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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.

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.

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 replication protein occupancy. propose asymmetric deposition newly synthesized chromatin proteins onto sister chromatids reflects differences in the processivity of leading and lagging strand synthesis.

The acquisition of iron is a crucial mechanism for the survival of pathogenic bacteria such as Pseudomonas aeruginosa in eukaryotic hosts. The key iron chelator in this organism is the siderophore pyoverdine, which was shown to be crucial for iron homeostasis. Pyoverdine is a non-ribosomal peptide with several maturation steps in the cytoplasm and others in the periplasmatic space. A key enzyme for its maturation is the acylase PvdQ. The inhibition of PvdQ stops the maturation of pyoverdine causing a significant imbalance in the iron homeostasis and hence can negatively influence the survival of P. aeruginosa. In this work, we successfully synthesized chromene-derived inhibitory molecules targeting PvdQ in a low micromolar range. In silico modeling as well as kinetic evaluations of the inhibitors suggest a competitive inhibition of the PvdQ function. Further, we evaluated the inhibitor in vivo on P. aeruginosa cells and report a dose-dependent reduction of pyoverdine formation. The compound also showed a protecting effect in a Galleria mellonella infection model.

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., Activity‐based labeling of lipoxygenase (LOX) activity using the novel one‐step labeling probe Labelox B enables detection of isoenzyme inhibitory selectivity in an ELISA‐based assay and visualization of the localization of LOX activity in living cells. The observed nuclear localization was rationalized by identification of a bromodomain‐like region in the 15‐LOX‐1 isoenzyme.

Histone acetylation is an important, reversible post-translational protein modification and a hallmark of epigenetic regulation. However, little is known about the dynamics of this process, due to the lack of analytical methods that can capture site-specific acetylation and deacetylation reactions. We present a new approach that combines metabolic and chemical labeling (CoMetChem) using uniformly 13C-labeled glucose and stable isotope-labeled acetic anhydride. Thereby, chemically equivalent, fully acetylated histone species are generated, enabling accurate relative quantification of site-specific lysine acetylation dynamics in tryptic peptides using high-resolution mass spectrometry. We show that CoMetChem enables site-specific quantification of the incorporation or loss of lysine acetylation over time, allowing the determination of reaction rates for acetylation and deacetylation. Thus, the CoMetChem methodology provides a comprehensive description of site-specific acetylation dynamics.

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., Targeting macrophage migration inhibitory factor (MIF) is challenging, as it is involved in diseases via protein‐protein interactions. Here, the first potent MIF‐directed PROTAC MD13 is reported, which eliminates MIF from its protein‐protein interaction network by inducing MIF degradation. MD13 suppresses the proliferation of A549 cells by reducing MIF protein levels and MIF‐related signaling, demonstrating the potential of PROTACs in cancer therapy.

Biaryl sulfonamides are excellent candidates for the azologization approach that yields photoswitchable drugs more active in their metastable cis state, compared to the stable trans state. Here we present the scope and limitations of this strategy for rational design in photopharmacology.

A practical, efficient, and selective electrochemicalN-demethylation method of tropane alkaloids to their nortropane derivatives is described. Nortropanes, such as noratropine and norscopolamine, are important intermediates for the semi-synthesis of the medicines ipratropium or oxitropium bromide, respectively. Synthesis was performed in a simple home-made electrochemical batch cell using a porous glassy carbon electrode. The reaction proceeds at room temperature in one step in a mixture of ethanol or methanol and water. The method avoids hazardous oxidizing agents such as H(2)O(2)orm-chloroperbenzoic acid (m-CPBA), toxic solvents such as chloroform, as well as metal-based catalysts. Various key parameters were investigated in electrochemical batch or flow cells, and the optimized conditions were used in batch and flow-cells at gram scale to synthesize noratropine in high yield and purity using a convenient liquid-liquid extraction method without any need for chromatographic purification. Mechanistic studies showed that the electrochemicalN-demethylation proceeds by the formation of an iminium intermediate which is converted by water as the nucleophile. The optimized method was further applied to scopolamine, cocaine, benzatropine, homatropine and tropacocaine, showing that this is a generic way ofN-demethylating tropane alkaloids to synthesize valuable precursors for pharmaceutical products.

Asthma is an inflammatory airway disease that is estimated to affect 339 million people globally. The symptoms of about 5-10% of patients with asthma are not adequately controlled with current therapy, and little success has been achieved in developing drugs that target the underlying mechanisms of asthma rather than suppressing symptoms. Over the past 3 years, well powered genetic studies of asthma have increased the number of independent asthma-associated genetic loci to 128. In this Series paper, we describe the immense progress in asthma genetics over the past 13 years and link asthma genetic variants to possible drug targets. Further studies are needed to establish the functional significance of gene variants associated with asthma in subgroups of patients and to describe the biological networks within which they function. The genomics-guided discovery of plausible drug targets for asthma could pave the way for the repurposing of existing drugs for asthma and the development of new treatments.

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

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.

The family of macrophage migration inhibitory factor (MIF) proteins in humans consist of MIF, its functional homolog D-dopachrome tautomerase (D-DT, also known as MIF-2) and the relatively unknown protein named DDT-like (DDTL). MIF is a pleiotropic cytokine with multiple properties in tissue homeostasis and pathology. MIF was initially found to associate with inflammatory responses and therefore established a reputation as a pro-inflammatory cytokine. However, increasing evidence demonstrates that MIF influences many different intra- and extracellular molecular processes important for the maintenance of cellular homeostasis, such as promotion of cellular survival, antioxidant signaling, and wound repair. In contrast, studies on D-DT are scarce and on DDTL almost nonexistent and their functions remain to be further investigated as it is yet unclear how similar they are compared to MIF. Importantly, the many and sometimes opposing functions of MIF suggest that targeting MIF therapeutically should be considered carefully, taking into account timing and severity of tissue injury. In this review, we focus on the latest discoveries regarding the role of MIF family members in tissue injury, inflammation and repair, and highlight the possibilities of interventions with therapeutics targeting or mimicking MIF family proteins.

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.

Affinity ligands such as antibodies are widely used in (bio)medical research for purifying proteins from complex biological samples. These ligands are generally immobilized onto solid supports which facilitate the separation of a captured protein from the sample matrix. Adsorptive microtiter plates are commonly used as solid supports prior to immunochemical detection (e.g., immunoassays) but hardly ever prior to liquid chromatography-mass spectrometry (LC-MS-)-based detection. Here, we describe the use of adsorptive microtiter plates for protein enrichment prior to LC-MS detection, and we discuss opportunities and challenges of corresponding workflows, based on examples of targeted (i.e., soluble receptor for advanced glycation end-products (sRAGE) in human serum) and discovery-based workflows (i.e., transcription factor p65 (NF-κB) in lysed murine RAW 264.7 macrophages and peptidyl-prolyl cis-trans isomerase FKBP5 (FKBP5) in lysed human A549 alveolar basal epithelial cells). Thereby, we aim to highlight the potential usefulness of adsorptive microtiter plates in affinity purification workflows prior to LC-MS detection, which could increase their usage in mass spectrometry-based protein research.

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.

Human 15-lipoxygenase-1 (15-LOX-1) is a mammalian lipoxygenase which plays an important regulatory role in several CNS and inflammatory lung diseases. To further explore the role of this enzyme in drug discovery, novel potent inhibitors with favorable physicochemical properties are required. In order to identify such new inhibitors, we established a combinatorial screening method based on acylhydrazone chemistry. This represents a novel application of combinatorial chemistry focusing on the improvement of physicochemical properties, rather than on potency. This strategy allowed us to efficiently screen 44 reaction mixtures of different hydrazides and our previously reported indole aldehyde core structure, without the need for individual synthesis of all possible combinations of building blocks. Our approach afforded three new inhibitors with IC50 values in the nanomolar range and improved lipophilic ligand efficiency.

Biaryl sulfonamides are excellent candidates for the azologization approach that yields photoswitchable drugs more active in their metastable cis state, compared to the stable trans state. Here we present the scope and limitations of this strategy for rational design in photopharmacology.

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

Histone acetylation is an important, reversible post-translational protein modification and a hallmark of epigenetic regulation. However, little is known about the dynamics of this process, due to the lack of analytical methods that can capture site-specific acetylation and deacetylation reactions. We present a new approach that combines metabolic and chemical labeling (CoMetChem) using uniformly 13C-labeled glucose and stable isotope labeled acetic anhydride. Thereby, chemically equivalent, fully acetylated histone species are generated enabling accurate relative quantification of site-specific lysine acetylation in tryptic peptides using high-resolution mass spectrometry. We show that CoMetChem enables site-specific quantification of the incorporation or loss of lysine acetylation over time, allowing the determination of reaction rates for acetylation and deacetylation. Thus, the CoMetChem methodology provides a comprehensive description of site-specific acetylation dynamics.

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

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.

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.

Brain tumours are among the deadliest tumours being highly resistant to currently available therapies. The proliferative behaviour of gliomas is strongly influenced by ion channel activity. Small-conductance calcium-activated potassium (SK/KCa) channels are a family of ion channels that are associated with cell proliferation and cell survival. A combined treatment of classical anti-cancer agents and pharmacological SK channel modulators has not been addressed yet. We used the gold-derivative auranofin to induce cancer cell death by targeting thioredoxin reductases in combination with CyPPA to activate SK channels in neuro- and glioblastoma cells. Combined treatment with auranofin and CyPPA induced massive mitochondrial damage and potentiated auranofin-induced toxicity in neuroblastoma cells in vitro. In particular, mitochondrial integrity, respiration and associated energy generation were impaired. These findings were recapitulated in patient-derived glioblastoma neurospheres yet not observed in non-cancerous HT22 cells. Taken together, integrating auranofin and SK channel openers to affect mitochondrial health was identified as a promising strategy to increase the effectiveness of anti-cancer agents and potentially overcome resistance.

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.

Histone acetyltransferases (HATs) are important mediators of epigenetic post-translational modifications of histones that play important roles in health and disease. A disturbance of these modifications can result in disease states, such as cancer or inflammatory diseases. Inhibitors of HATs (HATi) such as lysine (K) acetyltransferase 8 (KAT8), could be used to study the epigenetic processes in diseases related to these enzymes or to investigate HATs as therapeutic targets. However, the development of HATi is challenged by the difficulties in kinetic characterization of HAT enzymes and their inhibitors to enable calculation of a reproducible inhibitory potency. In this study, a fragment screening approach was used, enabling identification of 4-amino-1-naphthol, which potently inhibited KAT8. The inhibitor was investigated for enzyme inhibition using kinetic and calorimetric binding studies. This allowed for calculation of the K-i values for both the free enzyme as well as the acetylated intermediate. Importantly, it revealed a striking difference in binding affinity between the acetylated enzyme and the free enzyme, which could not be revealed by the IC50 value. This shows that kinetic characterization of inhibitors and calculation of K-i values is crucial for determining the binding constants of HAT inhibitors. We anticipate that more comprehensive characterization of enzyme inhibition, as described here, is needed to advance the field of HAT inhibitors. (C) 2017 Elsevier Masson SAS. All rights reserved.

Lysine acetylations are post-translational modifications of cellular proteins, that are crucial in the regulation of many cellular processes. Lysine acetylations on histone proteins are part of the epigenetic code regulating gene expression and are installed by histone acetyltransferases. Observations that inflammatory lung diseases, such as asthma and chronic obstructive pulmonary disease, are characterized by increased histone acetyltransferase activity indicate that development of small molecule inhibitors for these enzymes might be a valuable approach towards new therapies for these diseases. The 6-alkylsalicylate MG149 is a candidate to explore this hypothesis because it has been demonstrated to inhibit the MYST type histone acetyltransferases. In this study, we determined the K i value for inhibition of the MYST type histone acetyltransferase KAT8 by MG149 to be 39 ± 7.7 μM. Upon investigating whether the inhibition of histone acetyltransferases by MG149 correlates with inhibition of histone acetylation in murine precision-cut lung slices, inhibition of acetylation was observed using an LC-MS/MS based assay on histone H4 res 4–17, which contains the target lysine of KAT8. Following up on this, upon treatment with MG149, reduced pro-inflammatory gene expression was observed in lipopolysaccharide and interferon gamma stimulated murine precision-cut lung slices. Based on this, we propose that 6-alkylsalicylates such as MG149 have potential for development towards applications in the treatment of inflammatory lung diseases.

The implications of lysine-specific demethylase-1 (LSD1) in tumorigenesis have urged scientists to develop diagnostic tools in order to explore the function of this enzyme. In this work, we present our efforts on the development of tranylcypromine (TCP)-based functionalized probes for activity-based protein profiling (ABPP) of LSD1 activity. Biotinylated forms of selected compounds enabled dose-dependent enzyme labeling of recombinant LSD1. However, treatment with LSD1 inhibitors did not clearly reduce the LSD1 labeling efficiency thus indicating that labeling using these probes is not activity dependent. This calls for alternative strategies to develop probes for ABPP of the enzyme LSD1.

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.

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., Author summary Schistosomiasis mansoni is a chronic and debilitating tropical disease caused by the helminth Schistosoma mansoni. The control and treatment of the disease rely almost exclusively on praziquantel (PZQ). Thus, there is an urgent need to search for promising protein targets to develop new drugs. Drugs that inhibit enzymes that modify the chromatin structure have been developed for a number of diseases. We and others have shown that S. mansoni epigenetic enzymes are also potential therapeutic targets. Here we evaluated the potential of the S. mansoni histone demethylase LSD1 (SmLSD1) as a drug target. We reported the synthesis of a novel and potent LSD1 inhibitor, MC3935, and show that it selectively inhibited the enzymatic activity of SmLSD1. Treatment of juvenile or adult worms with MC3935 caused severe damage to the tegument of the parasites and compromised egg production. Importantly, MC3935 proved to be highly toxic to S. mansoni, culminating in the death of juvenile or adult worms within 96 h. Transcriptomic analysis of MC3935-treated parasites revealed changes in the gene expression of hundreds of genes involved in key biological processes. Importantly, SmLSD1 contains unique sequences within its polypeptide chain that could be explored to develop a S. mansoni selective drug.

International journal of biological macromolecules 146, 212 - 221 (2020). doi:10.1016/j.ijbiomac.2019.12.252, The biosynthesis of pyoverdine, the major siderophore of Pseudomonas aeruginosa, is a well-organized process involving a discrete number of enzyme-catalyzed steps. The final step of this process involves the PvdP tyrosinase, which converts ferribactin into pyoverdine. Thus, inhibition of the PvdP tyrosinase activity provides an attractive strategy to interfere with siderophore synthesis to manage P. aeruginosa infections. Here, we report phenylthiourea as a non-competitive inhibitor of PvdP for which we solved a crystal structure in complex with PvdP. The crystal structure indicates that phenylthiourea binds to an allosteric binding site and thereby interferes with its tyrosinase activity. We further provide proofs that PvdP tyrosinase inhibition by phenylthiourea requires the C-terminal lid region. This provides opportunities to develop inhibitors that target the allosteric site, which seems to be confined to fluorescent pseudomonads, and not the tyrosinase active site. Furthermore, increases the chances to identify PvdP inhibitors that selectively interfere with siderophore synthesis., Published by Elsevier, New York, NY [u.a.]

Around three million patients die due to airway inflammatory diseases each year. The most notable of these diseases are asthma and chronic obstructive pulmonary disease (COPD). Therefore, new therapies are urgently needed. Promising targets are histone deacetylases (HDACs), since they regulate posttranslational protein acetylation. Over a thousand proteins are reversibly acetylated, and acetylation critically influences aberrant intracellular signaling pathways in asthma and COPD. The diverse set of selective and non-selective HDAC inhibitors used in pre-clinical models of airway inflammation show promising results, but several challenges still need to be overcome. One such challenge is the design of HDAC inhibitors with unique selectivity profiles, such as selectivity towards specific HDAC complexes. Novel strategies to disrupt HDAC complexes should be developed to validate HDACs further as targets for new anti-inflammatory pulmonary treatments.

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.

Despite the rapid development of CRISPR/Cas9-mediated gene editing technology, the gene editing potential of CRISPR/Cas9 is hampered by low efficiency, especially for clinical applications. One of the major challenges is that chromatin compaction inevitably limits the Cas9 protein access to the target DNA. However, chromatin compaction is precisely regulated by histone acetylation and deacetylation. To overcome these challenges, we have comprehensively assessed the impacts of histone modifiers such as HDAC (1–9) inhibitors and HAT (p300/CBP, Tip60 and MOZ) inhibitors, on CRISPR/Cas9 mediated gene editing efficiency. Our findings demonstrate that attenuation of HDAC1, HDAC2 activity, but not other HDACs, enhances CRISPR/Cas9-mediated gene knockout frequencies by NHEJ as well as gene knock-in by HDR. Conversely, inhibition of HDAC3 decreases gene editing frequencies. Furthermore, our study showed that attenuation of HDAC1, HDAC2 activity leads to an open chromatin state, facilitates Cas9 access and binding to the targeted DNA and increases the gene editing frequencies. This approach can be applied to other nucleases, such as ZFN and TALEN.

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 plantArabidopsis thaliana. RecombinantArabidopsisMDLs (AtMDLs) share similar secondary structure characteristics with human MIF, yet only have minimal residual tautomerase activity using eitherp-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.

Non-alcoholic fatty liver disease (NAFLD) is a common liver disorder closely related to metabolic syndrome. NAFLD can progress to an inflammatory state called non-alcoholic steatohepatitis (NASH), which may result in the development of fibrosis and hepatocellular carcinoma. To develop therapeutic strategies against NAFLD, a better understanding of the molecular mechanism is needed. Current in vitro NAFLD models fail to capture the essential interactions between liver cell types and often do not reflect the pathophysiological status of patients. To overcome limitations of commonly used in vitro and in vivo models, precision-cut liver slices (PCLSs) were used in this study. PCLSs, prepared from liver tissue obtained from male Wistar rats, were cultured in supraphysiological concentrations of glucose, fructose, insulin, and palmitic acid to mimic metabolic syndrome. Accumulation of lipid droplets was visible and measurable after 24 h in PCLSs incubated with glucose, fructose, and insulin, both in the presence and absence of palmitic acid. Upregulation of acetyl-CoA carboxylase 1 and 2, and of sterol responsive element binding protein 1c, suggests increased de novo lipogenesis in PCLSs cultured under these conditions. Additionally, carnitine palmitoyltransferase 1 expression was reduced, which indicates impaired fatty acid transport and disrupted mitochondrial &beta, oxidation. Thus, steatosis was successfully induced in PCLSs with modified culture medium. This novel ex vivo NAFLD model could be used to investigate the multicellular and molecular mechanisms that drive NAFLD development and progression, and to study potential anti-steatotic drugs.

Antimicrobial resistance resulting in ineffective treatment of infectious diseases is an increasing global problem, particularly in infections with pathogenic bacteria. In some bacteria, such as Streptococcus pyogenes, the pathogenicity is strongly linked to the attachment of virulence factors. Their attachment to the cellular membrane is a transpeptidation reaction, catalyzed by sortase enzymes. As such, sortases pose an interesting target for the development of new antivirulence strategies that could yield novel antimicrobial drugs. Using the substitution-oriented fragment screening (SOS) approach, we discovered a potent and specific inhibitor (C10) of sortase A from S. pyogenes. The inhibitor C10 showed high specificity towards S. pyogenes sortase A, with an IC50 value of 10 μM and a Kd of 60 μM. We envision that this inhibitor could be employed as a starting point for further exploration of sortase's potential as therapeutic target for antimicrobial drug development.

Activation of inflammatory gene expression is regulated, among other factors, by post-translational modifications of histone proteins. The most investigated type of histone modifications is lysine acetylations. Histone deacetylases (HDACs) remove acetylations from lysines, thereby influencing (inflammatory) gene expression. Intriguingly, apart from histones, HDACs also target non-histone proteins. The nuclear factor kappa B (NF-kappa B) pathway is an important regulator in the expression of numerous inflammatory genes, and acetylation plays a crucial role in regulating its responses. Several studies have shed more light on the role of HDAC 1-3 in inflammation with a particular pro-inflammatory role for HDAC 3. Nevertheless, the HDAC-NF-kappa B interactions in inflammatory signalling have not been fully understood. An important challenge in targeting the regulatory role of HDACs in the NF-kappa B pathway is the development of highly potent small molecules that selectively target HDAC iso-enzymes. This review focuses on the role of HDAC 3 in (NF-kappa B-mediated) inflammation and NF-kappa B lysine acetylation. In addition, we address the application of frequently used small molecule HDAC inhibitors as an approach to attenuate inflammatory responses, and their potential as novel therapeutics. Finally, recent progress and future directions in medicinal chemistry efforts aimed at HDAC 3-selective inhibitors are discussed.

Graphical abstract, The increasing number of patients suffering from chronic obstructive pulmonary disease (COPD) represents a major and increasing health problem. Therefore, novel therapeutic approaches are needed. Class I HDACs 1, 2 and 3 play key roles in the regulation of inflammatory gene expression with a particular pro-inflammatory role for HDAC 3. HDAC 3 has been reported to be an important player in inflammation by deacetylating NF-κB p65, which has been implicated in the pathology of COPD. Here, we applied the pharmacological HDAC 3-selective inhibitor RGFP966, which attenuated pro-inflammatory gene expression in models for inflammatory lung diseases. Consistent with this, a robust decrease of the transcriptional activity of NF-κB p65 was observed. HDAC 3 inhibition affected neither the acetylation status of NF-κB p65 nor histone H3 or histone H4. This indicates that HDAC 3 inhibition does not inhibit NF-κB p65 transcriptional activity by affecting its deacetylation but rather by inhibiting enzymatic activity of HDAC 3. Taken together, our findings indicate that pharmacological HDAC 3-selective inhibition by inhibitors such as RGFP966 may provide a novel and effective approach toward development of therapeutics for inflammatory lung diseases.

We developed a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the site-specific quantification of lysine acetylation in the N-terminal region of histone H4 by combining chemical derivatization at the protein and peptide levels with digestion using chymotrypsin and trypsin. Unmodified ε-amino groups were first modified with propionic acid anhydride and the derivatized protein digested with trypsin and chymotrypsin. The newly formed peptide N-termini were subjected to a second derivatization step with d6- (heavy) or d0- (light) acetic acid anhydride. Samples were mixed at different ratios and peptides monitored by multiple reaction monitoring (MRM) LC-MS/MS. The method was validated in terms of linearity (R2 ≥ 0.94), precision (RSD ≤ 10 %), and accuracy (≤27 %) and used to assess the effect of the histone deacetylase (HDAC) inhibitors SAHA and MS-275 in the murine macrophage-like cell line RAW 264.7. SAHA and MS-275 showed site-specific effects on the acetylation levels of K5 and K8 with the K5(Ac)–K8 and K5–K8(Ac) peptides increasing 2.5-fold and 5-fold upon treatment with SAHA and MS-275, respectively. Assessing lysine acetylation in a site-specific manner is important for gaining a better understanding of the effects of HDAC inhibitors and for clarifying disease mechanisms where lysine acetylation plays a role. Electronic supplementary material The online version of this article (doi:10.1007/s00216-016-9431-1) contains supplementary material, which is available to authorized users.

Histone deacetylases (HDACs) have been used as pharmacological targets for the treatment of various diseases. Some non-selective HDAC inhibitors (HDACi) have been clinically-used as therapeutic agents for treatment of hematological cancers but their cytotoxic side effects are an important downside. The discovery of more selective inhibitors has certified the involvement of individual HDACs in pathological processes but the elucidation of the role of specific family members in inflammatory responses still remains a challenge. Here, we report the development of closely related, structural analogues of the clinically-used HDACi Entinostat via a chemical epigenetic approach. Three compounds were designed and synthesized in which the cap moiety of Entinostat was replaced by an azobenzene group that is either para, meta or ortho substituted. The compounds were then evaluated for selectivity towards HDACs 1-3 and their effect on pro-inflammatory gene expression in macrophages. One analogue, compound 4, lacked selectivity and demonstrated inhibition of NF-kappa B reporter gene activity and pro-inflammatory gene expression in RAW264.7 macrophages, thus indicating that there is a delicate balance between the selectivity of HDACi over specific family members and their pro-or anti-inflammatory effects.

Protein acylation is an abundant post-translational modification with a pivotal role in a plethora of biological processes. To date, metabolic labelling with functionalized precursors of acyl-CoA and subsequent bio-orthogonal ligation to a complementary detection tag has offered an attractive approach for monitoring endogenous protein acylation with excellent selectivity. This review focuses on the applications of alkyne- and alkene-based bioorthogonal chemistries in the study of enzyme activity in vitro and summarizes the carboxylate-type chemical reporters that have enabled the visualization and identification of cellular acylated proteins. However, despite their importance, serious limitations question the use of this two-step labelling method in the quantification of the protein acylome.

Histone deacetylases (HDACs) play important roles in inflammatory diseases like asthma and chronic obstructive pulmonary disease (COPD). Unravelling of and interfering with the functions of specific isoenzymes contributing to inflammation provides opportunities for drug development. Here we synthesize proteolysis targeting chimeras (PROTACs) for degradation of class I HDACs in which o-aminoanilide-based class I HDAC inhibitors are tethered to the cereblon ligand pomalidomide. One of these PROTACs, denoted HD-TAC7, showed promising degradation effects for HDAC3 with a DC50 value of 0.32 μM. In contrast to biochemical evidence using siRNA, HD-TAC7 showed a minimal effect on gene expression in LPS/IFNγ-stimulated RAW 264.7 macrophages. The lack of effect can be attributed to downregulation of the NF-κB subunit p65, which is a known side effect of pomalidomide treatment. Altogether, we describe a novel PROTAC that enables selective downregulation of HDAC3 levels, however we note that concomitant downregulation of the NF-κB subunit p65 can confound the biological outcome.

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.

Lipoxygenases metabolize polyunsaturated fatty acids into signalling molecules such as leukotrienes and lipoxins, which play a regulatory role in several inflammatory lung diseases such as asthma, chronic obstructive pulmonary disease (COPD) and chronic bronchitis. Human 15-lipoxygenase-1 (15-LOX-1) is an important mammalian lipoxygenase and plays a crucial role in the biosynthesis of these inflammatory signalling molecules. New classes of inhibitors are needed to explore the lipoxygenases as therapeutic targets. Here, we present the first study that identifies ruthenium(II) (Ru(II)) complexes as novel inhibitors of 15-LOX-1. Our study includes two novel Ru(II) complexes (C1a and C1b), bearing the sulfur macrocycle [9]aneS3, S-bonded dimethylsulfoxide (dmso-S), and chelate N,N- or N,O-donor ligands which were characterised by high-resolution NMR spectroscopy, X-ray crystallography and other standard physicochemical methods. These novel complexes and previously described Ru(II) complexes with the general formula [(η6-p-cymene)RuCl(O,O-ligand)]Cl were tested for inhibition of 15-LOX-1. This enabled identification of Ru(II) complexes that inhibit 15-LOX-1 with a potency in low micromolar range. Enzyme kinetic analysis was also performed, suggesting uncompetitive inhibition.