Inhibitors of the NAD⁺‑Dependent Protein Desuccinylase and Demalonylase Sirt5

NAD⁺-dependent histone deacetylases (sirtuins) play important roles in epigenetic regulation but also through nonhistone substrates for other key cellular events and have been linked to the pathogenesis of cancer, neurodegeneration, and metabolic diseases. The subtype Sirt5 has been shown recently to act as a desuccinylating and demalonylating enzyme. We have established an assay for biochemical testing of Sirt5 using a small labeled succinylated lysine derivative. We present a comparative study on the profiling of several established sirtuin inhibitors on Sirt1–3 as well as Sirt5 and also present initial results on a screening for new compounds that block Sirt5. Thiobarbiturates were identified as new Sirt5 inhibitors in the low micromolar range, which are selective over Sirt3 that can be found in the same cell compartment as Sirt5

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

Submitted by Nuzia Santos ([email protected]) on 2018-11-13T12:09:19Z No. of bitstreams: 1 Structural Basis for the Inhibition of Histone .pdf: 10051721 bytes, checksum: 36ced7239c061ef58937ef2728effa22 (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2018-11-13T12:45:56Z (GMT) No. of bitstreams: 1 Structural Basis for the Inhibition of Histone .pdf: 10051721 bytes, checksum: 36ced7239c061ef58937ef2728effa22 (MD5)Made available in DSpace on 2018-11-13T12:45:56Z (GMT). No. of bitstreams: 1 Structural Basis for the Inhibition of Histone .pdf: 10051721 bytes, checksum: 36ced7239c061ef58937ef2728effa22 (MD5) Previous issue date: 2013Département de Biologie Structurale Intégrative. Institut de Génétique et Biologie Moléculaire et Cellulaire.Université de Strasbourg. Illkirch, FranceInstitut für Pharmazie. Martin-Luther-Universität Halle-Wittenberg. Halle, GermanyInstitut für Pharmazeutische Wissenschaften. Albert-Ludwigs-Universität Freiburg. Freiburg, GermanyFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Instituto Nacional de Ciência e Tecnologia em Doenças Tropicais. Centro de Excelência em Bioinformática. Grupo de Genômica e Biologia Computacional. Belo Horizonte, MG, BrazilCenter for Infection and Immunity of Lille. Université Lille Nord de France. Institut Pasteur de Lille. Lille, FranceDépartement de Biologie Structurale Intégrative. Institut de Génétique et Biologie Moléculaire et Cellulaire. Université de Strasbourg. Illkirch, FranceInstitut für Pharmazeutische Wissenschaften. Albert-Ludwigs-Universität Freiburg. Freiburg, GermanyInstitut für Pharmazeutische Wissenschaften. Albert-Ludwigs-Universität Freiburg. Freiburg, GermanyCenter for Infection and Immunity of Lille. Université Lille Nord de France. Institut Pasteur de Lille. Lille, FranceFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Instituto Nacional de Ciência e Tecnologia em Doenças Tropicais. Centro de Excelência em Bioinformática. Grupo de Genômica e Biologia Computacional. Belo Horizonte, MG, BrazilDépartement de Biologie Structurale Intégrative. Institut de Génétique et Biologie Moléculaire et Cellulaire. Université de Strasbourg. Illkirch, FranceFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Instituto Nacional de Ciência e Tecnologia em Doenças Tropicais. Centro de Excelência em Bioinformática. Grupo de Genômica e Biologia Computacional. Belo Horizonte, MG, BrazilInstitut für Pharmazie, Martin-Luther-Universität Halle-Wittenberg, Halle, Germany, Freiburg Institute of Advanced Studies (FRIAS), Albert-Ludwigs-Universität Freiburg, Freiburg, GermanyInstitut für Pharmazeutische Wissenschaften. Albert-Ludwigs-Universität Freiburg. Freiburg, Germany/Freiburg Institute of Advanced Studies. Albert-Ludwigs-Universität Freiburg. Freiburg, GermanyDépartement de Biologie Structurale Intégrative. Institut de Génétique et Biologie Moléculaire et Cellulaire. Université de Strasbourg. Illkirch, FranceCenter for Infection and Immunity of Lille. Université Lille Nord de France. Institut Pasteur de Lille. Lille, FranceDépartement de Biologie Structurale Intégrative. Institut de Génétique et Biologie Moléculaire et Cellulaire. Université de Strasbourg. Illkirch, FranceThe 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 pathogen

Designed small-molecule inhibitors show decreased specificity towards human HDACs and induce apoptosis in schistosomes.

<p>(A) IC₅₀ values of SAHA, M344, J1038, J1037, and J1075 for smHDAC8 and human HDAC8, HDAC1, HDAC3, HDAC6 are plotted in graph. J1038 and J1075 show loss of specificities for human HDACs but not for smHDAC8. The results of three independent assays are shown, error bars represent the SD. (B,C) Merged TUNEL (pink) and DAPI (blue) staining of <i>S. mansoni</i> schistosomula incubated with DMSO alone (B) or with 100 µM J1075 dissolved in DMSO (C) for 96 h. (D) Quantification of TUNEL positivity of schistosomula incubated for 96 h with J1075 at 50 µM or 100 µM or with DMSO alone. The results of three independent assays are shown, error bars represent the SD. (E) Dose- and time-dependent mortality of schistosomula induced by J1075 inhibitor. <i>Schistosoma mansoni</i> schistosomula (1000 per well) were incubated in 1 mL of culture medium with varying quantities of J1075 inhibitor or the solvent (DMSO). The results of three independent assays are shown; error bars represent the SD. (F) J1075-triggered separation of <i>S. mansoni</i> adult worm pairs in culture. The paired status of male and female adult worms was assessed daily in the presence of varying quantities of J1075 or the solvent (DMSO). The results of three independent assays are shown, error bars represent the SD.</p

smHDAC8 adopts a canonical HDAC fold with specific external loops.

<p>(A) Structure-based sequence alignment of schistosome, mouse and human HDAC8 proteins. Sequences similarities are shown by levels of blue. Secondary structure elements found in smHDAC8 and hHDAC8 are shown above and below the alignment, respectively. Residues that could not be built in densities are depicted with a black dotted line. Important residues that participate in the specificity of the smHDAC8 active site are labeled with triangles. The numbering indicated above the alignment corresponds to smHDAC8. For clarity, the first thirteen residues of mouse and human HDAC8 have been removed. (B) Superposition of native smHDAC8 (green) and SAHA-inhibited hHDAC8 (blue; PDB 1T69) structures. Both enzymes adopt the same fold. smHDAC8 sequence insertions form specific external loops and C-terminus (colored in pink). The orange sphere represents the catalytic zinc ion (Zn). (C,D) Ribbon representations of smHDAC8 (C) and hHDAC8 (D) structures. Both enzymes adopt the same fold and their catalytic zinc ion is found at the same position.</p

smHDAC8 is a functional acetyl-L-lysine deacetylase that is essential for parasite infectivity.

<p>(A) Down-regulation of <i>smHDAC8</i> decreases the number of adult worms recovered from infected mice. (B) The number of recovered eggs from the livers of infected mice is decreased. ‘*’, p<0.05; ‘**’, p<0.01. (C) Comparison of smHDAC8 and hHDAC8 deacetylase activities. Data indicate the average of relative deacetylase activity (hHDAC8 = 100%). Error bars represent the standard deviations (SD). (D) Close-up view of hHDAC8 active site. D101 and Y306, which participate in the hHDAC8 catalytic mechanism, and M274, which is replaced by a histidine in smHDAC8, are displayed. (E) Deacetalyse activities of smHDAC8 wild-type (wt), D100A, Y341F, and H292M mutants. Data indicate the average of relative deacetylase activity (smHDAC8 wt = 100%).</p

Data collection and refinement statistics.

*<p>Values in parentheses are for highest-resolution shell.</p>#<p>The number of protein atoms varies slightly for each structure, depending on the quality of the electron density for poorly folded regions.</p

Flipping-out of smHDAC8 phenylalanine 151 (F151) cannot be accommodated by major human HDACs.

<p>Ribbon representation of the active sites of (A) smHDAC8, (B) hHDAC8 (PDB 1T67), (C) hHDAC3 (PDB 4A69), and (D) hHDAC7 (PDB 3COY). Residues participating in zinc binding, catalysis, and active site formation are shown as sticks. smHDAC8 F151 and its counterparts in human HDACs are shown as well as the residues that influence their conformation. Specifically, only the schistosome phenylalanine can adopt a favored flipped-out conformation. Of note, the active sites of hHDAC2 (PDB 3MAX) and hHDAC4 (PDB 2VQJ) have highly similar features as observed for hHDAC3 and hHDAC7, respectively.</p