Publications by authors named "Manfred Jung"

203 Publications

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.

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

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 Dec 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

Proteolysis targeting chimeras (PROTACs) for epigenetics research.

Curr Opin Chem Biol 2020 08 5;57:8-16. Epub 2020 Mar 5.

Institute of Pharmaceutical Sciences, University of Freiburg, Albertstraße 25, Freiburg, 79104, Germany; CIBSS - Centre for Integrative Biological Signalling Studies, University of Freiburg, Schänzlestraße 18, Freiburg, 79104, Germany. Electronic address:

Proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules and allow selective protein degradation by addressing the natural ubiquitin proteasome system. As this new strategy of chemically induced protein degradation can serve as a biological tool and provides new possibilities for drug discovery, it has been applied to a variety of targets including (nuclear) receptors, kinases, and epigenetic proteins. A lot of PROTACs have already been designed in the field of epigenetics, and their synthesis and characterization highly contributed to structural optimization and improved mechanistic understanding of these molecules. In this review, we will discuss and summarize recent advances in PROTAC discovery with focus on epigenetic targets.
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http://dx.doi.org/10.1016/j.cbpa.2020.01.010DOI Listing
August 2020

Structure-Based Design, Synthesis, and Biological Evaluation of Triazole-Based smHDAC8 Inhibitors.

ChemMedChem 2020 04 9;15(7):571-584. Epub 2020 Jan 9.

Department of Chemistry Institute of Organic Chemistry, University of Hamburg, Martin-Luther-King-Platz 6, 20146, Hamburg, Germany.

Schistosomiasis is a neglected tropical disease caused by parasitic flatworms of the genus Schistosoma, which affects over 200 million people worldwide and leads to at least 300,000 deaths every year. In this study, initial screening revealed the triazole-based hydroxamate 2 b (N-hydroxy-1-phenyl-1H-1,2,3-triazole-4-carboxamide) exhibiting potent inhibitory activity toward the novel antiparasitic target Schistosoma mansoni histone deacetylase 8 (smHDAC8) and promising selectivity over the major human HDACs. Subsequent crystallographic studies of the 2 b/smHDAC8 complex revealed key interactions between the inhibitor and the enzyme's active site, thus explaining the unique selectivity profile of the inhibitor. Further chemical modifications of 2 b led to the discovery of 4-fluorophenoxy derivative 21 (1-[5-chloro-2-(4-fluorophenoxy)phenyl]-N-hydroxy-1H-1,2,3-triazole-4-carboxamide), a nanomolar smHDAC8 inhibitor (IC =0.5 μM), exceeding the smHDAC8 inhibitory activity of 2 b and SAHA (vorinostat), while exhibiting an improved selectivity profile over the investigated human HDACs. Collectively, this study reveals specific interactions between smHDAC8 and the synthesized triazole-based inhibitors and demonstrates that these small molecules represent promising lead structures, which could be further developed in the search for novel drugs for the treatment of schistosomiasis.
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http://dx.doi.org/10.1002/cmdc.201900583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7187165PMC
April 2020

Exploring Structural Determinants of Inhibitor Affinity and Selectivity in Complexes with Histone Deacetylase 6.

J Med Chem 2020 01 19;63(1):295-308. Epub 2019 Dec 19.

Roy and Diana Vagelos Laboratories, Department of Chemistry , University of Pennsylvania , 231 South 34th Street , Philadelphia , Pennsylvania 19104-6323 , United States.

Inhibition of histone deacetylase 6 (HDAC6) has emerged as a promising therapeutic strategy for the treatment of cancer, chemotherapy-induced peripheral neuropathy, and neurodegenerative disease. The recent X-ray crystal structure determination of HDAC6 enables an understanding of structural features directing affinity and selectivity in the active site. Here, we present the X-ray crystal structures of five HDAC6-inhibitor complexes that illuminate key molecular features of the inhibitor linker and capping groups that facilitate and differentiate binding to HDAC6. In particular, aromatic and heteroaromatic linkers nestle within an aromatic cleft defined by F583 and F643, and different aromatic linkers direct the capping group toward shallow pockets defined by the L1 loop, the L2 loop, or somewhere in between these pockets. These results expand our understanding of factors contributing to the selective inhibition of HDAC6, particularly regarding interactions that can be targeted in the region of the L2 pocket.
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http://dx.doi.org/10.1021/acs.jmedchem.9b01540DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6952581PMC
January 2020

Validation of the Slow Off-Kinetics of Sirtuin-Rearranging Ligands (SirReals) by Means of Label-Free Electrically Switchable Nanolever Technology.

Chembiochem 2020 04 22;21(8):1161-1166. Epub 2020 Jan 22.

Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg, Albertstrasse 25, 79104, Freiburg im Breisgau, Germany.

We have discovered the sirtuin-rearranging ligands (SirReals) to be highly potent and selective inhibitors of the NAD -dependent lysine deacetylase Sirt2. Using a biotinylated SirReal in combination with biolayer interferometry, we previously observed a slow dissociation rate of the inhibitor-enzyme complex; this had been postulated to be the key to the high affinity and selectivity of SirReals. However, to attach biotin to the SirReal core, we introduced a triazole as a linking moiety; this was shown by X-ray co-crystallography to interact with Arg97 of the cofactor binding loop. Herein, we aim to elucidate whether the observed long residence time of the SirReals is induced mainly by triazole incorporation or is an inherent characteristic of the SirReal inhibitor core. We used the novel label-free switchSENSE® technology, which is based on electrically switchable DNA nanolevers, to prove that the long residence time of the SirReals is indeed caused by the core scaffold.
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http://dx.doi.org/10.1002/cbic.201900527DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7217041PMC
April 2020

Screening and Phenotypical Characterization of Histone Deacetylase 8 (HDAC8) Inhibitors as Multistage Antischistosomal Agents.

ACS Infect Dis 2020 01 12;6(1):100-113. Epub 2019 Nov 12.

Institute of Biochemistry and Cell Biology (IBBC) , National Research Council (CNR) , Campus A. Buzzati-Traverso, via E. Ramarini 32 , 00015 Monterotondo ( Rome ), Italy.

Schistosomiasis (also known as bilharzia) is a neglected tropical disease caused by platyhelminths of the genus . The disease is endemic in tropical and subtropical areas of the world where water is infested by the intermediate parasite host, the snail. More than 800 million people live in endemic areas and more than 200 million are infected and require treatment. Praziquantel (PZQ) is the drug of choice for schistosomiasis treatment and transmission control being safe and very effective against adult worms of all the clinically relevant species. Unfortunately, it is ineffective on immature, juvenile worms; therefore, it does not prevent reinfection. Moreover, the risk of development and spread of drug resistance because of the widespread use of a single drug in such a large population represents a serious threat. Therefore, research aimed at identifying novel drugs to be used alone or in combination with PZQ are needed. histone deacetylase 8 (HDAC8) is a class I zinc-dependent HDAC, which is abundantly expressed in all stages of its life cycle, thus representing an interesting target for drug discovery. Through virtual screening and phenotypical characterization of selected hits, we discovered two main chemical classes of compounds characterized by the presence of a hydroxamate-based metal binding group coupled to a spiroindoline or a tricyclic thieno[3,2-]indole core as capping groups. Some of the compounds of both classes were deeply investigated and showed to impair viability of larval, juvenile, and adult schistosomes, to impact egg production and/or to induce morphological alterations of the adult schistosome reproductive systems. Noteworthy, all of them inhibit the recombinant form of HDAC8 enzyme . Overall, we identified very interesting scaffolds, paving the way to the development of effective antischistosomal agents.
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http://dx.doi.org/10.1021/acsinfecdis.9b00224DOI Listing
January 2020

Lifespan-increasing drug nordihydroguaiaretic acid inhibits p300 and activates autophagy.

NPJ Aging Mech Dis 2019 2;5. Epub 2019 Oct 2.

1Buck Institute for Research on Aging, 8001 Redwood Boulevard, Novato, CA 94945-1400 USA.

Aging is characterized by the progressive loss of physiological function in all organisms. Remarkably, the aging process can be modulated by environmental modifications, including diet and small molecules. The natural compound nordihydroguaiaretic acid (NDGA) robustly increases lifespan in flies and mice, but its mechanism of action remains unclear. Here, we report that NDGA is an inhibitor of the epigenetic regulator p300. We find that NDGA inhibits p300 acetyltransferase activity in vitro and suppresses acetylation of a key p300 target in histones (i.e., H3K27) in cells. We use the cellular thermal shift assay to uniquely demonstrate NDGA binding to p300 in cells. Finally, in agreement with recent findings indicating that p300 is a potent blocker of autophagy, we show that NDGA treatment induces autophagy. These findings identify p300 as a target of NDGA and provide mechanistic insight into its role in longevity.
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http://dx.doi.org/10.1038/s41514-019-0037-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6775102PMC
October 2019

Azologization and repurposing of a hetero-stilbene-based kinase inhibitor: towards the design of photoswitchable sirtuin inhibitors.

Beilstein J Org Chem 2019 16;15:2170-2183. Epub 2019 Sep 16.

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

The use of light as an external trigger to change ligand shape and as a result its bioactivity, allows the probing of pharmacologically relevant systems with spatiotemporal resolution. A hetero-stilbene lead resulting from the screening of a compound that was originally designed as kinase inhibitor served as a starting point for the design of photoswitchable sirtuin inhibitors. Because the original stilbenoid structure exerted unfavourable photochemical characteristics it was remodelled to its heteroarylic diazeno analogue. By this intramolecular azologization, the shape of the molecule was left unaltered, whereas the photoswitching ability was improved. As anticipated, the highly analogous compound showed similar activity in its thermodynamically stable stretched-out ()-form. Irradiation of this isomer triggers isomerisation to the long-lived ()-configuration with a bent geometry causing a considerably shorter end-to-end distance. The resulting affinity shifts are intended to enable real-time photomodulation of sirtuins in vitro.
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http://dx.doi.org/10.3762/bjoc.15.214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6774072PMC
September 2019

A Chemical Probe for Tudor Domain Protein Spindlin1 to Investigate Chromatin Function.

J Med Chem 2019 10 15;62(20):9008-9025. Epub 2019 Oct 15.

Structural Genomics Consortium, Nuffield Department of Medicine , University of Oxford , OX3 7DQ Oxford , U.K.

Modifications of histone tails, including lysine/arginine methylation, provide the basis of a "chromatin or histone code". Proteins that contain "reader" domains can bind to these modifications and form specific effector complexes, which ultimately mediate chromatin function. The spindlin1 (SPIN1) protein contains three Tudor methyllysine/arginine reader domains and was identified as a putative oncogene and transcriptional coactivator. Here we report a SPIN1 chemical probe inhibitor with low nanomolar in vitro activity, exquisite selectivity on a panel of methyl reader and writer proteins, and with submicromolar cellular activity. X-ray crystallography showed that this Tudor domain chemical probe simultaneously engages Tudor domains 1 and 2 via a bidentate binding mode. Small molecule inhibition and siRNA knockdown of SPIN1, as well as chemoproteomic studies, identified genes which are transcriptionally regulated by SPIN1 in squamous cell carcinoma and suggest that SPIN1 may have a role in cancer related inflammation and/or cancer metastasis.
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http://dx.doi.org/10.1021/acs.jmedchem.9b00562DOI Listing
October 2019

Structure-Reactivity Relationships on Substrates and Inhibitors of the Lysine Deacylase Sirtuin 2 from (Sirt2).

J Med Chem 2019 10 20;62(19):8733-8759. Epub 2019 Sep 20.

Institute of Pharmaceutical Sciences , University of Freiburg , Albertstr. 25 , 79104 Freiburg , Germany.

The only drug currently available for treatment of the neglected disease Schistosomiasis is Praziquantel, and the possible emergence of resistance makes research on novel therapeutic agents necessary and urgent. To this end, the targeting of epigenetic enzymes, which regulate the parasitic life cycle, emerged as a promising approach. Due to the strong effects of human sirtuin inhibitors on parasite survival and reproduction, sirtuins were postulated as potential therapeutic targets. In vitro testing of synthetic substrates of sirtuin 2 (Sirt2) and kinetic experiments on a myristoylated peptide demonstrated lysine long-chain deacylation as an intrinsic Sirt2 activity in addition to its known deacetylase activity for the first time. Focused in vitro screening of the GSK Kinetobox library and structure-activity relationships of identified hits led to the first Sirt2 inhibitors with activity in the low micromolar range. Several Sirt2 inhibitors showed potency against both larval schistosomes (viability) and adult worms (pairing, egg laying) in culture without general toxicity to human cancer cells.
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http://dx.doi.org/10.1021/acs.jmedchem.9b00638DOI Listing
October 2019

Structure-Based Screening of Tetrazolylhydrazide Inhibitors versus KDM4 Histone Demethylases.

ChemMedChem 2019 11 10;14(21):1828-1839. Epub 2019 Oct 10.

Macromolecular Crystallography, Helmholtz-Zentrum Berlin für Materialien und Energie, Albert-Einstein-Str. 15, 12489, Berlin, Germany.

Human histone demethylases are known to play an important role in the development of several tumor types. Consequently, they have emerged as important medical targets for the treatment of human cancer. Herein, structural studies on tetrazolylhydrazide inhibitors as a new scaffold for a certain class of histone demethylases, the JmjC proteins, are reported. A series of compounds are structurally described and their respective binding modes to the KDM4D protein, which serves as a high-resolution model to represent the KDM4 subfamily in crystallographic studies, are examined. Similar to previously reported inhibitors, the compounds described herein are competitors for the natural KDM4 cofactor, 2-oxoglutarate. The tetrazolylhydrazide scaffold fills an important gap in KDM4 inhibition and newly described, detailed interactions of inhibitor moieties pave the way to the development of compounds with high target-binding affinity and increased membrane permeability, at the same time.
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http://dx.doi.org/10.1002/cmdc.201900441DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6899576PMC
November 2019

The Clinically Used Iron Chelator Deferasirox Is an Inhibitor of Epigenetic JumonjiC Domain-Containing Histone Demethylases.

ACS Chem Biol 2019 08 19;14(8):1737-1750. Epub 2019 Jul 19.

Institute of Pharmaceutical Sciences , Albert-Ludwigs-Universität Freiburg , Albertstraße 25 , 79104 Freiburg i.Br. , Germany.

Fe(II)- and 2-oxoglutarate (2OG)-dependent JumonjiC domain-containing histone demethylases (JmjC KDMs) are "epigenetic eraser" enzymes involved in the regulation of gene expression and are emerging drug targets in oncology. We screened a set of clinically used iron chelators and report that they potently inhibit JMJD2A (KDM4A) . Mode of action investigations revealed that one compound, deferasirox, is a active site-binding inhibitor as shown by kinetic and spectroscopic studies. Synthesis of derivatives with improved cell permeability resulted in significant upregulation of histone trimethylation and potent cancer cell growth inhibition. Deferasirox was also found to inhibit human 2OG-dependent hypoxia inducible factor prolyl hydroxylase activity. Therapeutic effects of clinically used deferasirox may thus involve transcriptional regulation through 2OG oxygenase inhibition. Deferasirox might provide a useful starting point for the development of novel anticancer drugs targeting 2OG oxygenases and a valuable tool compound for investigations of KDM function.
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http://dx.doi.org/10.1021/acschembio.9b00289DOI Listing
August 2019

Discovery of a Potent and Selective Fragment-like Inhibitor of Methyllysine Reader Protein Spindlin 1 (SPIN1).

J Med Chem 2019 10 24;62(20):8996-9007. Epub 2019 Jul 24.

Mount Sinai Center for Therapeutics Discovery, Departments of Pharmacological Sciences and Oncological Sciences, Tisch Cancer Institute , Icahn School of Medicine at Mount Sinai , New York , New York 10029 , United States.

By screening an epigenetic compound library, we identified that UNC0638, a highly potent inhibitor of the histone methyltransferases G9a and GLP, was a weak inhibitor of SPIN1 (spindlin 1), a methyllysine reader protein. Our optimization of this weak hit resulted in the discovery of a potent, selective, and cell-active SPIN1 inhibitor, compound (MS31). Compound potently inhibited binding of trimethyllysine-containing peptides to SPIN1, displayed high binding affinity, was highly selective for SPIN1 over other epigenetic readers and writers, directly engaged SPIN1 in cells, and was not toxic to nontumorigenic cells. The crystal structure of the SPIN1-compound complex indicated that it selectively binds tudor domain II of SPIN1. We also designed a structurally similar but inactive compound (MS31N) as a negative control. Our results have demonstrated for the first time that potent, selective, and cell-active fragment-like inhibitors can be generated by targeting a single tudor domain.
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http://dx.doi.org/10.1021/acs.jmedchem.9b00522DOI Listing
October 2019

KMT9 monomethylates histone H4 lysine 12 and controls proliferation of prostate cancer cells.

Nat Struct Mol Biol 2019 05 6;26(5):361-371. Epub 2019 May 6.

Klinik für Urologie und Zentrale Klinische Forschung, Klinikum der Albert-Ludwigs-Universität Freiburg, Freiburg, Germany.

Histone lysine methylation is generally performed by SET domain methyltransferases and regulates chromatin structure and gene expression. Here, we identify human C21orf127 (HEMK2, N6AMT1, PrmC), a member of the seven-β-strand family of putative methyltransferases, as a novel histone lysine methyltransferase. C21orf127 functions as an obligate heterodimer with TRMT112, writing the methylation mark on lysine 12 of histone H4 (H4K12) in vitro and in vivo. We characterized H4K12 recognition by solving the crystal structure of human C21orf127-TRMT112, hereafter termed 'lysine methyltransferase 9' (KMT9), in complex with S-adenosyl-homocysteine and H4K12me1 peptide. Additional analyses revealed enrichment for KMT9 and H4K12me1 at the promoters of numerous genes encoding cell cycle regulators and control of cell cycle progression by KMT9. Importantly, KMT9 depletion severely affects the proliferation of androgen receptor-dependent, as well as that of castration- and enzalutamide-resistant prostate cancer cells and xenograft tumors. Our data link H4K12 methylation with KMT9-dependent regulation of androgen-independent prostate tumor cell proliferation, thereby providing a promising paradigm for the treatment of castration-resistant prostate cancer.
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http://dx.doi.org/10.1038/s41594-019-0219-9DOI Listing
May 2019

Novel Sustainable-by-Design HDAC Inhibitors for the Treatment of Alzheimer's Disease.

ACS Med Chem Lett 2019 Apr 29;10(4):671-676. Epub 2019 Mar 29.

Department of Pharmacy and Biotechnology, Alma Mater Studiorum - University of Bologna, Via Belmeloro 6, I-40126 Bologna, Italy.

Alzheimer's disease (AD) represents a global problem, with an estimation of the majority of dementia patients in low- and middle-income countries by 2050. Thus, the development of sustainable drugs has attracted much attention in recent years. In light of this, taking inspiration from the HDAC inhibitor vorinostat (), we develop the first HDAC inhibitors derived from cashew nut shell liquid (CNSL), an inexpensive agro-food waste material. CNSL derivatives and display a HDAC inhibitory profile similar to , together with a more promising safety for compared to . Moreover, both compounds and particularly were able to effectively modulate glial cell-induced inflammation and to revert the pro-inflammatory phenotype. All these results demonstrate that the use of inexpensive food waste materials could be successfully applied for the development of accessible and sustainable drug candidates for the treatment of AD.
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http://dx.doi.org/10.1021/acsmedchemlett.9b00071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6466821PMC
April 2019

Tetrahydroindoles as Multipurpose Screening Compounds and Novel Sirtuin Inhibitors.

ChemMedChem 2019 04 21;14(8):853-864. Epub 2019 Mar 21.

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

Indoles are privileged structures in medicinal and bioorganic chemistry that are particularly well suited to serve as platforms for diversity. Among many other therapeutic areas, the indole scaffold has been used to design aromatic compounds useful to interfere with enzymes engaged in the regulation of substrate acylation status, such as sirtuins. However, the planarity of the indole ring is not necessarily optimal for all target enzymes, especially when functionalization with aromatic side chains is required. Replacement of flat scaffolds by nonplanar molecular cores dominated by sp hybridization is a common strategy to avoid the disadvantages associated with poor solubility and high promiscuity, while covering less-well-explored areas of chemical space. Thus, we synthesized fragment-like tetrahydroindoles suitable for fragment-based drug discovery as well as a well-characterized small library intended as multipurpose screening compounds. For proof of principle, these compounds were screened against sirtuins 1-3, enzymes known to be addressable by indoles. We found that 2,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indole-3-carboxamides are potent and selective SIRT2 inhibitors. Compound 16 t displayed an IC value of 0.98 μm and could serve as exquisite starting point for hit-to-lead profiling.
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http://dx.doi.org/10.1002/cmdc.201900054DOI Listing
April 2019

Cofactor Analogues as Active Site Probes in Lysine Acetyltransferases.

J Med Chem 2019 03 6;62(5):2582-2597. Epub 2019 Mar 6.

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

Lysine acetyltransferases (KATs, also termed histone acetyltransferases, HATs) catalyze the acetylation of substrate lysine residues by employing the cofactor acetyl-coenzyme A (AcCoA), thereby providing a dynamic control mechanism of protein function. Because of their major involvement in cell development and homeostasis, small-molecule modulators of KAT activity are urgently needed to assess their therapeutic potential and for probing their underlying biology. Recent advances in the field suggest that targeting the cofactor binding site represents a promising strategy for identifying potent and selective ligands. Here, we present the synthesis of two functional cofactor-based chemical probes and their usage as mechanistic tools in a broadly applicable assay platform. A fluorescence polarization (FP)-based binding assay was combined with biolayer interferometry competition analysis and a FP competition activity immunoassay to enable easy, reliable, and profound evaluation of ligands that target the KAT cofactor binding site.
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http://dx.doi.org/10.1021/acs.jmedchem.8b01887DOI Listing
March 2019