D-dopachrome tautomerase (D-DT), also known as macrophage migration inhibitory factor-2, is a member of the MIF cytokine family and plays a key role in cancer and inflammation. Molecules that bind to the D-DT or MIF-1 tautomerase site could block their biological activity. However, relatively few D-DT inhibitors have been reported. In this study, we designed, synthesized, and screened a focused compound library. This led to the identification of 4h, a furan-2-carboxylic acid derivative with IC50 values of 2.4 μM for D-DT and 9.8 μM for MIF-1. Subsequent SAR optimization yielded the more potent inhibitor 10b, exhibiting IC50 values of 1.5 μM for D-DT and 1.0 μM for MIF-1. The specific interactions of 4h with D-DT and MIF-1 were explored using 1H-15N NMR endpoint titrations. 4h also inhibited D-DT-induced ERK phosphorylation in A549 cells. Thus, 4h and 10b represent a new class of inhibitors that can be utilized as tools to investigate the biological functions of D-DT and MIF-1.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death globally, with progressive emphysema driven by repeated epithelial damage and impaired repair. Recently, we found that secretion of cytokine receptor agonist of nuclear factor κB signaling ligand (RANKL) is higher from lung fibroblasts of patients with COPD compared with control and that RANKL reduces lung epithelial cell death. However, the underlying mechanisms, their conservation across species, and the specific epithelial cell types involved remain unclear. To investigate how RANKL affects lung epithelial cells, we used in vitro and in vivo models. Primary lung organoids from human and mouse epithelial cells assessed progenitor activation and expansion. Furthermore, we used a murine model of elastase-induced alveolar injury to examine which epithelial cell types were affected by RANKL in vivo and whether this was altered upon lung damage. RANKL supplementation increased alveolar organoid formation in both murine and human cultures compared with vehicle-treated controls. In elastase-treated mice, RANKL administration during the early repair phase enhanced the proportion of EpCAM+ epithelial cells and increased transitional epithelial cell states characterized by keratin 8 (Krt8) and major histocompatibility complex II (MHCII) expression following elastase-induced injury. RANKL signaling promoted epithelial regeneration by expanding alveolar progenitor and transitional epithelial cell populations, with consistent effects across human and murine models. These findings identify RANKL as a novel modulator of epithelial repair and suggest that controlled activation of this pathway could enhance lung regeneration in diseases such as COPD.NEW & NOTEWORTHY This study describes for the first time a role for bone cytokine RANKL in lung epithelial repair following lung damage. Using both murine and human models, we describe how RANKL-mediated signaling promotes epithelial expansion by acting on epithelial transitional cell states. For diseases with substantial lung damage, modulating RANKL signaling during the early repair phase following lung injury could represent a strategy to enhance epithelial regeneration.

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.

ABSTRACTReceptor activator for NF-κβ (RANK) ligand (RANKL) is found in lung tissue and elevated in lung diseases like chronic obstructive pulmonary disease (COPD), cystic fibrosis and silica-induced pulmonary fibrosis. RANKL is a well-known stimulator of bone tissue degradation, which may explain the association between these lung diseases and osteoporosis. However, RANKL is also reported to be involved in epithelial cell regeneration in breast and thymus. We hypothesized that RANKL, which is produced directly in lung tissue, is involved in the regeneration of lung epithelial cells. Therefore, we aimed to clarify the specific role of RANKL in this process.Using an organoid model of lung epithelial development by co-culturing primary EpCAM+ lung epithelial cells with fibroblasts, we found higher numbers of alveolar organoids after soluble RANKL treatment compared to control. Importantly, this effect was similar in human RANKL-treated organoids derived from epithelial cells isolated from lung tissue of COPD patients. The effect of RANKL was abrogated upon addition of osteoprotegerin, the soluble inhibitor of RANKL. We also found that RANKL stimulated phosphorylation of Akt suggesting involvement of its receptor RANK in the signaling pathway. Moreover,in vivoRANKL administration resulted in more type II alveolar epithelial cells in lungs of mice with silica-induced pulmonary fibrosis.In conclusion, we found that RANKL promotes type II alveolar epithelial cell regeneration and may therefore be a novel contributor to lung tissue repair.New and NoteworthyOur study provides compelling evidence demonstrating an as of yet unknown function of receptor activator for NF-κβ ligand (RANKL) in lung tissue regeneration. We found that RANKL plays a role in the regeneration of lung epithelial cells, particularly type II alveolar epithelial cells. This may also have clinical implications as promotion of alveolar epithelial cell regeneration may enhance lung tissue repair, an important target in patients with lung diseases like COPD and fibrosis.

In proteins, the amino acids Phe, Tyr, and especially Trp are frequently involved in π interactions such as π-π, cation-π, and CH-π bonds. These interactions are often crucial for protein structure and protein-ligand binding. A powerful means to study these interactions is progressive fluorination of these aromatic residues to modulate the electrostatic component of the interaction. However, to date no protein expression platform is available to produce milligram amounts of proteins labeled with such fluorinated amino acids. Here, we present a Lactococcus lactis Trp auxotroph-based expression system for efficient incorporation (≥95%) of mono-, di-, tri-, and tetrafluorinated, as well as a methylated Trp analog. As a model protein we have chosen LmrR, a dimeric multidrug transcriptional repressor protein from L. lactis. LmrR binds aromatic drugs, like daunomycin and riboflavin, between Trp96 and Trp96' in the dimer interface. Progressive fluorination of Trp96 decreased the affinity for the drugs 6- to 70-fold, clearly establishing the importance of electrostatic π-π interactions for drug binding. Presteady state kinetic data of the LmrR-drug interaction support the enthalpic nature of the interaction, while high resolution crystal structures of the labeled protein-drug complexes provide for the first time a structural view of the progressive fluorination approach. The L. lactis expression system was also used to study the role of Trp68 in the binding of riboflavin by the membrane-bound riboflavin transport protein RibU from L. lactis. Progressive fluorination of Trp68 revealed a strong electrostatic component that contributed 15-20% to the total riboflavin-RibU binding energy.

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.

BACKGROUND:Emphysematous COPD is characterized by aberrant alveolar repair. Macrophage migration inhibitory factor (MIF) contributes to alveolar repair, but for its structural and functional homolog D-dopachrome tautomerase (DDT) this is unknown. MIF mediates its effects through CD74 and/or C-X-C chemokine receptors 2 (CXCR2), 4(CXCR4), and possibly 7 (ACKR3). DDT can also signal through CD74, but interactions with other receptors have not been described yet. We therefore aimed at investigating if and how DDT contributes to epithelial repair in COPD.METHODS:We studied effects of recombinant DDT on cell proliferation and survival by clonogenic assay and annexin V-PI staining respectively. DDT-induced signaling was investigated by Western blot. Effects on epithelial growth and differentiation was studied using lung organoid cultures with primary murine or human epithelial cells and incubating with DDT or an ACKR3-blocking nanobody. DDT-ACKR3 interactions were identified by ELISA and co-immunoprecipitation.FINDINGS:We found that DDT promoted proliferation of and prevented staurosporine-induced apoptosis in A549 lung epithelial cells. Importantly, DDT also stimulated growth of primary alveolar epithelial cells as DDT treatment resulted in significantly more and larger murine and human alveolar organoids compared to untreated controls. The anti-apoptotic effect of DDT and DDT-induced organoid growth were inhibited in the presence of an ACKR3-blocking nanobody. Furthermore, ELISA assay and co-immunoprecipitation suggested DDT complexes with ACKR3. DDT could activate the PI3K-Akt pathway and this activation was enhanced in ACKR3-overexpressing cells.INTERPRETATION:In conclusion, DDT contributes to alveolar epithelial repair via ACKR3 and may thus augment lung epithelial repair in COPD.FUNDING:As stated in the Acknowledgments.

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.

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.

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.

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.

Higher levels of receptor activator for NF-κβ ligand (RANKL) are found in serum of emphysema patients. RANKL is a well-known stimulator of bone tissue degradation, possibly explaining the association of COPD with osteoporosis. However, RANKL is also reported to be involved in epithelial cell regeneration in breast and thymus. Given RANKL is produced directly in lung tissue, we hypothesized a role for RANKL in lung epithelial cell regeneration. The aim of this study was to elucidate the role of RANKL in lung epithelial repair. Mouse soluble RANKL (sRANKL) treatment of murine precision-cut lung slices resulted in a higher number of proliferating cells compared to untreated controls. We also found that sRANKL stimulated proliferation of type II alveolar (A549 cells) but not airway (16HBE) epithelial cells. Using an organoid model of epithelial development by co-culturing primary EpCAM+ cells with fibroblasts, we found higher numbers of alveolar organoids in cultures derived from murine epithelial cells upon sRANKL treatment compared to control. Importantly, this effect was similar in RANKL-treated organoids derived from epithelial cells isolated from lung tissue of COPD patients. The effect of sRANKL was abrogated upon addition of osteoprotegerin, the soluble, inhibitory, receptor for RANKL. In conclusion, we found that sRANKL promotes type II alveolar epithelial cell proliferation and may therefore contribute to lung tissue repair. Our data suggest that the higher levels of RANKL found in emphysema, may reflect an attempt at epithelial repair by the lung to counteract the lung tissue destruction that characterizes emphysema.

Correction of a genetic mutation via homologous recombination (HR) is a promising approach to actually cure some genetic diseases. To increase HR effi ciency, DNA damage can be induced specifi cally to the target gene using DNA targeting agents such as triple helix forming oligonucleotides (TFOs), either alone or fused to DNA damaging agents. In this study, we report on sequence-specifi c TFOs conjugated to sequence-specifi c restriction endonucleases as double targeted nucleases. TFOs were designed for acid-α-glucosidase (GAA), the gene mutated in Pompe disease, and fused to camptothecin or restriction endonucleases. The agents were tested for target binding affi nity by BIAcore, for DNA damage by measuring γH2AX, and for HR induction using luciferase reporter plasmids. Co-transfecting TFO-MunI conjugates with a promoter-less reporter plasmid and a promoter-containing HR fragment resulted in restoration of luciferase activity to up to 24% of the promoter containing control. The data presented here demonstrate that TFOrestriction endonuclease conjugates are promising candidates for gene correction approaches for monogenetic disorders like Pompe disease.

Fibrosis is characterized by the progressive alteration of the tissue structure due to the excessive production of extracellular matrix (ECM). The signaling system encompassing Receptor Activator of Nuclear factor NF-κB Ligand (RANKL)/RANK/Osteoprotegerin (OPG) was discovered to play an important role in the regulation of ECM formation and degradation in bone tissue. However, whether and how this signaling pathway plays a role in liver or pulmonary ECM degradation is unclear up to now. Interestingly, increased decoy receptor OPG levels are found in fibrotic tissues. We hypothesize that RANKL can stimulate RANK on macrophages and initiate the process of ECM degradation. This process may be inhibited by highly expressed OPG in fibrotic conditions. In this case, RANKL mutants that can bind to RANK without binding to OPG might become promising therapeutic candidates. In this study, we built a structure-based library containing 44 RANKL mutants and found that the Q236 residue of RANKL is important for OPG binding. We show that RANKL_Q236D can activate RAW cells to initiate the process of ECM degradation and is able to escape from the obstruction by exogenous OPG. We propose that the generation of RANKL mutants with reduced affinity for OPG is a promising strategy for the exploration of new therapeutics against fibrosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis that may be a promising agent in cancer therapy due to its selectivity toward tumor cells. However, many cancer cells are resistant to TRAIL due to defects in apoptosis signaling or activation of survival pathways. We hypothesized that a disruption of pro-survival signaling cascades with the multi-tyrosine kinase inhibitor sunitinib would be an effective strategy to enhance TRAIL-mediated apoptosis. Here we demonstrate that sunitinib significantly augments the anticancer activity of TRAIL in models of colon cancer. The therapeutic benefit of the TRAIL/sunitinib combination was associated with increased apoptosis marked by enhanced caspase-3 cleavage and DNA fragmentation. Overexpression of the anti-apoptotic factor B-cell lymphoma 2 (BCL-2) in HCT116 cells reduced TRAIL/sunitinib-mediated apoptosis, further supporting that sunitinib enhances the anticancer activity of TRAIL via augmented apoptosis. Analysis of pro-survival factors identified that the combination of TRAIL and sunitinib significantly downregulated the anti-apoptotic protein X-linked inhibitor of apoptosis protein (XIAP) through a c-Jun N-terminal kinase (JNK)-mediated mechanism. Short hairpin RNA (shRNA)-mediated knockdown of JNK confirmed its key role in the regulation of sensitivity to this combination as cells with suppressed JNK expression exhibited significantly reduced TRAIL/sunitinib-mediated apoptosis. Importantly, the therapeutic benefit of the TRAIL/sunitinib combination was validated in the HCT116-Luc and HCT15 colon cancer xenograft models, which both demonstrated significant anti-tumor activity in response to combination treatment. Collectively, our data demonstrate that sunitinib enhances TRAIL-mediated apoptosis by heightened JNK activation, diminished XIAP levels, and augmented apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis that may be a promising agent in cancer therapy due to its selectivity toward tumor cells. However, many cancer cells are resistant to TRAIL due to defects in apoptosis signaling or activation of survival pathways. We hypothesized that a disruption of pro-survival signaling cascades with the multi-tyrosine kinase inhibitor sunitinib would be an effective strategy to enhance TRAIL-mediated apoptosis. Here we demonstrate that sunitinib significantly augments the anticancer activity of TRAIL in models of colon cancer. The therapeutic benefit of the TRAIL/sunitinib combination was associated with increased apoptosis marked by enhanced caspase-3 cleavage and DNA fragmentation. Overexpression of the anti-apoptotic factor B-cell lymphoma 2 (BCL-2) in HCT116 cells reduced TRAIL/sunitinib-mediated apoptosis, further supporting that sunitinib enhances the anticancer activity of TRAIL via augmented apoptosis. Analysis of pro-survival factors identified that the combination of TRAIL and sunitinib significantly downregulated the anti-apoptotic protein X-linked inhibitor of apoptosis protein (XIAP) through a c-Jun N-terminal kinase (JNK)-mediated mechanism. Short hairpin RNA (shRNA)-mediated knockdown of JNK confirmed its key role in the regulation of sensitivity to this combination as cells with suppressed JNK expression exhibited significantly reduced TRAIL/sunitinib-mediated apoptosis. Importantly, the therapeutic benefit of the TRAIL/sunitinib combination was validated in the HCT116-Luc and HCT15 colon cancer xenograft models, which both demonstrated significant anti-tumor activity in response to combination treatment. Collectively, our data demonstrate that sunitinib enhances TRAIL-mediated apoptosis by heightened JNK activation, diminished XIAP levels, and augmented apoptosis.

The HLA class II histocompatibility antigen gamma chain, also known as HLA-DR antigen-associated invariant chain or CD74, has been shown to be involved in many biological processes amongst which antigen loading and transport of MHC class II molecules from the endoplasmic reticulum to the Golgi complex. It is also part of a receptor complex for macrophage migration inhibitory factor (MIF), and participates in inflammatory signaling. The inhibition of MIF-CD74 complex formation is regarded as a potentially attractive therapeutic target in inflammation, cancer and immune diseases. In order to be able to produce large quantities of the extracellular moiety of human CD74, which has been reported to be unstable and protease-sensitive, different constructs were made as fusions with two solubility enhancers: the well-known maltose-binding domain and Fh8, a small protein secreted by the parasite Fasciola hepatica. The fusion proteins could be purified with high yields from Escherichia coli and were demonstrated to be active in binding to MIF. Moreover, our results strongly suggest that the MIF binding site is located in the sequence between the transmembrane and the membrane-distal trimerisation domain of CD74, and comprises at least amino acids 113-125 of CD74.

The ability to target DNA specifically at any given position within the genome allows many intriguing possibilities and has inspired scientists for decades. Early gene-targeting efforts exploited chemicals or DNA oligonucleotides to interfere with the DNA at a given location in order to inactivate a gene or to correct mutations. We here describe an example towards correcting a genetic mutation underlying Pompe's disease using a nucleotide-fused nuclease (TFO-MunI). In addition to the promise of gene correction, scientists soon realized that genes could be inactivated or even re-activated without inducing potentially harmful DNA damage by targeting transcriptional modulators to a particular gene. However, it proved difficult to fuse protein effector domains to the first generation of programmable DNA-binding agents. The engineering of gene-targeting proteins (zinc finger proteins (ZFPs), transcription activator-like effectors (TALEs)) circumvented this problem. The disadvantage of protein-based gene targeting is that a fusion protein needs to be engineered for every locus. The recent introduction of CRISPR/Cas offers a flexible approach to target a (fusion) protein to the locus of interest using cheap designer RNA molecules. Many research groups now exploit this platform and the first human clinical trials have been initiated: CRISPR/Cas has kicked off a new era of gene targeting and is revolutionizing biomedical sciences. This article is part of a discussion meeting issue ‘Frontiers in epigenetic chemical biology’.

Macrophage migration inhibitory factor (MIF) is an important cytokine for which an increasing number of functions is being described in the pathogenesis of inflammation and cancer. Nevertheless, the availability of potent and druglike MIF inhibitors that are well-characterized in relevant disease models remains limited. Development of highly potent and selective small-molecule MIF inhibitors and validation of their use in relevant disease models will advance drug discovery. In this review, we provide an overview of recent advances in the identification of MIF as a pharmacological target in the pathogenesis of inflammatory diseases and cancer. We also give an overview of the current developments in the discovery and design of small-molecule MIF inhibitors and define future aims in this field.

Activated hepatic stellate cells (HSCs) are known to play a central role in liver fibrosis and their elimination is a crucial step toward the resolution and reversion of liver fibrosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a molecule that may contribute to the apoptotic removal of activated HSC through binding to its dedicated receptors. In the present study, we investigated the potential application of recombinant receptor-specific TRAIL proteins in the efficient elimination of activated HSCs. Our finding revealed differential contribution of TRAIL receptors among HSCs populations with activated hepatic stellate cells expresses more TRAIL receptors DR5. In vitro treatment of activated HSCs with DR5-specific or wild-type TRAIL variants induced a significant reduction in viability and extracellular matrix production, whereas no significant decrease in viability was associated with the treatment of cells by DR4-specific TRAIL. Our analysis indicate the successful application of the DR5 receptor-specific TRAIL variant in the targeted elimination of activated HSCs via interference with collagen production and simultaneous induction of apoptosis via activation of the caspase pathway. DR5 receptor-specific TRAIL may thus represent a new therapeutic compound for the treatment of liver fibrosis.