proteins or MDLs) of the model plant Arabidopsis thaliana Recombinant Arabidopsis MDLs (AtMDLs) share similar secondary structure characteristics with human MIF, yet only have minimal residual tautomerase activity using either p-hydroxyphenylpyruvate or dopachrome methyl ester as substrate. Site-specific mutagenesis suggests that this is due to a distinct amino acid difference at the catalytic cavity-defining residue Asn-98. Surprisingly, AtMDLs bind to the human MIF receptors CD74 and CXCR4. Moreover, they activate CXCR4-dependent signaling in a receptor-specific yeast reporter system and in CXCR4-expressing human HEK293 transfectants. Notably, plant MDLs exert dose-dependent chemotactic activity toward human monocytes and T cells. A small molecule MIF inhibitor and an allosteric CXCR4 inhibitor counteract this function, revealing its specificity. Our results indicate cross-kingdom conservation of the receptor signaling and leukocyte recruitment capacities of human MIF by its plant orthologs. This may point toward a previously unrecognized interplay between plant proteins and the human innate immune system.

Discovery of novel multidrug resistance protein 4 (MRP4) inhibitors as active agents reducing resistance to anticancer drug 6-Mercaptopurine (6-MP) by structure and ligand-based virtual screening.

Multidrug resistance protein 4 (MRP4/ABCC4) is an ATP-binding cassette (ABC) transporter. It is associated with multidrug resistance (MDR), which is becoming a growing challenge to the treatment of cancer and infections. In the context of several types of cancer in which MRP4 is overexpressed, MRP4 inhibition manifests striking effects against cancer progression and drug resistance. In this study, we combined ligand-based and structure-based drug design strategy, by searching the SPECS chemical library to find compounds that are most likely to bind to MRP4. Clustering analysis based on a two-dimensional fingerprint was performed to help with visual selection of potential compounds. Cell viability assays with potential inhibitors and the anticancer drug 6-MP were carried out to identify their bioactivity. As a result, 39 compounds were tested and seven of them reached inhibition above 55% with 6-MP. Then compound Cpd23 was discovered to improve HEK293/MRP4 cell sensibility to 6-MP dramatically, and low concentration Cpd23 (5 µM) achieved the equivalent effect of 50 µM MK571. The accumulation of 6-MP was determined by validated high-performance liquid chromatography methods, and pretreatment of the HEK293/MRP4 cells with 50 µM MK571 or Cpd23 resulted in significantly increased accumulation of 6-MP by approximately 1.5 times. This compound was first reported with a novel scaffold compared with previously known MRP4 inhibitors, which is a hopeful molecular tool that can be used for overcoming multidrug resistance research.

Annealing novel nucleobase-lipids with oligonucleotides or plasmid DNA based on H-bonding or π-π interaction: Assemblies and transfections.

Lipid derivatives of nucleoside analogs have been highlighted for their potential for effective gene delivery. A novel class of nucleobase-lipids are rationally designed and readily synthesized, comprising thymine/cytosine, an ester/amide linker and an oleyl lipid. The diversity of four nucleobase-lipids termed DXBAs (DOTA, DNTA, DOCA and DNCA) is investigated. Besides, DNCA is demonstrated to be an effective neutral transfection material for nucleic acid delivery, which enbles to bind to oligonucleotides via H-bonding and π-π stacking with reduced toxicity in vitro and in vivo. Several kinds of nucleic acid drugs including aptamer, ssRNA, antisense oligonucleotide, and plasmid DNAs can be delivered by DXBAs, especially DNCA. In particular, G4-aptamer AS1411 encapsulated by DNCA exhibits cellular uptake enhancement, lysosome degradation reduction, cell apoptosis promotion, cell cycle phase alteration in vitro and duration prolongation in vivo, resulting in significant anti-proliferative activity. Our results demonstrate that DNCA is a promising transfection agent for G4-aptamers and exhibites bright application prospects in the permeation improvement of single-stranded oligonucleotides or plasmid DNAs.