Publications by authors named "Karin Schmidtkunz"

25 Publications

  • Page 1 of 1

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

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

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

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

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

Synthesis and Biological Investigation of Phenothiazine-Based Benzhydroxamic Acids as Selective Histone Deacetylase 6 Inhibitors.

J Med Chem 2019 02 1;62(3):1138-1166. Epub 2019 Feb 1.

Department of Pharmacy-Center for Drug Research , Ludwig-Maximilians University Munich , Butenandtstr. 5-13 , 81377 Munich , Germany.

The phenothiazine system was identified as a favorable cap group for potent and selective histone deacetylase 6 (HDAC6) inhibitors. Here, we report the preparation and systematic variation of phenothiazines and their analogues containing a benzhydroxamic acid moiety as the zinc-binding group. We evaluated their ability to selectively inhibit HDAC6 by a recombinant HDAC enzyme assay, 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. Structure-activity relationship studies revealed that incorporation of a nitrogen atom into the phenothiazine framework results in increased potency and selectivity for HDAC6 (more than 500-fold selectivity relative to the inhibition of HDAC1, HDAC4, and HDAC8), as rationalized by molecular modeling and docking studies. The binding mode was confirmed by co-crystallization of the potent azaphenothiazine inhibitor with catalytic domain 2 from Danio rerio HDAC6.
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http://dx.doi.org/10.1021/acs.jmedchem.8b01090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6519732PMC
February 2019

Characterization of Histone Deacetylase 8 (HDAC8) Selective Inhibition Reveals Specific Active Site Structural and Functional Determinants.

J Med Chem 2018 11 8;61(22):10000-10016. Epub 2018 Nov 8.

Institut de Génétique et Biologie Moléculaire et Cellulaire (IGBMC), Département de Biologie Structurale Intégrative , Université de Strasbourg, Centre National de la Recherche Scientifique (CNRS UMR7104), Institut National de la Santé et de la Recherche Médicale (INSERM U1258) , 1 rue Laurent Fries , 67404 Illkirch Cedex , France.

Metal-dependent histone deacetylases (HDACs) are key epigenetic regulators that represent promising therapeutic targets for the treatment of numerous human diseases. Yet the currently FDA-approved HDAC inhibitors nonspecifically target at least several of the 11 structurally similar but functionally different HDAC isozymes, which hampers their broad usage in clinical settings. Selective inhibitors targeting single HDAC isozymes are being developed, but precise understanding in molecular terms of their selectivity remains sparse. Here, we show that HDAC8-selective inhibitors adopt a L-shaped conformation required for their binding to a HDAC8-specific pocket formed by HDAC8 catalytic tyrosine and HDAC8 L1 and L6 loops. In other HDAC isozymes, a L1-L6 lock sterically prevents L-shaped inhibitor binding. Shielding of the HDAC8-specific pocket by protein engineering decreases potency of HDAC8-selective inhibitors and affects catalytic activity. Collectively, our results unravel key HDAC8 active site structural and functional determinants important for the design of next-generation chemical probes and epigenetic drugs.
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http://dx.doi.org/10.1021/acs.jmedchem.8b01087DOI Listing
November 2018

Synthesis, Crystallization Studies, and in vitro Characterization of Cinnamic Acid Derivatives as SmHDAC8 Inhibitors for the Treatment of Schistosomiasis.

ChemMedChem 2018 08 4;13(15):1517-1529. Epub 2018 Jul 4.

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

Schistosomiasis is a neglected parasitic disease that affects more than 265 million people worldwide and for which the control strategy relies on mass treatment with only one drug: praziquantel. Based on the 3-chlorobenzothiophene-2-hydroxamic acid J1075, a series of hydroxamic acids with different scaffolds were prepared as potential inhibitors of Schistosoma mansoni histone deacetylase 8 (SmHDAC8). The crystal structures of SmHDAC8 with four inhibitors provided insight into the binding mode and orientation of molecules in the binding pocket as well as the orientation of its flexible amino acid residues. The compounds were evaluated in screens for inhibitory activity against schistosome and human HDACs. The most promising compounds were further investigated for their activity toward the major human HDAC isotypes. The most potent inhibitors were additionally screened for lethality against the schistosome larval stage using a fluorescence-based assay. Two of the compounds 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/10.1002/cmdc.201800238DOI Listing
August 2018

A Novel Class of Schistosoma mansoni Histone Deacetylase 8 (HDAC8) Inhibitors Identified by Structure-Based Virtual Screening and In Vitro Testing.

Molecules 2018 Mar 2;23(3). Epub 2018 Mar 2.

Department of Pharmaceutical Chemistry, University Halle-Wittenberg, 06120 Halle/Saale, Germany.

A promising means in the search of new small molecules for the treatment of schistosomiasis (amongst other parasitic ailments) is by targeting the parasitic epigenome. In the present study, a docking based virtual screening procedure using the crystal structure of histone deacetylase 8 from (smHDAC8) was designed. From the developed screening protocol, we were able to identify eight novel -(2,5-dioxopyrrolidin-3-yl)--alkylhydroxamate derivatives as smHDAC8 inhibitors with IC values ranging from 4.4-20.3 µM against smHDAC8. These newly identified inhibitors were further tested against human histone deacetylases (hsHDAC1, 6 and 8), and were found also to be exerting interesting activity against them. In silico prediction of the docking pose of the compounds was confirmed by the resolved crystal structure of one of the identified hits. This confirmed these compounds were able to chelate the catalytic zinc ion in a bidentate fashion, whilst showing an inverted binding mode of the hydroxamate group when compared to the reported smHDAC8/hydroxamates crystal structures. Therefore, they can be considered as new potential scaffold for the development of new smHDAC8 inhibitors by further investigation of their structure-activity relationship.
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http://dx.doi.org/10.3390/molecules23030566DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6017931PMC
March 2018

Structure-Based Design and Biological Characterization of Selective Histone Deacetylase 8 (HDAC8) Inhibitors with Anti-Neuroblastoma Activity.

J Med Chem 2017 12 15;60(24):10188-10204. Epub 2017 Dec 15.

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

Histone deacetylases (HDACs) are important modulators of epigenetic gene regulation and additionally control the activity of non-histone protein substrates. While for HDACs 1-3 and 6 many potent selective inhibitors have been obtained, for other subtypes much less is known on selective inhibitors and the consequences of their inhibition. The present report describes the development of substituted benzhydroxamic acids as potent and selective HDAC8 inhibitors. Docking studies using available crystal structures have been used for structure-based optimization of this series of compounds. Within this study, we have investigated the role of HDAC8 in the proliferation of cancer cells and optimized hits for potency and selectivity, both in vitro and in cell culture. The combination of structure-based design, synthesis, and in vitro screening to cellular testing resulted in potent and selective HDAC8 inhibitors that showed anti-neuroblastoma activity in cellular testing.
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http://dx.doi.org/10.1021/acs.jmedchem.7b01447DOI Listing
December 2017

Structure-Activity Relationship of Propargylamine-Based HDAC Inhibitors.

ChemMedChem 2017 12 30;12(24):2044-2053. Epub 2017 Nov 30.

Organic and Bioorganic Chemistry, Bielefeld University, Universitätsstrasse 25, 33615, Bielefeld, Germany.

As histone deacetylases (HDACs) play an important role in the treatment of cancer, their selective inhibition has been the subject of various studies. These continuous investigations have given rise to a large collection of pan- and selective HDAC inhibitors, containing diverse US Food and Drug Administration (FDA)-approved representatives. In previous studies, a class of alkyne-based HDAC inhibitors was presented. We modified this scaffold in two previously neglected regions and compared their cytotoxicity and affinity toward HDAC1, HDAC6, and HDAC8. We were able to show that R-configured propargylamines contribute to increased selectivity for HDAC6. Docking studies on available HDAC crystal structures were carried out to rationalize the observed selectivity of the compounds. Substitution of the aromatic portion by a thiophene derivative results in high affinity and low cytotoxicity, indicating an improved drug tolerance.
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http://dx.doi.org/10.1002/cmdc.201700550DOI Listing
December 2017

Metabolic Response to XD14 Treatment in Human Breast Cancer Cell Line MCF-7.

Int J Mol Sci 2016 Oct 24;17(10). Epub 2016 Oct 24.

Center for Biological Systems Analysis ZBSA, Albert-Ludwigs-University Freiburg, 79104 Freiburg, Germany.

XD14 is a 4-acyl pyrrole derivative, which was discovered by a high-throughput virtual screening experiment. XD14 inhibits bromodomain and extra-terminal domain (BET) proteins (BRD2, BRD3, BRD4 and BRDT) and consequently suppresses cell proliferation. In this study, metabolic profiling reveals the molecular effects in the human breast cancer cell line MCF-7 (Michigan Cancer Foundation-7) treated by XD14. A three-day time series experiment with two concentrations of XD14 was performed. Gas chromatography-mass spectrometry (GC-MS) was applied for untargeted profiling of treated and non-treated MCF-7 cells. The gained data sets were evaluated by several statistical methods: analysis of variance (ANOVA), clustering analysis, principle component analysis (PCA), and partial least squares discriminant analysis (PLS-DA). Cell proliferation was strongly inhibited by treatment with 50 µM XD14. Samples could be discriminated by time and XD14 concentration using PLS-DA. From the 117 identified metabolites, 67 were significantly altered after XD14 treatment. These metabolites include amino acids, fatty acids, Krebs cycle and glycolysis intermediates, as well as compounds of purine and pyrimidine metabolism. This massive intervention in energy metabolism and the lack of available nucleotides could explain the decreased proliferation rate of the cancer cells.
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http://dx.doi.org/10.3390/ijms17101772DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5085796PMC
October 2016

Tofacitinib and analogs as inhibitors of the histone kinase PRK1 (PKN1).

Future Med Chem 2016 09 30;8(13):1537-51. Epub 2016 Aug 30.

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

Aim: The histone kinase PRK1 has been identified as a potential target to combat prostate cancer but selective PRK1 inhibitors are lacking. The US FDA -approved JAK1-3 inhibitor tofacitinib also potently inhibits PRK1 in vitro.

Results: We show that tofacitinib also inhibits PRK1 in a cellular setting. Using tofacitinib as a starting point for structure-activity relationship studies, we identified a more potent and another more selective PRK1 inhibitor compared with tofacitinib. Furthermore, we found two potential PRK1/JAK3-selectivity hotspots.

Conclusion: The identified inhibitors and the selectivity hotspots lay the basis for the development of selective PRK1 inhibitors. The identification of PRK1, but also of other cellular tofacitinib targets, has implications on its clinical use and on future development of tofacitinib-like JAK inhibitors. [Formula: see text].
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http://dx.doi.org/10.4155/fmc-2016-0132DOI Listing
September 2016

Identification of Highly Potent Protein Kinase C-Related Kinase 1 Inhibitors by Virtual Screening, Binding Free Energy Rescoring, and in vitro Testing.

ChemMedChem 2016 09 29;11(18):2084-94. Epub 2016 Jul 29.

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

Despite the considerable interest in protein kinase C-related kinase 1 (PRK1) as a target in cancer research, there is still a lack of PRK1 inhibitors with suitable selectivity profiles and physicochemical properties. To identify new PRK1 inhibitors we applied a virtual screening approach, which combines ensemble docking, minimization of the protein-ligand complex, binding free energy calculations, and application of quantitative structure-activity relationship (QSAR) models for predicting in vitro activity. The developed approach was then applied in a prospective manner to screen available libraries of kinase inhibitors from Selleck and GlaxoSmithKline (GSK). Compounds that showed favorable prediction were then tested in vitro for PRK1 inhibition. Some of the hits were found to inhibit PRK1 in the low-nanomolar range. Three in vitro hits were additionally tested in a mass-spectrometry-based cellular kinase profiling assay to examine selectivity. Our findings show that nanomolar and drug-like inhibitors can be identified by the virtual screening approach presented herein. The identified inhibitors are valuable tools for gaining a better understanding of PRK1 inhibition, and the identified hits can serve as starting points for further chemical optimization.
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http://dx.doi.org/10.1002/cmdc.201600284DOI Listing
September 2016

Preparation data of the bromodomains BRD3(1), BRD3(2), BRD4(1), and BRPF1B and crystallization of BRD4(1)-inhibitor complexes.

Data Brief 2016 Jun 11;7:1370-4. Epub 2016 Apr 11.

Albert-Ludwigs-Universität Freiburg, Institut für Biochemie, Albertstr. 21, D-79104 Freiburg, Germany.

This article presents detailed purification procedures for the bromodomains BRD3(1), BRD3(2), BRD4(1), and BRPF1B. In addition we provide crystallization protocols for apo BRD4(1) and BRD4(1) in complex with numerous inhibitors. The protocols described here were successfully applied to obtain affinity data by isothermal titration calorimetry (ITC) and by differential scanning fluorimetry (DSF) as well as structural characterizations of BRD4(1) inhibitor complexes (PDB codes: PDB: 4LYI, PDB: 4LZS, PDB: 4LYW, PDB: 4LZR, PDB: 4LYS, PDB: 5D24, PDB: 5D25, PDB: 5D26, PDB: 5D3H, PDB: 5D3J, PDB: 5D3L, PDB: 5D3N, PDB: 5D3P, PDB: 5D3R, PDB: 5D3S, PDB: 5D3T). These data have been reported previously and are discussed in more detail elsewhere [1], [2].
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http://dx.doi.org/10.1016/j.dib.2016.04.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4845076PMC
June 2016

Identification of a small-molecule ligand of the epigenetic reader protein Spindlin1 via a versatile screening platform.

Nucleic Acids Res 2016 05 17;44(9):e88. Epub 2016 Feb 17.

Institute of Pharmaceutical Sciences, University of Freiburg, Freiburg 79104, Germany BIOSS Centre of Biological Signalling Studies, University of Freiburg, 79106 Freiburg, Germany German Cancer Research Centre (DKFZ), Heidelberg, Germany

Epigenetic modifications of histone tails play an essential role in the regulation of eukaryotic transcription. Writer and eraser enzymes establish and maintain the epigenetic code by creating or removing posttranslational marks. Specific binding proteins, called readers, recognize the modifications and mediate epigenetic signalling. Here, we present a versatile assay platform for the investigation of the interaction between methyl lysine readers and their ligands. This can be utilized for the screening of small-molecule inhibitors of such protein-protein interactions and the detailed characterization of the inhibition. Our platform is constructed in a modular way consisting of orthogonal in vitro binding assays for ligand screening and verification of initial hits and biophysical, label-free techniques for further kinetic characterization of confirmed ligands. A stability assay for the investigation of target engagement in a cellular context complements the platform. We applied the complete evaluation chain to the Tudor domain containing protein Spindlin1 and established the in vitro test systems for the double Tudor domain of the histone demethylase JMJD2C. We finally conducted an exploratory screen for inhibitors of the interaction between Spindlin1 and H3K4me3 and identified A366 as the first nanomolar small-molecule ligand of a Tudor domain containing methyl lysine reader.
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http://dx.doi.org/10.1093/nar/gkw089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4872087PMC
May 2016

Selective Sirt2 inhibition by ligand-induced rearrangement of the active site.

Nat Commun 2015 Feb 12;6:6263. Epub 2015 Feb 12.

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

Sirtuins are a highly conserved class of NAD(+)-dependent lysine deacylases. The human isotype Sirt2 has been implicated in the pathogenesis of cancer, inflammation and neurodegeneration, which makes the modulation of Sirt2 activity a promising strategy for pharmaceutical intervention. A rational basis for the development of optimized Sirt2 inhibitors is lacking so far. Here we present high-resolution structures of human Sirt2 in complex with highly selective drug-like inhibitors that show a unique inhibitory mechanism. Potency and the unprecedented Sirt2 selectivity are based on a ligand-induced structural rearrangement of the active site unveiling a yet-unexploited binding pocket. Application of the most potent Sirtuin-rearranging ligand, termed SirReal2, leads to tubulin hyperacetylation in HeLa cells and induces destabilization of the checkpoint protein BubR1, consistent with Sirt2 inhibition in vivo. Our structural insights into this unique mechanism of selective sirtuin inhibition provide the basis for further inhibitor development and selective tools for sirtuin biology.
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http://dx.doi.org/10.1038/ncomms7263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4339887PMC
February 2015

The discovery of a highly selective 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4(3H)-one SIRT2 inhibitor that is neuroprotective in an in vitro Parkinson's disease model.

ChemMedChem 2015 Jan 13;10(1):69-82. Epub 2014 Nov 13.

Department of Chemistry, Imperial College London, St. Kensington Campus, London SW7 2AZ, (UK).

Sirtuins, NAD(+) -dependent histone deacetylases (HDACs), have recently emerged as potential therapeutic targets for the treatment of a variety of diseases. The discovery of potent and isoform-selective inhibitors of this enzyme family should provide chemical tools to help determine the roles of these targets and validate their therapeutic value. Herein, we report the discovery of a novel class of highly selective SIRT2 inhibitors, identified by pharmacophore screening. We report the identification and validation of 3-((2-methoxynaphthalen-1-yl)methyl)-7-((pyridin-3-ylmethyl)amino)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4(3H)-one (ICL-SIRT078), a substrate-competitive SIRT2 inhibitor with a Ki value of 0.62 ± 0.15 μM and more than 50-fold selectivity against SIRT1, 3 and 5. Treatment of MCF-7 breast cancer cells with ICL-SIRT078 results in hyperacetylation of α-tubulin, an established SIRT2 biomarker, at doses comparable with the biochemical IC50 data, while suppressing MCF-7 proliferation at higher concentrations. In concordance with the recent reports that suggest SIRT2 inhibition is a potential strategy for the treatment of Parkinson's disease, we find that compound ICL-SIRT078 has a significant neuroprotective effect in a lactacystin-induced model of Parkinsonian neuronal cell death in the N27 cell line. These results encourage further investigation into the effects of ICL-SIRT078, or an optimised derivative thereof, as a candidate neuroprotective agent in in vivo models of Parkinson's disease.
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http://dx.doi.org/10.1002/cmdc.201402431DOI Listing
January 2015

Discovery of inhibitors of Schistosoma mansoni HDAC8 by combining homology modeling, virtual screening, and in vitro validation.

J Chem Inf Model 2014 Oct 2;54(10):3005-19. Epub 2014 Oct 2.

Department of Pharmaceutical Chemistry, University Halle-Wittenberg , 06120 Halle/Saale, Germany.

Schistosomiasis, caused by S. mansoni, is a tropical disease that affects over 200 million people worldwide. A novel approach for targeting eukaryotic parasites is to tackle their dynamic epigenetic machinery that is necessary for the extensive phenotypic changes during their life cycle. We recently identified S. mansoni histone deacetylase 8 (smHDAC8) as a potential target for antiparasitic therapy. Here we present results from a virtual screening campaign on smHDAC8. Besides hydroxamates, several sulfonamide-thiazole derivatives were identified by a target-based virtual screening using a homology model of smHDAC8. In vitro testing of 75 compounds identified 8 hydroxamates as potent and lead-like inhibitors of the parasitic HDAC8. Solving of the crystal structure of smHDAC8 with two of the virtual screening hits confirmed the predicted binding mode.
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http://dx.doi.org/10.1021/ci5004653DOI Listing
October 2014

Total synthesis of (18S)- and (18R)-homolargazole by rhodium-catalyzed hydrocarboxylation.

Chemistry 2014 Feb 29;20(8):2164-8. Epub 2014 Jan 29.

Institut für Organische Chemie, Albert-Ludwigs-Universität Freiburg, Albertstrasse 21, 79104 Freiburg i. Bg. (Germany), Fax: (+49) 761-203-8715.

Homolargazole derivatives, in which the macrocycle of natural largazole is extended by one methylene group, were prepared by the recently developed rhodium-catalyzed hydrocarboxylation reaction onto allenes. This strategy gives access to both the (18S)- and (18R)-stereoisomers in high stereoselectivity under ligand control.
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http://dx.doi.org/10.1002/chem.201303300DOI Listing
February 2014

4-Acyl pyrroles: mimicking acetylated lysines in histone code reading.

Angew Chem Int Ed Engl 2013 Dec 24;52(52):14055-9. Epub 2013 Nov 24.

Pharmazeutische Bioinformatik, Institut für Pharmazeutische, Wissenschaften, Albert-Ludwigs-Universität, Hermann-Herder-Strasse 9, 79104 Freiburg (Germany) http://www.pharmaceutical-bioinformatics.org.

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http://dx.doi.org/10.1002/anie.201307652DOI Listing
December 2013

Structural basis for the inhibition of histone deacetylase 8 (HDAC8), a key epigenetic player in the blood fluke Schistosoma mansoni.

PLoS Pathog 2013 26;9(9):e1003645. Epub 2013 Sep 26.

Département de Biologie Structurale Intégrative, Institut de Génétique et Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UDS), CNRS, INSERM, Illkirch, France.

The treatment of schistosomiasis, a disease caused by blood flukes parasites of the Schistosoma genus, depends on the intensive use of a single drug, praziquantel, which increases the likelihood of the development of drug-resistant parasite strains and renders the search for new drugs a strategic priority. Currently, inhibitors of human epigenetic enzymes are actively investigated as novel anti-cancer drugs and have the potential to be used as new anti-parasitic agents. Here, we report that Schistosoma mansoni histone deacetylase 8 (smHDAC8), the most expressed class I HDAC isotype in this organism, is a functional acetyl-L-lysine deacetylase that plays an important role in parasite infectivity. The crystal structure of smHDAC8 shows that this enzyme adopts a canonical α/β HDAC fold, with specific solvent exposed loops corresponding to insertions in the schistosome HDAC8 sequence. Importantly, structures of smHDAC8 in complex with generic HDAC inhibitors revealed specific structural changes in the smHDAC8 active site that cannot be accommodated by human HDACs. Using a structure-based approach, we identified several small-molecule inhibitors that build on these specificities. These molecules exhibit an inhibitory effect on smHDAC8 but show reduced affinity for human HDACs. Crucially, we show that a newly identified smHDAC8 inhibitor has the capacity to induce apoptosis and mortality in schistosomes. Taken together, our biological and structural findings define the framework for the rational design of small-molecule inhibitors specifically interfering with schistosome epigenetic mechanisms, and further support an anti-parasitic epigenome targeting strategy to treat neglected diseases caused by eukaryotic pathogens.
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http://dx.doi.org/10.1371/journal.ppat.1003645DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3784479PMC
April 2014

The Discovery of Novel 10,11-Dihydro-5H-dibenz[b,f]azepine SIRT2 Inhibitors.

Medchemcomm 2012 Mar(3)

Department of Chemistry, Imperial College London, London, United Kingdom.

Isoform selective inhibitors of the sirtuins (NAD-dependent histone deacetylases) should enable an in depth study of the molecular biology underpinning these targets and how they are deregulated in diseases such as cancer and neurodegeneration. Herein, we present the discovery of structurally novel SIRT2 inhibitors. Hit molecule was discovered through the chemical synthesis and biological characterization of a small-molecule compound library based around the 10,11-dihydro-5H-dibenz[b,f]azepine scaffold. screening assays revealed compound to have an IC of 18 μM against SIRT2 and to exhibit more than 30-fold selectivity compared to SIRT1. Cellular assays, performed on MCF-7 cells, confirmed the selectivity and showed hit to have antiproliferative activity at a concentration of 30 μM. Computational studies were performed to predict the SIRT2 binding mode and to rationalise the observed selectivity.
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http://dx.doi.org/10.1039/C2MD00290FDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3856871PMC
March 2012

Targeting the c-Myc coiled coil with interfering peptides.

J Pept Sci 2008 Sep;14(9):1022-31

Institute for Biology III, Albert-Ludwigs University of Freiburg, Schaenzlestrasse 1, Freiburg, Germany.

c-Myc is one of the most frequently deregulated oncogenes in human cancers, and recent studies showed that even brief inactivation of Myc can be sufficient to induce tumor regression or loss. Consequently, inactivation of Myc provides a novel therapeutic opportunity and challenge, as the dimerization of Myc with Max is crucial for its function. We applied two strategies to specifically target this coiled coil mediated interaction with interfering peptides: a dominant-negative human Max sequence (Max) and a peptide selected from a genetic library (Mip). Both peptides form coiled coils and were fused to an acidic extension interacting with the basic DNA-binding region of human Myc. The genetic library was obtained by semi-rational design randomizing residues important for interaction, and selection was carried out using a protein-fragment complementation assay. The peptides Max and Mip easily outcompeted the human Myc:Max interaction and successfully interfered with the DNA binding of the complex. Both interfering peptides exhibited higher T(m) (DeltaT(m) = 13 and 15 degrees C) upon interaction with Myc compared to wt Max. The inhibitory effect of the two interfering peptides on human Myc:Max activity makes them promising molecules for analytical and therapeutic Myc-directed research.
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http://dx.doi.org/10.1002/psc.1038DOI Listing
September 2008
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