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.

First-in-Class Selective Inhibitors of the Lysine Acetyltransferase KAT8.

KAT8 is a lysine acetyltransferase primarily catalyzing the acetylation of Lys16 of histone H4 (H4K16). KAT8 dysregulation is linked to the development and metastatization of many cancer types, including non-small cell lung cancer (NSCLC) and acute myeloid leukemia (AML). Few KAT8 inhibitors have been reported so far, none of which displaying selective activity. Based on the KAT3B/KDAC inhibitor C646, we developed a series of N-phenyl-5-pyrazolone derivatives and identified compounds 19 and 34 as low-micromolar KAT8 inhibitors selective over a panel of KATs and KDACs. Western blot, immunofluorescence, and CETSA experiments demonstrated that both inhibitors selectively target KAT8 in cells. Moreover, 19 and 34 exhibited mid-micromolar antiproliferative activity in different cancer cell lines, including NSCLC and AML, without impacting the viability of nontransformed cells. Overall, these compounds are valuable tools for elucidating KAT8 biology, and their simple structures make them promising candidates for future optimization studies.

Discovery of highly potent HDAC8 PROTACs with anti-tumor activity.

Various diseases are deeply associated with aberrations in HDAC8 functions. These aberrations can be assigned to either structural functions or catalytic functions of HDAC8. Therefore, development of HDAC8 degradation inducers might be more promising than HDAC8 inhibitors. We employed the proteolysis targeting chimera (PROTAC) strategy to develop a selective and potent HDAC8 degradation inducer CT-4 with single-digit nanomolar DC50 values and over 95% Dmax in both triple-negative breast cancer MDA-MB-231 cells and T-cell leukemia cells. Notably, CT-4 demonstrated potent anti-migration activity and limited anti-proliferative activity in MDA-MB-231 cells. In contrast, CT-4 effectively induced apototic cell death in Jurkat cells, as assessed by a caspase 3/7 activity assay and flow cytometry. Our findings suggest that the development of HDAC8 degradation inducers holds great potential for the treatment of HDAC8-related diseases.

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.

Deposition Bias of Chromatin Proteins Inverts under DNA Replication Stress Conditions.

Following DNA replication, equal amounts of chromatin proteins are distributed over sister chromatids by re-deposition of parental chromatin proteins and deposition of newly synthesized chromatin proteins. Molecular mechanisms balancing the allocation of new and old chromatin proteins remain largely unknown. Here, we studied the genome-wide distribution of new chromatin proteins relative to parental DNA template strands and replication initiation zones using the double-click-seq. Under control conditions, new chromatin proteins were preferentially found on DNA replicated by the lagging strand machinery. Strikingly, replication stress induced by hydroxyurea or curaxin treatment and inhibition of ataxia telangiectasia and Rad3-related protein (ATR) or p53 inactivation inverted the observed chromatin protein deposition bias to the strand replicated by the leading strand polymerase in line with previously reported effects on replication protein A occupancy. We propose that asymmetric deposition of newly synthesized chromatin proteins onto sister chromatids reflects differences in the processivity of leading and lagging strand synthesis.

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.

Epigenetic regulation in macrophage migration inhibitory factor (MIF)-mediated signaling in cancer and inflammation.

Epigenetic mechanisms are important for the regular development and maintenance of the tissue-specific expression of cytokine genes. One of the crucial cytokines involved in cancer and inflammation is macrophage migration inhibitory factor (MIF), which triggers the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) signaling pathways by binding to CD74 and other receptors. Altered expression of this cytokine and altered activity states of the connected pathways are linked to inflammatory disease and cancer. Therapeutic strategies based on epigenetic mechanisms have the potential to regulate MIF-mediated signaling in cancer and inflammation.

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.

Pharmacological inhibition of lysine-specific demethylase 1 (LSD1) induces global transcriptional deregulation and ultrastructural alterations that impair viability in Schistosoma mansoni.

Treatment and control of schistosomiasis still rely on only one effective drug, praziquantel (PZQ) and, due to mass treatment, the increasing risk of selecting for schistosome strains that are resistant to PZQ has alerted investigators to the urgent need to develop novel therapeutic strategies. The histone-modifying enzymes (HMEs) represent promising targets for the development of epigenetic drugs against Schistosoma mansoni. In the present study, we targeted the S. mansoni lysine-specific demethylase 1 (SmLSD1), a transcriptional corepressor, using a novel and selective synthetic inhibitor, MC3935, which was used to treat schistosomula and adult worms in vitro. By using cell viability assays and optical and electron microscopy, we showed that treatment with MC3935 affected parasite motility, egg-laying, tegument, and cellular organelle structures, culminating in the death of schistosomula and adult worms. In silico molecular modeling and docking analysis suggested that MC3935 binds to the catalytic pocket of SmLSD1. Western blot analysis revealed that MC3935 inhibited SmLSD1 demethylation activity of H3K4me1/2. Knockdown of SmLSD1 by RNAi recapitulated MC3935 phenotypes in adult worms. RNA-Seq analysis of MC3935-treated parasites revealed significant differences in gene expression related to critical biological processes. Collectively, our findings show that SmLSD1 is a promising drug target for the treatment of schistosomiasis and strongly support the further development and in vivo testing of selective schistosome LSD1 inhibitors.

A novel histone acetyltransferase inhibitor A485 improves sensitivity of non-small-cell lung carcinoma cells to TRAIL.

Transcriptional coactivators p300 and CBP catalyze the acetylation of lysine residues in histone proteins. Upregulation of p300 and CBP has been associated with lung, colorectal and hepatocellular cancer, indicating an important role of p300 and CBP in tumorigenesis. Recently, the novel p300 and CBP-selective inhibitor A485 became available, which was shown to inhibit proliferation of 124 different cancer cell lines. Here, we found that downregulation of EP300 or CREBBP enhances apoptosis upon TRAIL stimulation in non-small-cell lung cancer (NSCLC) cells. A485 upregulates pro- and anti-apoptotic genes at the mRNA level, implying an apoptosis-modulating effect in NSCLC cells. However, A485 alone does not induce apoptosis. Interestingly, we observed that the number of apoptotic cells increases upon combined treatment with A485 and TRAIL. Therefore, A485, as a TRAIL-sensitizer, was used in combination with TRAIL in wild type of NSCLC cell lines (HCC827 and H1650) and cells with acquired erlotinib resistance (HCC827-ER and H1650-ER). Our results show that the combination of A485 and TRAIL synergistically increases cell death and inhibits long-term cell proliferation. Furthermore, this combination inhibits the growth of 3D spheroids of EGFR-TKI-resistant cells. Taken together, we demonstrate a successful combination of A485 and TRAIL in EGFR-TKI-sensitive and resistant NSCLC cells.

Improving TRAIL-induced apoptosis in cancers by interfering with histone modifications.

Epigenetic regulation refers to alterations to the chromatin template that collectively establish differential patterns of gene transcription. Post-translational modifications of the histones play a key role in epigenetic regulation of gene transcription. In this review, we provide an overview of recent studies on the role of histone modifications in carcinogenesis. Since tumour-selective ligands such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) are well-considered as promising anti-tumour therapies, we summarise strategies for improving TRAIL sensitivity by inhibiting aberrant histone modifications in cancers. In this perspective we also discuss new epigenetic drug targets for enhancing TRAIL-mediated apoptosis.

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.

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.

Synthesis of Peptoid-Based Class I-Selective Histone Deacetylase Inhibitors with Chemosensitizing Properties.

There is increasing evidence that histone deacetylase (HDAC) inhibitors can (re)sensitize cancer cells for chemotherapeutics via "epigenetic priming". In this work, we describe the synthesis of a series of class I-selective HDAC inhibitors with 2-aminoanilides as zinc-binding groups. Several of the synthesized compounds revealed potent inhibition of the class I HDAC isoforms HDAC1, HDAC2, and/or HDAC3 and promising antiproliferative effects in the human ovarian cancer cell line A2780 and the human squamous carcinoma cell line Cal27. Selected compounds were investigated in a cellular model of platinum resistance. In particular, compound 2a revealed potent chemosensitizing properties and full reversal of cisplatin resistance in Cal27CisR cells. This effect is related to a synergistic increase in caspase 3/7 activation and induction of apoptosis. Thus, this work demonstrates that pan-HDAC inhibition or dual class I/class IIb inhibition is not required for full reversal of cisplatin resistance.

Light-controlled inhibition of BRAFV600E kinase.

Metastatic melanoma is amongst the most difficult types of cancer to treat, with current therapies mainly relying on the inhibition of the BRAFV600E mutant kinase. However, systemic inhibition of BRAF by small molecule drugs in cancer patients results - paradoxically - in increased wild-type BRAF activity in healthy tissue, causing side-effects and even the formation of new tumors. Here we show the development of BRAFV600E kinase inhibitors of which the activity can be switched on and off reversibly with light, offering the possibility to overcome problems of systemic drug activity by selectively activating the drug at the desired site of action. Based on a known inhibitor, eight photoswitchable effectors containing an azobenzene photoswitch were designed, synthesized and evaluated. The most promising inhibitor showed an approximately 10-fold increase in activity upon light-activation. This research offers inspiration for the development of therapies for metastatic melanoma in which tumor tissue is treated with an active BRAFV600E inhibitor with high spatial and temporal resolution, thus limiting the damage to other tissues.

Inhibitory selectivity among class I HDACs has a major impact on inflammatory gene expression in macrophages.

Histone deacetylases (HDACs) play an important role in cancer, degenerative diseases and inflammation. The currently applied HDAC inhibitors in the clinic lack selectivity among HDAC isoforms, which limits their application for novel indications such as inflammatory diseases. Recent, literature indicates that HDAC 3 plays an important role among class I HDACs in gene expression in inflammation. In this perspective, the development and understanding of inhibitory selectivity among HDACs 1, 2 and 3 and their respective influence on gene expression need to be characterized to facilitate drug discovery. Towards this aim, we synthesized nine structural analogues of the class I HDAC inhibitor Entinostat and investigated their selectivity profile among HDACs 1, 2 and 3. We found that we can explain the observed structure activity relationships by small structural and conformational differences between HDAC 1 and HDAC 3 in the 'lid' interacting region. Cell-based studies indicated, however, that application of inhibitors with improved HDAC 3 selectivity did not provide an anti-inflammatory response in contrast to expectations from biochemical evidence in literature. Altogether, in this study, we identified structure activity relationships among class I HDACs and we connected isoform selectivity among class I HDACs with pro- and anti-inflammatory gene transcription in macrophages.

Replacement of an Indole Scaffold Targeting Human 15-Lipoxygenase-1 Using Combinatorial Chemistry.

Human 15-lipoxygenase-1 (15-LOX-1) belongs to the class of lipoxygenases, which catalyze oxygenation of polyunsaturated fatty acids, such as arachidonic and linoleic acid. Recent studies have shown that 15-LOX-1 plays an important role in physiological processes linked to several diseases such as airway inflammation disease, coronary artery disease, and several types of cancer such as rectal, colon, breast and prostate cancer. In this study, we aimed to extend the structural diversity of 15-LOX-1 inhibitors, starting from the recently identified indolyl core. In order to find new scaffolds, we employed a combinatorial approach using various aromatic aldehydes and an aliphatic hydrazide tail. This scaffold-hopping study resulted in the identification of the 3-pyridylring as a suitable replacement of the indolyl core with an inhibitory activity in the micromolar range (IC 50=16±6 µm) and a rapid and efficient structure-activity relationship investigation.