Publications by authors named "Manfred Jung"

220 Publications

First fluorescent acetylspermidine deacetylation assay for HDAC10 identifies selective inhibitors with cellular target engagement.

Chembiochem 2022 May 24. Epub 2022 May 24.

University of Freiburg, Institute of Pharmaceutical Sciences, Albertstr. 25, 71904, Freiburg, GERMANY.

Histone deacetylases (HDACs) are key epigenetic regulators involved in many diseases, especially cancer. Five HDAC inhibitors have been approved as drugs and many are in clinical trials. Among the 11 zinc-dependent HDACs, HDAC10 has received relatively little attention by drug discovery campaigns, despite its involvement, e.g., in the pathogenesis of neuroblastoma. This is due in part to a lack of robust enzymatic conversion assays. HDAC10 has strong preferences for deacetylation of oligoamine substrates like acetyl-putrescine or -spermidine. Hence, it is also termed as a polyamine deacetylase (PDAC). Here, we present the first fluorescent conversion assay for HDAC10 using an aminocoumarin-labelled acetyl-spermidine derivative to measure its PDAC activity. Using this assay, we identified potent inhibitors of HDAC10-mediated spermidine deacetylation in vitro. Based on the oligoamine preference of HDAC10, we also designed inhibitors with a basic moiety in appropriate distance to the zinc binding hydroxamate that showed potent inhibition of HDAC10 with high selectivity, and we solved a HDAC10-inhibitor structure using X-ray crystallography. We could demonstrate selective cellular target engagement for HDAC10 but a lysosomal phenotype in neuroblastoma cells that was previously associated with HDAC10 inhibition was not observed. Thus, we have developed new chemical probes for HDAC10 that allow further clarification of the biological role of this enzyme.
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http://dx.doi.org/10.1002/cbic.202200180DOI Listing
May 2022

Azetidin-2-one-based small molecules as dual hHDAC6/HDAC8 inhibitors: Investigation of their mechanism of action and impact of dual inhibition profile on cell viability.

Eur J Med Chem 2022 May 1;238:114409. Epub 2022 May 1.

Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg, 79104, Freiburg, Germany.

The search of new therapeutic tools for the treatment of cancer is being a challenge for medicinal chemists. Due to their role in different pathological conditions, histone deacetylase (HDAC) enzymes are considered valuable therapeutic targets. HDAC6 is a well-investigated HDAC-class IIb enzyme mainly characterized by a cytoplasmic localization; HDAC8 is an epigenetic eraser, unique HDAC-class I member that displays some aminoacidic similarity to HDAC6. New polypharmacological agents for cancer treatment, based on a dual hHDAC6/hHDAC8 inhibition profile were developed. The dual inhibitor design investigated the diphenyl-azetidin-2-one scaffold, typified in three different structural families, that, combined to a slender benzyl linker (6c, 6i, and 6j), displays nanomolar inhibition potency against hHDAC6 and hHDAC8 isoforms. Notably, their selective action was also corroborated by measuring their low inhibitory potency towards hHDAC1 and hHDAC10. Selectivity of these compounds was further demonstrated in human cell-based western blots experiments, by testing the acetylation of the non-histone substrates alpha-tubulin and SMC3. Furthermore, the compounds reduced the proliferation of colorectal HCT116 and leukemia U937 cells, after 48 h of treatment. The toxicity of the compounds was evaluated in rat perfused heart and in zebrafish embryos. In this latter model we also validated the efficacy of the dual hHDAC6/hHDAC8 inhibitors against their common target acetylated-alpha tubulin. Finally, the metabolic stability was verified in rat, mouse, and human liver microsomes.
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http://dx.doi.org/10.1016/j.ejmech.2022.114409DOI Listing
May 2022

Identification of histone deacetylase 10 (HDAC10) inhibitors that modulate autophagy in transformed cells.

Eur J Med Chem 2022 Apr 11;234:114272. Epub 2022 Mar 11.

Institute of Pharmacy, Martin-Luther University of Halle-Wittenberg, Wolfgang-Langenbeck-Str. 2-4, 06120, Halle/Saale, Germany. Electronic address:

Histone deacetylases (HDACs) are a family of 18 epigenetic modifiers that fall into 4 classes. Histone deacetylase inhibitors (HDACi) are valid tools to assess HDAC functions. HDAC6 and HDAC10 belong to the class IIb subgroup of the HDAC family. The targets and biological functions of HDAC10 are ill-defined. This lack of knowledge is due to a lack of specific and potent HDAC10 inhibitors with cellular activity. Here, we have synthesized and characterized piperidine-4-acrylhydroxamates as potent and highly selective inhibitors of HDAC10. This was achieved by targeting the acidic gatekeeper residue Glu274 of HDAC10 with a basic piperidine moiety that mimics the interaction of the polyamine substrate of HDAC10. We have confirmed the binding modes of selected inhibitors using X-ray crystallography. Promising candidates were selected based on their specificity by in vitro profiling using recombinant HDACs. The most promising HDAC10 inhibitors 10c and 13b were tested for specificity in acute myeloid leukemia (AML) cells with the FLT3-ITD oncogene. By immunoblot experiments we assessed the hyperacetylation of histones and tubulin-α, which are class I and HDAC6 substrates, respectively. As validated test for HDAC10 inhibition we used flow cytometry assessing autolysosome formation in neuroblastoma and AML cells. We demonstrate that 10c and 13b inhibit HDAC10 with high specificity over HDAC6 and with no significant impact on class I HDACs. The accumulation of autolysosomes is not a consequence of apoptosis and 10c and 13b are not toxic for normal human kidney cells. These data show that 10c and 13b are nanomolar inhibitors of HDAC10 with high specificity. Thus, our new HDAC10 inhibitors are tools to identify the downstream targets and functions of HDAC10 in cells.
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http://dx.doi.org/10.1016/j.ejmech.2022.114272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9007901PMC
April 2022

Comparison of Cellular Target Engagement Methods for the Tubulin Deacetylases Sirt2 and HDAC6: NanoBRET, CETSA, Tubulin Acetylation, and PROTACs.

ACS Pharmacol Transl Sci 2022 Feb 27;5(2):138-140. Epub 2022 Jan 27.

Department of Chemistry and Pharmacy, Medicinal Chemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Nikolaus-Fiebiger-Straße 10, 91058 Erlangen, Germany.

The tubulin deacetylases Sirt2 and HDAC6 have been associated with the development of various diseases. Herein, we discuss recent approaches that enable cellular target engagement studies for these deacetylases and thus play a critical role in the evaluation of small molecule inhibitors of Sirt2 or HDAC6 as potential therapeutic agents.
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http://dx.doi.org/10.1021/acsptsci.2c00004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8844959PMC
February 2022

Identification and characterization of sirtuin enzymes in cestodes and evaluation of sirtuin inhibitors as new cestocidal molecules.

Int J Parasitol 2022 04 9;52(5):317-329. Epub 2022 Feb 9.

Departamento de Microbiología, Parasitología e Inmunología, Facultad de Medicina, Universidad de Buenos Aires (UBA), Ciudad Autónoma de Buenos Aires, Argentina; Universidad de Buenos Aires (UBA), CONICET, Instituto de Investigaciones en Microbiología y Parasitología Médica (IMPaM), Ciudad Autónoma de Buenos Aires, Argentina. Electronic address:

Anti-parasitic treatment of neglected tropical diseases caused by cestodes such as echinococcosis and cysticercosis relies on a small number of approved anthelmintic drugs. Furthermore, the treatment is usually prolonged and often partially effective and not well tolerated by some patients. Therefore, the identification of novel drug targets and their associated compounds is critical. In this study, we identified and characterised sirtuin enzymes in cestodes and evaluated the cestocidal potential of sirtuin inhibitors as new cestocidal molecules. Sirtuins are a highly conserved family of nicotinamide-adenine dinucleotide-lysine deacylases involved in multiple cellular functions. Here, we described the full repertoire of sirtuin-encoding genes in several cestode species. We identified six sirtuin-encoding genes that were classified into sirtuins Class I (SIRT1, SIRT2, and SIRT3), Class III (SIRT5), and Class IV (SIRT6 and SIRT7). In Echinococcus spp., sirtuin genes showed transcriptional expression throughout several developmental stages, sirtuin 2 (SIRT2) being the most expressed. To evaluate the potential of sirtuin inhibitors as new cestocidal molecules, we determined the in vitro effect of several Class I sirtuin inhibitors by motility assay. Of those, the selective SIRT2 inhibitor Mz25 showed a strong cestocidal activity in Mesocestoides vogae (syn. Mesocestoides corti) tetrathyridia at various concentrations. The Mz25 cestocidal activity was time- and dose-dependent with a half-maximal inhibitory concentration value significantly lower than that of albendazole. Additionally, Mz25 induced extensive damage in the general morphology with marked alterations in the tegument and ultrastructural features. By homology modelling, we found that cestode SIRT2s showed a high conservation of the canonical sirtuin structure as well as in the residues related to Mz25 binding. Interestingly, some non-conservative mutations were found on the selectivity pocket (an Mz25-induced structural rearrangement on the active site), which represent a promising lead for developing selective cestode SIRT2 inhibitors derived from Mz25. Nevertheless, the Mz25 molecular target in M. vogae is unknown and remains to be determined. This report provides the basis for further studies of sirtuins to understand their roles in cestode biology and to develop selective sirtuin inhibitors to treat these neglected tropical diseases.
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http://dx.doi.org/10.1016/j.ijpara.2021.12.002DOI Listing
April 2022

Ex vivo propagation in a novel 3D high-throughput co-culture system for multiple myeloma.

J Cancer Res Clin Oncol 2022 May 24;148(5):1045-1055. Epub 2022 Jan 24.

Department of Internal Medicine I, Faculty of Medicine and Medical Center, University of Freiburg, Hugstetterstr. 53, 79106, Freiburg, Germany.

Purpose: Multiple myeloma (MM) remains an incurable hematologic malignancy which ultimately develops drug resistance and evades treatment. Despite substantial therapeutic advances over the past years, the clinical failure rate of preclinically promising anti-MM drugs remains substantial. More realistic in vitro models are thus required to better predict clinical efficacy of a preclinically active compound.

Methods: Here, we report on the establishment of a conical agarose 3D co-culture platform for the preclinical propagation of primary MM cells ex vivo. Cell growth was compared to yet established 2D and liquid overlay systems. MM cell lines (MMCL: RPMI-8226, U266, OPM-2) and primary patient specimens were tested. Drug sensitivity was examined by exploring the cytotoxic effect of bortezomib and the deubiquitinase inhibitor auranofin under various conditions.

Results: In contrast to 2D and liquid overlay, cell proliferation in the 3D array followed a sigmoidal curve characterized by an initial growth delay but more durable proliferation of MMCL over 12 days of culture. Primary MM specimens did not expand in ex vivo monoculture, but required co-culture support by a human stromal cell line (HS-5, MSP-1). HS-5 induced a > fivefold increase in cluster volume and maintained long-term viability of primary MM cells for up to 21 days. Bortezomib and auranofin induced less cytotoxicity under 3D vs. 2D condition and in co- vs. monoculture, respectively.

Conclusions: This study introduces a novel model that is capable of long-term propagation and drug testing of primary MM specimens ex vivo overcoming some of the pitfalls of currently available in vitro models.
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http://dx.doi.org/10.1007/s00432-021-03854-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9016043PMC
May 2022

BET Proteins Regulate Expression of Osr1 in Early Kidney Development.

Biomedicines 2021 Dec 10;9(12). Epub 2021 Dec 10.

III Department of Medicine, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany.

In utero renal development is subject to maternal metabolic and environmental influences affecting long-term renal function and the risk of developing chronic kidney failure and cardiovascular disease. Epigenetic processes have been implicated in the orchestration of renal development and prenatal programming of nephron number. However, the role of many epigenetic modifiers for kidney development is still unclear. Bromodomain and extra-terminal domain (BET) proteins act as histone acetylation reader molecules and promote gene transcription. BET family members Brd2, Brd3 and Brd4 are expressed in the nephrogenic zone during kidney development. Here, the effect of the BET inhibitor JQ1 on renal development is evaluated. Inhibition of BET proteins via JQ1 leads to reduced growth of metanephric kidney cultures, loss of the nephron progenitor cell population, and premature and disturbed nephron differentiation. Gene expression of key nephron progenitor transcription factor Osr1 is downregulated after 24 h BET inhibition, while Lhx1 and Pax8 expression is increased. Mining of BRD4 ChIP-seq and gene expression data identify Osr1 as a key factor regulated by BRD4-controlled gene activation. Inhibition of BRD4 by BET inhibitor JQ1 leads to downregulation of Osr1, thereby causing a disturbance in the balance of nephron progenitor cell self-renewal and premature differentiation of the nephron, which ultimately leads to kidney hypoplasia and disturbed nephron development. This raises questions about the potential teratogenic effects of BET inhibitors for embryonic development. In summary, our work highlights the role of BET proteins for prenatal programming of nephrogenesis and identifies Osr1 as a potential target of BET proteins.
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http://dx.doi.org/10.3390/biomedicines9121878DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8698285PMC
December 2021

Species-selective targeting of pathogens revealed by the atypical structure and active site of Trypanosoma cruzi histone deacetylase DAC2.

Cell Rep 2021 12;37(12):110129

Université de Strasbourg, CNRS, INSERM, Institut de Génétique et de Biologie Moléculaire et Cellulaire, UMR 7104, U 1258, 67404 Illkirch, France; IGBMC, Department of Integrated Structural Biology, 1 rue Laurent Fries, B.P. 10142, 67404 Illkirch Cedex, France. Electronic address:

Writing and erasing of posttranslational modifications are crucial to phenotypic plasticity and antigenic variation of eukaryotic pathogens. Targeting pathogens' modification machineries, thus, represents a valid approach to fighting parasitic diseases. However, identification of parasitic targets and the development of selective anti-parasitic drugs still represent major bottlenecks. Here, we show that the zinc-dependent histone deacetylases (HDACs) of the protozoan parasite Trypanosoma cruzi are key regulators that have significantly diverged from their human counterparts. Depletion of T. cruzi class I HDACs tcDAC1 and tcDAC2 compromises cell-cycle progression and division, leading to cell death. Notably, tcDAC2 displays a deacetylase activity essential to the parasite and shows major structural differences with human HDACs. Specifically, tcDAC2 harbors a modular active site with a unique subpocket targeted by inhibitors showing substantial anti-parasitic effects in cellulo and in vivo. Thus, the targeting of the many atypical HDACs in pathogens can enable anti-parasitic selective chemical impairment.
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http://dx.doi.org/10.1016/j.celrep.2021.110129DOI Listing
December 2021

Designed membrane protein heterodimers and control of their affinity by binding domain and membrane linker properties.

Nanoscale 2021 Dec 16;13(48):20692-20702. Epub 2021 Dec 16.

BIOSS Centre for Biological Signalling Studies, University of Freiburg, Germany.

Many membrane proteins utilize dimerization to transmit signals across the cell membrane regulation of the lateral binding affinity. The complexity of natural membrane proteins hampers the understanding of this regulation on a biophysical level. We designed simplified membrane proteins from well-defined soluble dimerization domains with tunable affinities, flexible linkers, and an inert membrane anchor. Live-cell single-molecule imaging demonstrates that their dimerization affinity indeed depends on the strength of their binding domains. We confirm that as predicted, the 2-dimensional affinity increases with the 3-dimensional binding affinity of the binding domains and decreases with linker lengths. Models of extended and coiled linkers delineate an expected range of 2-dimensional affinities, and our observations for proteins with medium binding strength agree well with the models. Our work helps in understanding the function of membrane proteins and has important implications for the design of synthetic receptors.
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http://dx.doi.org/10.1039/d1nr06574bDOI Listing
December 2021

Lineage-Selective Disturbance of Early Human Hematopoietic Progenitor Cell Differentiation by the Commonly Used Plasticizer Di-2-ethylhexyl Phthalate via Reactive Oxygen Species: Fatty Acid Oxidation Makes the Difference.

Cells 2021 10 9;10(10). Epub 2021 Oct 9.

Institute of Precision Medicine, Medical and Life Sciences Faculty, Furtwangen University, Jakob-Kienzle-Straße 17, 78054 Villingen-Schwenningen, Germany.

Exposure to ubiquitous endocrine-disrupting chemicals (EDCs) is a major public health concern. We analyzed the physiological impact of the EDC, di-2-ethylhexyl phthalate (DEHP), and found that its metabolite, mono-2-ethylhexyl phthalate (MEHP), had significant adverse effects on myeloid hematopoiesis at environmentally relevant concentrations. An analysis of the underlying mechanism revealed that MEHP promotes increases in reactive oxygen species (ROS) by reducing the activity of superoxide dismutase in all lineages, possibly via its actions at the aryl hydrocarbon receptor. This leads to a metabolic shift away from glycolysis toward the pentose phosphate pathway and ultimately results in the death of hematopoietic cells that rely on glycolysis for energy production. By contrast, cells that utilize fatty acid oxidation for energy production are not susceptible to this outcome due to their capacity to uncouple ATP production. These responses were also detected in non-hematopoietic cells exposed to alternate inducers of ROS.
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http://dx.doi.org/10.3390/cells10102703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8534767PMC
October 2021

Kappa but not delta or mu opioid receptors form homodimers at low membrane densities.

Cell Mol Life Sci 2021 Dec 17;78(23):7557-7568. Epub 2021 Oct 17.

Institute of Internal Medicine IV, Medical Center of the University of Freiburg, Freiburg, Germany.

Opioid receptors (ORs) have been observed as homo- and heterodimers, but it is unclear if the dimers are stable under physiological conditions, and whether monomers or dimers comprise the predominant fraction in a cell. Here, we use three live-cell imaging approaches to assess dimerization of ORs at expression levels that are 10-100 × smaller than in classical biochemical assays. At membrane densities around 25/µm, a split-GFP assay reveals that κOR dimerizes, while µOR and δOR stay monomeric. At receptor densities < 5/µm, single-molecule imaging showed no κOR dimers, supporting the concept that dimer formation depends on receptor membrane density. To directly observe the transition from monomers to dimers, we used a single-molecule assay to assess membrane protein interactions at densities up to 100 × higher than conventional single-molecule imaging. We observe that κOR is monomeric at densities < 10/µm and forms dimers at densities that are considered physiological. In contrast, µOR and δOR stay monomeric even at the highest densities covered by our approach. The observation of long-lasting co-localization of red and green κOR spots suggests that it is a specific effect based on OR dimerization and not an artefact of coincidental encounters.
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http://dx.doi.org/10.1007/s00018-021-03963-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8629795PMC
December 2021

Hypoxia-activated KDAC inhibitor: Taking a breath from untargeted therapy.

Cell Chem Biol 2021 09;28(9):1255-1257

Institute of Pharmaceutical Sciences, University of Freiburg, 79104 Freiburg, Germany; Centre for Integrative Biological Signalling Studies (CIBSS), University of Freiburg, 79104 Freiburg, Germany. Electronic address:

Use of hypoxia-activated prodrugs has emerged as a strategy for selectively targeting tumors in hypoxic conditions harboring reductive environments. In this issue of Cell Chemical Biology, Skwarska et al. (2021) report a hypoxia-activated prodrug targeting histone deacetylases (lysine deacetylases, KDACs) selectively over normoxic cells with activity in an animal model.
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http://dx.doi.org/10.1016/j.chembiol.2021.08.016DOI Listing
September 2021

Synthesis, structure-activity relationships, cocrystallization and cellular characterization of novel smHDAC8 inhibitors for the treatment of schistosomiasis.

Eur J Med Chem 2021 Dec 8;225:113745. Epub 2021 Aug 8.

Institute of Pharmacy, Martin-Luther University of Halle-Wittenberg, 06120, Halle/Saale, Germany. Electronic address:

Schistosomiasis is a major neglected parasitic disease that affects more than 265 million people worldwide and for which the control strategy consists of mass treatment with the only available drug, praziquantel. In this study, we chemically optimized our previously reported benzhydroxamate-based inhibitors of Schistosoma mansoni histone deacetylase 8 (smHDAC8). Crystallographic analysis provided insights into the inhibition mode of smHDAC8 activity by the highly potent inhibitor 5o. Structure-based optimization of the novel inhibitors was carried out using the available crystal structures as well as docking studies on smHDAC8. The compounds were evaluated in screens for inhibitory activity against schistosome and human HDACs (hHDAC). The in vitro and docking results were used for detailed structure activity relationships. The synthesized compounds were further investigated for their lethality against the schistosome larval stage using a fluorescence-based assay. The most promising inhibitor 5o showed significant dose-dependent killing of the schistosome larvae and markedly impaired egg laying of adult worm pairs maintained in culture.
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http://dx.doi.org/10.1016/j.ejmech.2021.113745DOI Listing
December 2021

Harnessing the Role of HDAC6 in Idiopathic Pulmonary Fibrosis: Design, Synthesis, Structural Analysis, and Biological Evaluation of Potent Inhibitors.

J Med Chem 2021 07 12;64(14):9960-9988. Epub 2021 Jul 12.

Department of Biotechnology, Chemistry and Pharmacy, DoE Department of Excellence 2018-2022, University of Siena, via Aldo Moro 2, 53100 Siena, Italy.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by a progressive-fibrosing phenotype. IPF has been associated with aberrant HDAC activities confirmed by our immunohistochemistry studies on HDAC6 overexpression in IPF lung tissues. We herein developed a series of novel HDAC6 inhibitors, having low inhibitory potency over HDAC1 and HDAC8, as potential pharmacological tools for IPF treatment. Their inhibitory potency was combined with low and toxicity. Structural analysis of and structure-activity relationship studies contributed to the optimization of the binding mode of the new molecules. The best-performing analogues were tested for their efficacy in inhibiting fibrotic sphere formation and cell viability, proving their capability in reverting the IPF phenotype. The efficacy of analogue was also determined in a validated human lung model of TGF-β1-dependent fibrogenesis. The results highlighted in this manuscript may pave the way for the identification of first-in-class molecules for the treatment of IPF.
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http://dx.doi.org/10.1021/acs.jmedchem.1c00184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8300879PMC
July 2021

Structure-Based Design, Docking and Binding Free Energy Calculations of A366 Derivatives as Spindlin1 Inhibitors.

Int J Mol Sci 2021 May 31;22(11). Epub 2021 May 31.

Institute of Pharmacy, Martin Luther University of Halle-Wittenberg, Kurt-Mothes-Str.3, 06120 Halle/Saale, Germany.

The chromatin reader protein Spindlin1 plays an important role in epigenetic regulation, through which it has been linked to several types of malignant tumors. In the current work, we report on the development of novel analogs of the previously published lead inhibitor . In an effort to improve the activity and explore the structure-activity relationship (SAR), a series of 21 derivatives was synthesized, tested in vitro, and investigated by means of molecular modeling tools. Docking studies and molecular dynamics (MD) simulations were performed to analyze and rationalize the structural differences responsible for the Spindlin1 activity. The analysis of MD simulations shed light on the important interactions. Our study highlighted the main structural features that are required for Spindlin1 inhibitory activity, which include a positively charged pyrrolidine moiety embedded into the aromatic cage connected via a propyloxy linker to the 2-aminoindole core. Of the latter, the amidine group anchor the compounds into the pocket through salt bridge interactions with Asp184. Different protocols were tested to identify a fast in silico method that could help to discriminate between active and inactive compounds within the series. Rescoring the docking poses with MM-GBSA calculations was successful in this regard. Because is known to be a G9a inhibitor, the most active developed Spindlin1 inhibitors were also tested over G9a and GLP to verify the selectivity profile of the analogs. This resulted in the discovery of diverse selective compounds, among which and showed Spindlin1 activity in the nanomolar range and selectivity over G9a and GLP. Finally, future design hypotheses were suggested based on our findings.
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http://dx.doi.org/10.3390/ijms22115910DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8199216PMC
May 2021

Effects of Environmental Conditions on Nephron Number: Modeling Maternal Disease and Epigenetic Regulation in Renal Development.

Int J Mol Sci 2021 Apr 16;22(8). Epub 2021 Apr 16.

III Department of Medicine, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany.

A growing body of evidence suggests that low nephron numbers at birth can increase the risk of chronic kidney disease or hypertension later in life. Environmental stressors, such as maternal malnutrition, medication and smoking, can influence renal size at birth. Using metanephric organ cultures to model single-variable environmental conditions, models of maternal disease were evaluated for patterns of developmental impairment. While hyperthermia had limited effects on renal development, fetal iron deficiency was associated with severe impairment of renal growth and nephrogenesis with an all-proximal phenotype. Culturing kidney explants under high glucose conditions led to cellular and transcriptomic changes resembling human diabetic nephropathy. Short-term high glucose culture conditions were sufficient for long-term alterations in DNA methylation-associated epigenetic memory. Finally, the role of epigenetic modifiers in renal development was tested using a small compound library. Among the selected epigenetic inhibitors, various compounds elicited an effect on renal growth, such as HDAC (entinostat, TH39), histone demethylase (deferasirox, deferoxamine) and histone methyltransferase (cyproheptadine) inhibitors. Thus, metanephric organ cultures provide a valuable system for studying metabolic conditions and a tool for screening for epigenetic modifiers in renal development.
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http://dx.doi.org/10.3390/ijms22084157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8073167PMC
April 2021

The potential for histone deacetylase (HDAC) inhibitors as cestocidal drugs.

PLoS Negl Trop Dis 2021 03 3;15(3):e0009226. Epub 2021 Mar 3.

Instituto de Microbiología y Parasitología Médica, Universidad de Buenos Aires-Consejo Nacional de Investigaciones Científicas y Tecnológicas (IMPaM, UBA-CONICET). Facultad de Medicina, Paraguay 2155, piso 13, C1121ABG, Buenos Aires, Argentina.

Background: Echinococcosis and cysticercosis are neglected tropical diseases caused by cestode parasites (family Taeniidae). Not only there is a small number of approved anthelmintics for the treatment of these cestodiases, but also some of them are not highly effective against larval stages, such that identifying novel drug targets and their associated compounds is critical. Histone deacetylase (HDAC) enzymes are validated drug targets in cancers and other diseases, and have been gaining relevance for developing new potential anti-parasitic treatments in the last years. Here, we present the anthelmintic profile for a panel of recently developed HDAC inhibitors against the model cestode Mesocestoides vogae (syn. M. corti).

Methodology/principal Findings: Phenotypic screening was performed on M. vogae by motility measurements and optical microscopic observations. Some HDAC inhibitors showed potent anthelmintic activities; three of them -entinostat, TH65, and TH92- had pronounced anthelmintic effects, reducing parasite viability by ~100% at concentrations of ≤ 20 μM. These compounds were selected for further characterization and showed anthelmintic effects in the micromolar range and in a time- and dose-dependent manner. Moreover, these compounds induced major alterations on the morphology and ultrastructural features of M. vogae. The potencies of these compounds were higher than albendazole and the anthelmintic effects were irreversible. Additionally, we evaluated pairwise drug combinations of these HDAC inhibitors and albendazole. The results suggested a positive interaction in the anthelmintic effect for individual pairs of compounds. Due to the maximum dose approved for entinostat, adjustments in the dose regime and/or combinations with currently-used anthelmintic drugs are needed, and the selectivity of TH65 and TH92 towards parasite targets should be assessed.

Conclusion, Significance: The results presented here suggest that HDAC inhibitors represent novel and potent drug candidates against cestodes and pave the way to understanding the roles of HDACs in these parasites.
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http://dx.doi.org/10.1371/journal.pntd.0009226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7959350PMC
March 2021

4-Acyl Pyrroles as Dual BET-BRD7/9 Bromodomain Inhibitors Address BETi Insensitive Human Cancer Cell Lines.

J Med Chem 2020 12 4;63(24):15603-15620. Epub 2020 Dec 4.

Institut für Pharmazeutische Wissenschaften, Albert-Ludwigs-Universität Freiburg, Hermann-Herder-Str. 8, D-79104 Freiburg, Germany.

Various malignant human diseases show disturbed signaling pathways due to increased activity of proteins within the epigenetic machinery. Recently, various novel inhibitors for epigenetic regulation have been introduced which promise a great therapeutic benefit. Inhibitors for the bromo- and extra-terminal domain (BET) family were of particular interest after inhibitors had shown a strong antiproliferative effect. More recently, the focus has increasingly shifted to bromodomains (BDs) outside the BET family. Based on previously developed inhibitors, we have optimized a small series of 4-acyl pyrroles, which we further analyzed by ITC, X-ray crystallography, selectivity studies, the NCI60 cell-panel, and GI determinations for several cancer cell lines. The inhibitors address both, BET and BRD7/9 BDs, with very high affinity and show a strong antiproliferative effect on various cancer cell lines that could not be observed for BD family selective inhibitors. Furthermore, a synergistic effect on breast cancer (MCF-7) and melanoma (SK-MEL-5) was proven.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00478DOI Listing
December 2020

Spiroindoline-Capped Selective HDAC6 Inhibitors: Design, Synthesis, Structural Analysis, and Biological Evaluation.

ACS Med Chem Lett 2020 Nov 29;11(11):2268-2276. Epub 2020 Sep 29.

Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6323, United States.

Histone deacetylase inhibitors (HDACi) have emerged as promising therapeutics for the treatment of neurodegeneration, cancer, and rare disorders. Herein, we report the development of a series of spiroindoline-based HDAC6 isoform-selective inhibitors based on the X-ray crystal studies of the hit . We identified compound as the most potent and selective HDAC6 inhibitor of the series. Biological investigation of compounds , , and demonstrated their antiproliferative activity against several cancer cell lines. Western blotting studies indicated that they were able to increase tubulin acetylation, without significant variation in histone acetylation state, and induced PARP cleavage indicating their apoptotic potential at the molecular level. induced HDAC6-dependent pSTAT3 inhibition.
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http://dx.doi.org/10.1021/acsmedchemlett.0c00395DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7667836PMC
November 2020

Novel quinolone-based potent and selective HDAC6 inhibitors: Synthesis, molecular modeling studies and biological investigation.

Eur J Med Chem 2021 Feb 7;212:112998. Epub 2020 Nov 7.

Department of Biotechnology, Chemistry and Pharmacy, DoE Department of Excellence 2018-2022, University of Siena, Via Aldo Moro 2, 53100, Siena, Italy. Electronic address:

In this work we describe the synthesis of potent and selective quinolone-based histone deacetylase 6 (HDAC6) inhibitors. The quinolone moiety has been exploited as an innovative bioactive cap-group for HDAC6 inhibition; its synthesis was achieved by applying a multicomponent reaction. The optimization of potency and selectivity of these products was performed by employing computational studies which led to the discovery of the diethylaminomethyl derivatives 7g and 7k as the most promising hit molecules. These compounds were investigated in cellular studies to evaluate their anticancer effect against colon (HCT-116) and histiocytic lymphoma (U9347) cancer cells, showing good to excellent potency, leading to tumor cell death by apoptosis induction. The small molecules 7a, 7g and 7k were able to strongly inhibit the cytoplasmic and slightly the nuclear HDAC enzymes, increasing the acetylation of tubulin and of the lysine 9 and 14 of histone 3, respectively. Compound 7g was also able to increase Hsp90 acetylation levels in HCT-116 cells, thus further supporting its HDAC6 inhibitory profile. Cytotoxicity and mutagenicity assays of these molecules showed a safe profile; moreover, the HPLC analysis of compound 7k revealed good solubility and stability profile.
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http://dx.doi.org/10.1016/j.ejmech.2020.112998DOI Listing
February 2021

Identification of the subtype-selective Sirt5 inhibitor balsalazide through systematic SAR analysis and rationalization via theoretical investigations.

Eur J Med Chem 2020 Nov 1;206:112676. Epub 2020 Aug 1.

Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians University, Butenandtstraße 5-13, 81377, Munich, Germany. Electronic address:

We report here an extensive structure-activity relationship study of balsalazide, which was previously identified in a high-throughput screening as an inhibitor of Sirt5. To get a closer understanding why this compound is able to inhibit Sirt5, we initially performed docking experiments comparing the binding mode of a succinylated peptide as the natural substrate and balsalazide with Sirt5 in the presence of NAD. Based on the evidence gathered here, we designed and synthesized 13 analogues of balsalazide, in which single functional groups were either deleted or slightly altered to investigate which of them are mandatory for high inhibitory activity. Our study confirms that balsalazide with all its given functional groups is an inhibitor of Sirt5 in the low micromolar concentration range and structural modifications presented in this study did not increase potency. While changes on the N-aroyl-β-alanine side chain eliminated potency, the introduction of a truncated salicylic acid part minimally altered potency. Calculations of the associated reaction paths showed that the inhibition potency is very likely dominated by the stability of the inhibitor-enzyme complex and not the type of inhibition (covalent vs. non-covalent). Further in-vitro characterization in a trypsin coupled assay determined that the tested inhibitors showed no competition towards NAD or the synthetic substrate analogue ZKsA. In addition, investigations for subtype selectivity revealed that balsalazide is a subtype-selective Sirt5 inhibitor, and our initial SAR and docking studies pave the way for further optimization.
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http://dx.doi.org/10.1016/j.ejmech.2020.112676DOI Listing
November 2020

Metabolite Patterns in Human Myeloid Hematopoiesis Result from Lineage-Dependent Active Metabolic Pathways.

Int J Mol Sci 2020 Aug 24;21(17). Epub 2020 Aug 24.

Institute of Precision Medicine, Medical and Life Sciences Faculty, Furtwangen University, Jakob-Kienzle-Straße 17, 78054 Villingen-Schwenningen, Germany.

Assessment of hematotoxicity from environmental or xenobiotic compounds is of notable interest and is frequently assessed via the colony forming unit (CFU) assay. Identification of the mode of action of single compounds is of further interest, as this often enables transfer of results across different tissues and compounds. Metabolomics displays one promising approach for such identification, nevertheless, suitability with current protocols is restricted. Here, we combined a hematopoietic stem and progenitor cell (HSPC) expansion approach with distinct lineage differentiations, resulting in formation of erythrocytes, dendritic cells and neutrophils. We examined the unique combination of pathway activity in glycolysis, glutaminolysis, polyamine synthesis, fatty acid oxidation and synthesis, as well as glycerophospholipid and sphingolipid metabolism. We further assessed their interconnections and essentialness for each lineage formation. By this, we provide further insights into active metabolic pathways during the differentiation of HSPC into different lineages, enabling profound understanding of possible metabolic changes in each lineage caused by exogenous compounds.
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http://dx.doi.org/10.3390/ijms21176092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7504406PMC
August 2020

Epigenetics meets GPCR: inhibition of histone H3 methyltransferase (G9a) and histamine H receptor for Prader-Willi Syndrome.

Sci Rep 2020 08 11;10(1):13558. Epub 2020 Aug 11.

Institute of Pharmaceutical and Medicinal Chemistry, Heinrich Heine University Duesseldorf, Universitaetsstr. 1, 40225, Duesseldorf, Germany.

The role of epigenetic regulation is in large parts connected to cancer, but additionally, its therapeutic claim in neurological disorders has emerged. Inhibition of histone H3 lysine N-methyltransferase, especially G9a, has been recently shown to restore candidate genes from silenced parental chromosomes in the imprinting disorder Prader-Willi syndrome (PWS). In addition to this epigenetic approach, pitolisant as G-protein coupled histamine H receptor (HR) antagonist has demonstrated promising therapeutic effects for Prader-Willi syndrome. To combine these pioneering principles of drug action, we aimed to identify compounds that combine both activities, guided by the pharmacophore blueprint for both targets. However, pitolisant as selective HR inverse agonist with FDA and EMA-approval did not show the required inhibition at G9a. Pharmacological characterization of the prominent G9a inhibitor A-366, that is as well an inhibitor of the epigenetic reader protein Spindlin1, revealed its high affinity at HR while showing subtype selectivity among subsets of the histaminergic and dopaminergic receptor families. This work moves prominent G9a ligands forward as pharmacological tools to prove for a potentially combined, symptomatic and causal, therapy in PWS by bridging the gap between drug development for G-protein coupled receptors and G9a as an epigenetic effector in a multi-targeting approach.
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http://dx.doi.org/10.1038/s41598-020-70523-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7419559PMC
August 2020

Syntheses of Thailandepsin B Pseudo-Natural Products: Access to New Highly Potent HDAC Inhibitors via Late-Stage Modification.

Chemistry 2020 Dec 3;26(69):16241-16245. Epub 2020 Nov 3.

Institut für Organische Chemie, Albert-Ludwigs-Universität Freiburg, Albertstr. 21, 79104, Freiburg, Germany.

New Thailandepsin B pseudo-natural products have been prepared. Our synthetic strategy offers the possibility to introduce varying warheads via late stage modification. Additionally, it gives access to the asymmetric branched allylic ester moiety of the natural product in a highly diastereoselective manner applying rhodium-catalyzed hydrooxycarbonylation. The newly developed pseudo-natural products are extremely potent and selective HDAC inhibitors. The non-proteinogenic amino acid d-norleucine was obtained enantioselectively by a recently developed method of rhodium-catalyzed hydroamination.
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http://dx.doi.org/10.1002/chem.202002449DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7756392PMC
December 2020

HaloTag-Targeted Sirtuin-Rearranging Ligand (SirReal) for the Development of Proteolysis-Targeting Chimeras (PROTACs) against the Lysine Deacetylase Sirtuin 2 (Sirt2)*.

Chembiochem 2020 12 27;21(23):3371-3376. Epub 2020 Aug 27.

Institute of Pharmaceutical Sciences, University of Freiburg, Albertstraße 25, 79104, Freiburg im Breisgau, Germany.

We have discovered the sirtuin-rearranging ligands (SirReals) as a novel class of highly potent and selective inhibitors of the NAD -dependent lysine deacetylase sirtuin 2 (Sirt2). In previous studies, conjugation of a SirReal with a ligand for the E3 ubiquitin ligase cereblon to form a so-called proteolysis-targeting chimera (PROTAC) enabled small-molecule-induced degradation of Sirt2. Herein, we report the structure-based development of a chloroalkylated SirReal that induces the degradation of Sirt2 mediated by Halo-tagged E3 ubiquitin ligases. Using this orthogonal approach for Sirt2 degradation, we show that other E3 ligases than cereblon, such as the E3 ubiquitin ligase parkin, can also be harnessed for small-molecule-induced Sirt2 degradation, thereby emphasizing the great potential of parkin to be used as an E3 ligase for new PROTACs approaches. Thus, our study provides new insights into targeted protein degradation in general and Sirt2 degradation in particular.
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http://dx.doi.org/10.1002/cbic.202000351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7754454PMC
December 2020

Design, synthesis, and biological evaluation of dual targeting inhibitors of histone deacetylase 6/8 and bromodomain BRPF1.

Eur J Med Chem 2020 Aug 18;200:112338. Epub 2020 May 18.

Institute of Pharmacy, Martin-Luther University of Halle-Wittenberg, 06120, Halle/Saale, Germany. Electronic address:

Histone modifying proteins, specifically histone deacetylases (HDACs) and bromodomains, have emerged as novel promising targets for anticancer therapy. In the current work, based on available crystal structures and docking studies, we designed dual inhibitors of both HDAC6/8 and the bromodomain and PHD finger containing protein 1 (BRPF1). Biochemical and biophysical tests showed that compounds 23a,b and 37 are nanomolar inhibitors of both target proteins. Detailed structure-activity relationships were deduced for the synthesized inhibitors which were supported by extensive docking and molecular dynamics studies. Cellular testing in acute myeloid leukemia (AML) cells showed only a weak effect, most probably because of the poor permeability of the inhibitors. We also aimed to analyse the target engagement and the cellular activity of the novel inhibitors by determining the protein acetylation levels in cells by western blotting (tubulin vs histone acetylation), and by assessing their effects on various cancer cell lines.
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http://dx.doi.org/10.1016/j.ejmech.2020.112338DOI Listing
August 2020

Sirtuin 1 Inhibiting Thiocyanates (S1th)-A New Class of Isotype Selective Inhibitors of NAD Dependent Lysine Deacetylases.

Front Oncol 2020 30;10:657. Epub 2020 Apr 30.

Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmaceutical Sciences, University of Freiburg, Freiburg im Breisgau, Germany.

Sirtuin 1 (Sirt1) is a NAD dependent lysine deacetylase associated with the pathogenesis of various diseases including cancer. In many cancer types Sirt1 expression is increased and higher levels have been associated with metastasis and poor prognosis. However, it was also shown, that Sirt1 can have tumor suppressing properties and in some instances even a dual role for the same cancer type has been reported. Increased Sirt1 activity has been linked to extension of the life span of cells, respectively, organisms by promoting DNA repair processes and downregulation of tumor suppressor proteins. This may have the downside of enhancing tumor growth and metastasis. In mice embryonic fibroblasts depletion of Sirt1 was shown to decrease levels of the DNA damage sensor histone H2AX. Impairment of DNA repair mechanisms by Sirt1 can promote tumorigenesis but also lower chemoresistance toward DNA targeting therapies. Despite many biological studies, there is currently just one small molecule Sirt1 inhibitor in clinical trials. Selisistat (EX-527) reached phase III clinical trials for treatment of Huntington's Disease. New small molecule Sirt1 modulators are crucial for further investigation of the contradicting roles of Sirt1 in cancer. We tested a small library of commercially available compounds that were proposed by virtual screening and docking studies against Sirt1, 2 and 3. A thienopyrimidone featuring a phenyl thiocyanate moiety was found to selectively inhibit Sirt1 with an IC of 13 μM. Structural analogs lacking the thiocyanate function did not show inhibition of Sirt1 revealing this group as key for the selectivity and affinity toward Sirt1. Further analogs with higher solubility were identified through iterative docking studies and testing. The most active compounds (down to 5 μM IC) were further studied in cells. The ratio of phosphorylated γH2AX to unmodified H2AX is lower when Sirt1 is depleted or inhibited. Our new Sirtuin 1 inhibiting thiocyanates (S1th) lead to similarly lowered γH2AX/H2AX ratios in mouse embryonic fibroblasts as Sirt1 knockout and treatment with the reference inhibitor EX-527. In addition to that we were able to show antiproliferative activity, inhibition of migration and colony forming as well as hyperacetylation of Sirt1 targets p53 and H3 by the S1th in cervical cancer cells (HeLa). These results reveal thiocyanates as a promising new class of selective Sirt1 inhibitors.
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http://dx.doi.org/10.3389/fonc.2020.00657DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7203344PMC
April 2020

Activation of Sirtuin 2 Inhibitors Employing Photoswitchable Geometry and Aqueous Solubility.

ChemMedChem 2020 08 7;15(15):1480-1489. Epub 2020 May 7.

Institute of Pharmacy, University of Greifswald, Friedrich-Ludwig-Jahn-Str. 17, 17489, Greifswald, Germany.

Because isoenzymes of the experimentally and therapeutically extremely relevant sirtuin family show high similarity, addressing the unique selectivity pocket of sirtuin 2 is a promising strategy towards selective inhibitors. An unrelated approach towards selective inhibition of isoenzymes with varied tissue distribution is targeted drug delivery or spatiotemporal activation by photochemical activation. Azologization of two nicotinamide-mimicking lead structures was undertaken to combine both approaches and yielded a set of 33 azobenzenes and azopyridines that have been evaluated for their photochemical behaviour and bioactivity. For some compounds, inhibitory activity reached the sub-micromolar range in their thermodynamically favoured E form and could be decreased by photoisomerization to the metastable Z form. Besides, derivatization with long-chain fatty acids yielded potent sirtuin 2 inhibitors, featuring another intriguing aspect of azo-based photoswitches. In these compounds, switching to the Z isomer increased aqueous solubility and thereby enhanced biological activity by up to a factor of 21. The biological activity of two compounds was confirmed by hyperacetylation of sirtuin specific histone proteins in a cell-based activity assay.
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http://dx.doi.org/10.1002/cmdc.202000148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7496931PMC
August 2020

Nitroreductase-Mediated Release of Inhibitors of Lysine-Specific Demethylase 1 (LSD1) from Prodrugs in Transfected Acute Myeloid Leukaemia Cells.

Chembiochem 2020 08 27;21(16):2329-2347. Epub 2020 Apr 27.

Department of Chemistry and Pharmacy, University of Freiburg, Institute of Pharmaceutical Sciences, Albertstrasse 25, 79104, Freiburg, Germany.

Lysine-specific demethylase 1 (LSD1) has evolved as a promising therapeutic target for cancer treatment, especially in acute myeloid leukaemia (AML). To approach the challenge of site-specific LSD1 inhibition, we developed an enzyme-prodrug system with the bacterial nitroreductase NfsB (NTR) that was expressed in the virally transfected AML cell line THP1-NTR . The cellular activity of the NTR was proven with a new luminescent NTR probe. We synthesised a diverse set of nitroaromatic prodrugs that by design do not affect LSD1 and are reduced by the NTR to release an active LSD1 inhibitor. The emerging side products were differentially analysed using negative controls, thereby revealing cytotoxic effects. The 2-nitroimidazolyl prodrug of a potent LSD1 inhibitor emerged as one of the best prodrug candidates with a pronounced selectivity window between wild-type and transfected THP1 cells. Our prodrugs are selectively activated and release the LSD1 inhibitor locally, proving their suitability for future targeting approaches.
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http://dx.doi.org/10.1002/cbic.202000138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7497180PMC
August 2020

Chemical Genetics Screen Identifies Epigenetic Mechanisms Involved in Dopaminergic and Noradrenergic Neurogenesis in Zebrafish.

Front Genet 2020 25;11:80. Epub 2020 Feb 25.

Developmental Biology, Faculty of Biology, Institute Biology 1, Albert Ludwigs University Freiburg, Freiburg, Germany.

The cell type diversity and complexity of the nervous system is generated by a network of signaling events, transcription factors, and epigenetic regulators. Signaling and transcriptional control have been easily amenable to forward genetic screens in model organisms like zebrafish. In contrast, epigenetic mechanisms have been somewhat elusive in genetic screens, likely caused by broad action in multiple developmental pathways that masks specific phenotypes, but also by genetic redundancies of epigenetic factors. Here, we performed a screen using small molecule inhibitors of epigenetic mechanisms to reveal contributions to specific aspects of neurogenesis in zebrafish. We chose development of dopaminergic and noradrenergic neurons from neural progenitors as target of epigenetic regulation. We performed the screen in two phases: First, we tested a small molecule inhibitor library that targets a broad range of epigenetic protein classes and mechanisms, using expression of the dopaminergic and noradrenergic marker as readout. We identified 10 compounds, including HDAC, Bromodomain and HAT inhibitors, which interfered with dopaminergic and noradrenergic development in larval zebrafish. In the second screening phase, we aimed to identify neurogenesis stages affected by these 10 inhibitors. We analyzed treated embryos for effects on neural stem cells, growth progression of the retina, and apoptosis in neural tissues. In addition, we analyzed effects on expressing neuronal populations to determine potential selectivity of compounds for transmitter phenotypes. In summary, our targeted screen of epigenetic inhibitors identified specific compounds, which reveal chromatin regulator classes that contribute to dopaminergic and noradrenergic neurogenesis .
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http://dx.doi.org/10.3389/fgene.2020.00080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052299PMC
February 2020
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