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Carol A. Fierke - One of the best experts on this subject based on the ideXlab platform.
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Phosphorylation of Histone Deacetylase 8: Structural and Mechanistic Analysis of the Phosphomimetic S39E Mutant.
Biochemistry, 2019Co-Authors: Katherine R. Welker Leng, David W. Christianson, Carol Ann Castañeda, Christophe Decroos, Barira Islam, Shozeb Haider, Carol A. FierkeAbstract:Histone Deacetylase (HDAC) enzymes that catalyze removal of acetyl-lysine post-translational modifications are frequently post-translationally modified. HDAC8 is phosphorylated within the Deacetylase domain at conserved residue serine 39, which leads to decreased catalytic activity. HDAC8 phosphorylation at S39 is unique in its location and function and may represent a novel mode of deacetylation regulation. To better understand the impact of phosphorylation of HDAC8 on enzyme structure and function, we performed crystallographic, kinetic, and molecular dynamics studies of the S39E HDAC8 phosphomimetic mutant. This mutation decreases the level of deacetylation of peptides derived from acetylated nuclear and cytoplasmic proteins. However, the magnitude of the effect depends on the peptide sequence and the identity of the active site metal ion [Zn(II) vs Fe(II)], with the value of kcat/KM for the mutant decreasing 9- to >200-fold compared to that of wild-type HDAC8. Furthermore, the dissociation rate constant of the active site metal ion increases by ∼10-fold. S39E HDAC8 was crystallized in complex with the inhibitor Droxinostat, revealing that phosphorylation of S39, as mimicked by the glutamate side chain, perturbs local structure through distortion of the L1 loop. Molecular dynamics simulations of both S39E and phosphorylated S39 HDAC8 demonstrate that the perturbation of the L1 loop likely occurs because of the lost hydrogen bond between D29 and S39. Furthermore, the S39 perturbation causes structural changes that propagate through the protein scaffolding to influence function in the active site. These data demonstrate that phosphorylation plays an important regulatory role for HDAC8 by affecting ligand binding, catalytic efficiency, and substrate selectivity.
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phosphorylation of Histone Deacetylase 8 structural and mechanistic analysis of the phosphomimetic s39e mutant
Biochemistry, 2019Co-Authors: Katherine Welker R Leng, David W. Christianson, Carol A. Fierke, Carol Ann Castañeda, Christophe Decroos, Barira Islam, Shozeb HaiderAbstract:Histone Deacetylase (HDAC) enzymes that catalyze removal of acetyl-lysine post-translational modifications are frequently post-translationally modified. HDAC8 is phosphorylated within the Deacetylase domain at conserved residue serine 39, which leads to decreased catalytic activity. HDAC8 phosphorylation at S39 is unique in its location and function and may represent a novel mode of deacetylation regulation. To better understand the impact of phosphorylation of HDAC8 on enzyme structure and function, we performed crystallographic, kinetic, and molecular dynamics studies of the S39E HDAC8 phosphomimetic mutant. This mutation decreases the level of deacetylation of peptides derived from acetylated nuclear and cytoplasmic proteins. However, the magnitude of the effect depends on the peptide sequence and the identity of the active site metal ion [Zn(II) vs Fe(II)], with the value of kcat/KM for the mutant decreasing 9- to >200-fold compared to that of wild-type HDAC8. Furthermore, the dissociation rate consta...
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hdac8 substrates identified by genetically encoded active site photocrosslinking
Journal of the American Chemical Society, 2017Co-Authors: Jeffrey E. Lopez, Sarah E Haynes, Jaimeen D Majmudar, Brent R Martin, Carol A. FierkeAbstract:The Histone Deacetylase family comprises 18 enzymes that catalyze deacetylation of acetylated lysine residues; however, the specificity and substrate profile of each isozyme remains largely unknown. Due to transient enzyme–substrate interactions, conventional co-immunoprecipitation methods frequently fail to identify enzyme-specific substrates. Additionally, compensatory mechanisms often limit the ability of knockdown or chemical inhibition studies to achieve significant fold changes observed by acetylation proteomics methods. Furthermore, measured alterations do not guarantee a direct link between enzyme and substrate. Here we present a chemical crosslinking strategy that incorporates a photoreactive, non-natural amino acid, p-benzoyl-l-phenylalanine, into various positions of the structurally characterized isozyme Histone Deacetylase 8 (HDAC8). After covalent capture, co-immunoprecipitation, and mass spectrometric analysis, we identified a subset of HDAC8 substrates from human cell lysates, which were f...
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Active Site Metal Identity Alters Histone Deacetylase 8 Substrate Selectivity: A Potential Novel Regulatory Mechanism.
Biochemistry, 2017Co-Authors: Carol Ann Castañeda, Jeffrey E. Lopez, Caleb G. Joseph, Michael D. Scholle, Milan Mrksich, Carol A. FierkeAbstract:Histone Deacetylase 8 (HDAC8) is a well-characterized member of the class I acetyl-lysine Deacetylase (HDAC) family. Previous work has shown that the efficiency of HDAC8-catalyzed deacetylation of a methylcoumarin peptide varies depending on the identity of the divalent metal ion in the HDAC8 active site. Here we demonstrate that both HDAC8 activity and substrate selectivity for a diverse range of peptide substrates depend on the identity of the active site metal ion. Varied Deacetylase activities of Fe(II)- and Zn(II)-HDAC8 toward an array of peptide substrates were identified using self-assembled monolayers for matrix-assisted laser desorption ionization (SAMDI) mass spectrometry. Subsequently, the metal dependence of deacetylation of peptides of biological interest was measured using an in vitro peptide assay. While Fe(II)-HDAC8 is generally more active than Zn(II)-HDAC8, the Fe(II)/Zn(II) HDAC8 activity ratio varies widely (from 2 to 150) among the peptides tested. These data provide support for the h...
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Ion Mobility-Mass Spectrometry Reveals Evidence of Specific Complex Formation between Human Histone Deacetylase 8 and Poly-r(C)-binding Protein 1.
International journal of mass spectrometry, 2016Co-Authors: Shuai Niu, Byungchul Kim, Carol A. Fierke, Brandon T. RuotoloAbstract:Histone Deacetylase 8, part of a broad class of proteins responsible for regulating transcription and many other cellular processes and directly linked to a host of human disease through its mis-function, has been canonically described as a zinc-based mettalo-enzyme for many years. Recent evidence, however, has linked this protein to iron incorporation, loaded through transient interactions with the poly r(C)-binding protein 1, a metallo-chaperone and storage protein. In this report, we construct and deploy an electrospray-mass spectrometry based assay aimed at quantifying the interaction strength between these two weakly-associated proteins, as well as the zinc and iron associated form of the Histone Deacetylase. Despite challenges derived from artifact protein complexes derived from the electrospray process, we use carefully-constructed positive and negative control experiments, along with detailed measurements of protein ionization efficiency to validate our dissociation constant measurements for protein dimers in this size range. Furthermore, our data strongly support that complexes between Histone Deacetylase 8 and poly r(C)-binding protein 1 are specific, and that they are equally strong when both zinc and iron-loaded proteins are involved, or perhaps mildly promoted in the latter case, suggesting an in vivo role for the non-canonical, iron-incorporated Histone Deacetylase.
David W. Christianson - One of the best experts on this subject based on the ideXlab platform.
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Structural analysis of Histone Deacetylase 8 mutants associated with Cornelia de Lange Syndrome spectrum disorders.
Journal of structural biology, 2020Co-Authors: Jeremy D Osko, Nicholas J Porter, Christophe Decroos, Matthew S Lee, Paris R Watson, Sarah E Raible, Ian D Krantz, Matthew A Deardorff, David W. ChristiansonAbstract:Cornelia de Lange Syndrome (CdLS) and associated spectrum disorders are characterized by one or more congenital anomalies including distinctive facial features, upper limb abnormalities, intellectual disability, and other symptoms. The molecular genetic basis of CdLS is linked to defects in cohesin, a protein complex that functions in sister chromatid cohesion, chromatin organization, and transcriptional regulation. Histone Deacetylase 8 (HDAC8) plays an important role in cohesin function by catalyzing the deacetylation of SMC3, which is required for efficient recycling of the cohesin complex. Missense mutations in HDAC8 have been identified in children diagnosed with CdLS spectrum disorders, and here we outline structure-function relationships for four of these mutations. Specifically, we report the 1.50 Å-resolution structure of the I45T HDAC8-suberoylanilide hydroxamic acid complex, the 1.84 Å-resolution structure of E66D/Y306F HDAC8 complexed with a peptide assay substrate, and the 2.40 Å-resolution structure of G320R HDAC8 complexed with the inhibitor M344. Additionally, we present a computationally generated model of D176G HDAC8. These structures illuminate new structure-function relationships for HDAC8 and highlight the importance of long-range interactions in the protein scaffold that can influence catalytic function.
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Phosphorylation of Histone Deacetylase 8: Structural and Mechanistic Analysis of the Phosphomimetic S39E Mutant.
Biochemistry, 2019Co-Authors: Katherine R. Welker Leng, David W. Christianson, Carol Ann Castañeda, Christophe Decroos, Barira Islam, Shozeb Haider, Carol A. FierkeAbstract:Histone Deacetylase (HDAC) enzymes that catalyze removal of acetyl-lysine post-translational modifications are frequently post-translationally modified. HDAC8 is phosphorylated within the Deacetylase domain at conserved residue serine 39, which leads to decreased catalytic activity. HDAC8 phosphorylation at S39 is unique in its location and function and may represent a novel mode of deacetylation regulation. To better understand the impact of phosphorylation of HDAC8 on enzyme structure and function, we performed crystallographic, kinetic, and molecular dynamics studies of the S39E HDAC8 phosphomimetic mutant. This mutation decreases the level of deacetylation of peptides derived from acetylated nuclear and cytoplasmic proteins. However, the magnitude of the effect depends on the peptide sequence and the identity of the active site metal ion [Zn(II) vs Fe(II)], with the value of kcat/KM for the mutant decreasing 9- to >200-fold compared to that of wild-type HDAC8. Furthermore, the dissociation rate constant of the active site metal ion increases by ∼10-fold. S39E HDAC8 was crystallized in complex with the inhibitor Droxinostat, revealing that phosphorylation of S39, as mimicked by the glutamate side chain, perturbs local structure through distortion of the L1 loop. Molecular dynamics simulations of both S39E and phosphorylated S39 HDAC8 demonstrate that the perturbation of the L1 loop likely occurs because of the lost hydrogen bond between D29 and S39. Furthermore, the S39 perturbation causes structural changes that propagate through the protein scaffolding to influence function in the active site. These data demonstrate that phosphorylation plays an important regulatory role for HDAC8 by affecting ligand binding, catalytic efficiency, and substrate selectivity.
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phosphorylation of Histone Deacetylase 8 structural and mechanistic analysis of the phosphomimetic s39e mutant
Biochemistry, 2019Co-Authors: Katherine Welker R Leng, David W. Christianson, Carol A. Fierke, Carol Ann Castañeda, Christophe Decroos, Barira Islam, Shozeb HaiderAbstract:Histone Deacetylase (HDAC) enzymes that catalyze removal of acetyl-lysine post-translational modifications are frequently post-translationally modified. HDAC8 is phosphorylated within the Deacetylase domain at conserved residue serine 39, which leads to decreased catalytic activity. HDAC8 phosphorylation at S39 is unique in its location and function and may represent a novel mode of deacetylation regulation. To better understand the impact of phosphorylation of HDAC8 on enzyme structure and function, we performed crystallographic, kinetic, and molecular dynamics studies of the S39E HDAC8 phosphomimetic mutant. This mutation decreases the level of deacetylation of peptides derived from acetylated nuclear and cytoplasmic proteins. However, the magnitude of the effect depends on the peptide sequence and the identity of the active site metal ion [Zn(II) vs Fe(II)], with the value of kcat/KM for the mutant decreasing 9- to >200-fold compared to that of wild-type HDAC8. Furthermore, the dissociation rate consta...
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Preparation of a new construct of human Histone Deacetylase 8 for the crystallization of enzyme-inhibitor complexes.
Methods in Enzymology, 2019Co-Authors: Nicholas J Porter, David W. ChristiansonAbstract:Abstract The metal-dependent Histone Deacetylases (HDACs) are critical regulatory enzymes that modulate myriad cellular processes. Implicated in cancer, neurodegenerative diseases, and other clinical disorders, various HDAC isozymes serve as validated drug targets. However, structural similarities among the HDAC isozymes challenge efforts in targeting a single isozyme for therapeutic intervention with an inhibitor. X-ray crystallography remains the premiere technique for studying the chemistry of isozyme-selective inhibition. While crystal structures of many HDAC-inhibitor complexes have been determined, especially with the class I isozyme HDAC8, the study of complexes with large inhibitors is complicated by flexible regions of the protein structure that can hinder crystallization. Here, we outline an approach for the identification of regions in HDAC8 that may hinder crystallization. We also describe protocols for the design and preparation of a truncated HDAC8 construct, HDAC8 374 , that enabled the successful crystallization and structure determination of the HDAC8-Trapoxin A complex at 1.24 A resolution.
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Structural and Functional Influence of the Glycine-Rich Loop G302GGGY on the Catalytic Tyrosine of Histone Deacetylase 8
Biochemistry, 2016Co-Authors: Nicholas J Porter, Christophe Decroos, Nicolas H. Christianson, David W. ChristiansonAbstract:Histone Deacetylase 8 (HDAC8) catalyzes the hydrolysis of acetyl-l-lysine to yield products l-lysine and acetate through a mechanism in which a nucleophilic water molecule is activated by a histidine general base and a catalytic metal ion (Zn2+ or Fe2+). Acetyl-l-lysine also requires activation by metal coordination and a hydrogen bond with catalytic tyrosine Y306, which also functions in transition state stabilization. Interestingly, Y306 is located in the conserved glycine-rich loop G302GGGY. The potential flexibility afforded by the tetraglycine segment may facilitate induced-fit conformational changes in Y306 between “in” and “out” positions, as observed in related Deacetylases. To probe the catalytic importance of the glycine-rich loop in HDAC8, we rigidified this loop by preparing the G302A, G303A, G304A, and G305A mutants and measured their steady state kinetics and determined their X-ray crystal structures. Substantial losses of catalytic efficiency are observed (10–500-fold based on kcat/KM), par...
Jelena Melesina - One of the best experts on this subject based on the ideXlab platform.
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characterization of Histone Deacetylase 8 hdac8 selective inhibition reveals specific active site structural and functional determinants
Journal of Medicinal Chemistry, 2018Co-Authors: Martin Marek, Tajith B Shaik, Tino Heimburg, Alokta Chakrabarti, Ramos E Morales, C Da Veiga, Dmitrii V Kalinin, Jelena Melesina, Julien Lancelot, Dina RobaaAbstract: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...
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a novel class of schistosoma mansoni Histone Deacetylase 8 hdac8 inhibitors identified by structure based virtual screening and in vitro testing
Molecules, 2018Co-Authors: Conrad V Simoben, Tajith B Shaik, Alokta Chakrabarti, Jelena Melesina, Karin Schmidtkunz, Julien Lancelot, Dina Robaa, Srinivasaraghavan Kannan, Martin Marek, Raymond J. PierceAbstract: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 Schistosoma mansoni (smHDAC8) was designed. From the developed screening protocol, we were able to identify eight novel N-(2,5-dioxopyrrolidin-3-yl)-n-alkylhydroxamate derivatives as smHDAC8 inhibitors with IC50 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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structure based design and biological characterization of selective Histone Deacetylase 8 hdac8 inhibitors with anti neuroblastoma activity
Journal of Medicinal Chemistry, 2017Co-Authors: Tino Heimburg, Tajith B Shaik, Jelena Melesina, Karin Schmidtkunz, Fiona R Kolbinger, Patrik Zeyen, Ehab Ghazy, Daniel Herp, Frank Erdmann, Matthias SchmidtAbstract: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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isophthalic acid based hdac inhibitors as potent inhibitors of hdac8 from schistosoma mansoni
Archiv Der Pharmazie, 2017Co-Authors: Katharina Stenzel, Alokta Chakrabarti, Jelena Melesina, Finn K Hansen, Simon Herkenhohner, Beate Lungerich, Julien Lancelot, Christophe Romier, Martin Marek, Raymond J. PierceAbstract:Schistosoma mansoni Histone Deacetylase 8 (SmHDAC8) has been recently identified as a new potential target for the treatment of schistosomiasis. A series of newly designed and synthesized alkoxyamide-based and hydrazide-based HDAC inhibitors were tested for inhibitory activity against SmHDAC8 and human HDACs 1, 6, and 8. The front runner compounds showed submicromolar activity against SmHDAC8 and modest preference for SmHDAC8 over its human orthologue hHDAC8. Docking studies provided insights into the putative binding mode in SmHDAC8 and allowed rationalization of the observed selectivity profile.
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Targeting Histone Deacetylase 8 as a therapeutic approach to cancer and neurodegenerative diseases.
Future medicinal chemistry, 2016Co-Authors: Alokta Chakrabarti, Jelena Melesina, Fiona R Kolbinger, Ina Oehme, Olaf Witt, Wolfgang Sippl, Johanna Senger, Manfred JungAbstract:Histone Deacetylase 8 (HDAC8), a unique class I zinc-dependent HDAC, is an emerging target in cancer and other diseases. Its substrate repertoire extends beyond Histones to many nonHistone proteins. Besides being a Deacetylase, HDAC8 also mediates signaling via scaffolding functions. Aberrant expression or deregulated interactions with transcription factors are critical in HDAC8-dependent cancers. Many potent HDAC8-selective inhibitors with cellular activity and anticancer effects have been reported. We present HDAC8 as a druggable target and discuss inhibitors of different chemical scaffolds with cellular effects. Furthermore, we review HDAC8 activators that revert activity of mutant enzymes. Isotype-selective HDAC8 targeting in patients with HDAC8-relevant cancers is challenging, however, is promising to avoid adverse side effects as observed with pan-HDAC inhibitors.
Martin Marek - One of the best experts on this subject based on the ideXlab platform.
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characterization of Histone Deacetylase 8 hdac8 selective inhibition reveals specific active site structural and functional determinants
Journal of Medicinal Chemistry, 2018Co-Authors: Martin Marek, Tajith B Shaik, Tino Heimburg, Alokta Chakrabarti, Ramos E Morales, C Da Veiga, Dmitrii V Kalinin, Jelena Melesina, Julien Lancelot, Dina RobaaAbstract: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...
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a novel class of schistosoma mansoni Histone Deacetylase 8 hdac8 inhibitors identified by structure based virtual screening and in vitro testing
Molecules, 2018Co-Authors: Conrad V Simoben, Tajith B Shaik, Alokta Chakrabarti, Jelena Melesina, Karin Schmidtkunz, Julien Lancelot, Dina Robaa, Srinivasaraghavan Kannan, Martin Marek, Raymond J. PierceAbstract: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 Schistosoma mansoni (smHDAC8) was designed. From the developed screening protocol, we were able to identify eight novel N-(2,5-dioxopyrrolidin-3-yl)-n-alkylhydroxamate derivatives as smHDAC8 inhibitors with IC50 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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isophthalic acid based hdac inhibitors as potent inhibitors of hdac8 from schistosoma mansoni
Archiv Der Pharmazie, 2017Co-Authors: Katharina Stenzel, Alokta Chakrabarti, Jelena Melesina, Finn K Hansen, Simon Herkenhohner, Beate Lungerich, Julien Lancelot, Christophe Romier, Martin Marek, Raymond J. PierceAbstract:Schistosoma mansoni Histone Deacetylase 8 (SmHDAC8) has been recently identified as a new potential target for the treatment of schistosomiasis. A series of newly designed and synthesized alkoxyamide-based and hydrazide-based HDAC inhibitors were tested for inhibitory activity against SmHDAC8 and human HDACs 1, 6, and 8. The front runner compounds showed submicromolar activity against SmHDAC8 and modest preference for SmHDAC8 over its human orthologue hHDAC8. Docking studies provided insights into the putative binding mode in SmHDAC8 and allowed rationalization of the observed selectivity profile.
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Abstract B80: New inhibitors of Histone Deacetylase 8 (HDAC8) as anticancer agents
Epigenetic Targets, 2015Co-Authors: Manfred Jung, Alokta Chakrabarti, Ina Oehme, Christophe Romier, Martin Marek, Wolfgang Sippl, Tino Heimbach, Olaf WittAbstract:The protein Deacetylase HDAC8 has been recognized to be a potential target for the treatment of certain types of cancer, e.g. CNS cancer or T-cell leukemia. Starting out froom HDAC8 inhibitors with potency in the low micromolar region we have prepared a series of analogues and subjected them to iterative cycles of in-vitro testing using a fluorescence based assay and subsequent synthetic variation. Molecular docking to available X-ray structures of human HDAC8 further supported the process. We have obtained potent HDAC8 inhibitors in-vitro (potency down to 26 nM) and high selectivity over hHDAC1 and 6(>200fold). Selected inhibitors block the proliferation of different cancer cell lines in culture (SH-SY5Y glioblastoma, Jurkat T-cell leukemia) and show hyperacetylation (shown by Western blotting) of the HDAC8 target Ac-SMC3. Thus, we have obtained potent and selective HDAC8 inhibitors with cellular activity and target engagement. Mechanistic studies are underway to further optimized selected compounds. Citation Format: Manfred Jung, Alokta Chakrabarti, Tino Heimbach, Wolfgang Sippl, Martin Marek, Christophe Romier, Ina Oehme, Olaf Witt. New inhibitors of Histone Deacetylase 8 (HDAC8) as anticancer agents. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B80.
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discovery of inhibitors of schistosoma mansoni hdac8 by combining homology modeling virtual screening and in vitro validation
Journal of Chemical Information and Modeling, 2014Co-Authors: Srinivasaraghavan Kannan, Tino Heimburg, Alokta Chakrabarti, Jelena Melesina, Alexanderthomas Hauser, Karin Schmidtkunz, Raymond J. Pierce, Martin Marek, Alexandra Walter, Christophe RomierAbstract: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.
Raymond J. Pierce - One of the best experts on this subject based on the ideXlab platform.
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a novel class of schistosoma mansoni Histone Deacetylase 8 hdac8 inhibitors identified by structure based virtual screening and in vitro testing
Molecules, 2018Co-Authors: Conrad V Simoben, Tajith B Shaik, Alokta Chakrabarti, Jelena Melesina, Karin Schmidtkunz, Julien Lancelot, Dina Robaa, Srinivasaraghavan Kannan, Martin Marek, Raymond J. PierceAbstract: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 Schistosoma mansoni (smHDAC8) was designed. From the developed screening protocol, we were able to identify eight novel N-(2,5-dioxopyrrolidin-3-yl)-n-alkylhydroxamate derivatives as smHDAC8 inhibitors with IC50 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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Analysis of the interactome of Schistosoma mansoni Histone Deacetylase 8.
PLoS neglected tropical diseases, 2017Co-Authors: Stephanie Caby, Raymond J. Pierce, Julien Lancelot, Lucile Pagliazzo, Jean-michel Saliou, Nicolas Bertheaume, Emmanuel RogerAbstract:Background Histone Deacetylase 8 from Schistosoma mansoni (SmHDAC8) is essential to parasite growth and development within the mammalian host and is under investigation as a target for the development of selective inhibitors as novel schistosomicidal drugs. Although some protein substrates and protein partners of human HDAC8 have been characterized, notably indicating a role in the function of the cohesin complex, nothing is known of the partners and biological function of SmHDAC8. Methodology/Principal findings We therefore employed two strategies to characterize the SmHDAC8 interactome. We first used SmHDAC8 as a bait protein in yeast two-hybrid (Y2H) screening of an S. mansoni cDNA library. This allowed the identification of 49 different sequences encoding proteins. We next performed co-immunoprecipitation (Co-IP) experiments on parasite extracts with an anti-SmHDAC8 antibody. Mass spectrometry (MS) analysis allowed the identification of 160 different proteins. Conclusions/Significance SmHDAC8 partners are involved in about 40 different processes, included expected functions such as the cohesin complex, cytoskeleton organization, transcriptional and translational regulation, metabolism, DNA repair, the cell cycle, protein dephosphorylation, proteolysis, protein transport, but also some proteasome and ribosome components were detected. Our results show that SmHDAC8 is a versatile Deacetylase, potentially involved in both cytosolic and nuclear processes.
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isophthalic acid based hdac inhibitors as potent inhibitors of hdac8 from schistosoma mansoni
Archiv Der Pharmazie, 2017Co-Authors: Katharina Stenzel, Alokta Chakrabarti, Jelena Melesina, Finn K Hansen, Simon Herkenhohner, Beate Lungerich, Julien Lancelot, Christophe Romier, Martin Marek, Raymond J. PierceAbstract:Schistosoma mansoni Histone Deacetylase 8 (SmHDAC8) has been recently identified as a new potential target for the treatment of schistosomiasis. A series of newly designed and synthesized alkoxyamide-based and hydrazide-based HDAC inhibitors were tested for inhibitory activity against SmHDAC8 and human HDACs 1, 6, and 8. The front runner compounds showed submicromolar activity against SmHDAC8 and modest preference for SmHDAC8 over its human orthologue hHDAC8. Docking studies provided insights into the putative binding mode in SmHDAC8 and allowed rationalization of the observed selectivity profile.
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hdac8 a multifaceted target for therapeutic interventions
Trends in Pharmacological Sciences, 2015Co-Authors: Alokta Chakrabarti, Raymond J. Pierce, Ina Oehme, Guilherme Oliveira, Olaf Witt, Christophe Romier, Wolfgang Sippl, Manfred JungAbstract:Histone Deacetylase 8 (HDAC8) is a class I Histone Deacetylase implicated as a therapeutic target in various diseases, including cancer, X-linked intellectual disability, and parasitic infections. It is a structurally well-characterized enzyme that also deacetylates nonHistone proteins. In cancer, HDAC8 is a major ‘epigenetic player’ that is linked to deregulated expression or interaction with transcription factors critical to tumorigenesis. In the parasite Schistosoma mansoni and in viral infections, HDAC8 is a novel target to subdue infection. The current challenge remains in the development of potent selective inhibitors that would specifically target HDAC8 with fewer adverse effects compared with pan-HDAC inhibitors. Here, we review HDAC8 as a drug target and discuss inhibitors with respect to their structural features and therapeutic interventions.
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discovery of inhibitors of schistosoma mansoni hdac8 by combining homology modeling virtual screening and in vitro validation
Journal of Chemical Information and Modeling, 2014Co-Authors: Srinivasaraghavan Kannan, Tino Heimburg, Alokta Chakrabarti, Jelena Melesina, Alexanderthomas Hauser, Karin Schmidtkunz, Raymond J. Pierce, Martin Marek, Alexandra Walter, Christophe RomierAbstract: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.