Karin Schmidtkunz

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

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

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

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

4-Acyl Pyrroles: Mimicking Acetylated Lysines in Histone Code Reading†

Chromatin remodeling is a key epigenetic mechanism of gene expression regulation controlled through the posttranscriptional modification of histones. Several enzymes, including histone deacetylases and lysine methyltransferases, add or remove functional groups at a variety of residues on histone tails. The recognition of this histone code by “reader” proteins, such as bromodomains (BRDs) and tudor domains, has a critical impact in the regulation of gene expression. The human genome encodes up to 61 different BRDs present in transcriptional co-regulators and chromatin modifying enzymes, including histone acetyl transferases and the bromodomain extra-terminal domain (BET) family. They specifically recognize e-N-acetylated lysine residues (Kac). BRDs fold into an evolutionary conserved four anti-parallel helix motif, linked by diverse loop regions of variable length (ZA and BC loops), which define the Kac binding site. [2] In most BRDs, this site features an asparagine residue mainly responsible for substrate recognition. The biological function of BRDs and their potential as therapeutic targets have been thoroughly reviewed. The large amount of crystallographic data available for most BRDs has recently shown the druggability of human BRDs, including the BET subfamily, namely BRD2, BRD3, BRD4, and BRDT, which modulate gene expression by recruiting transcriptional regulators to specific genomic locations. BRD2 and BRD4 have crucial roles in cell cycle control of mammalian cells. Along with BRD3, they are functionally linked to pathways important for cellular viability and cancer signaling and are co-regulators in obesity and inflammation. Specifically, BRD4 has been characterized as a key determinant in acute myeloid leukemia, multiple myeloma, Burkitt s lymphoma, NUT midline carcinoma, colon cancer, and inflammatory disease. Because of its continued association with Kac in mitotic chromosomes, BRD4 has been postulated to be important for the maintenance of epigenetic memory. Small molecules that inhibit BRD4 have potential as antiinflammatory, antiviral, and anticancer agents. Anticancer activity is mainly due to down-regulation of the key oncogene c-MYC. Recently, cytotoxicity in LAC cells by BRD4 inhibition has been related to suppression of the oncogenic transcription factor FOSL1 and its targets. Currently, two 1,4-diazepine derivatives, namely (+)-JQ1 and I-BET, are in preclinical development in cancer and inflammation, respectively, as potent antagonists of the BET bromodomains BRD2, BRD3, and BRD4. Recent fragment-based screenings and QSAR-based lead optimizations for the discovery of small molecules with BRD4 inhibitory activity have shed a few new relevant chemical scaffolds, including Compound 6a, an isoxazole derivative at an initial developmental stage in the clinics. During recent years, a large amount of structural knowledge about the binding features of inhibitors of BRD4 has become available by means of X-ray crystallography, providing an invaluable resource for drug discovery. The three key areas of interaction in ligand-BET bromodomain complexes are the acetyl-lysine recognition site, the WPF shelf, and the ZA channel (Figure 1c). On this basis, we performed a highthroughput virtual screening experiment using a library containing more than 7 million small molecules, aiming at the identification of novel inhibitors of the first bromodomain of BRD4 (BRD4(1); Supporting information).We selected 22

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

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

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

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.

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

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

Targeting the c-Myc coiled coil with interfering peptides.

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

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

Homolargazole derivatives, in which the macrocycle of natural largazole is extended by one methylene group, were prepared by the recently developed rhodium-catalyzed hydrocarboxylation reaction onto allenes. This strategy gives access to both the (18S)- and (18R)-stereoisomers in high stereoselectivity under ligand control.

The discovery of novel 10,11-dihydro-5H-dibenz[b,f]azepine SIRT2 inhibitors

Isoform selective inhibitors of the sirtuins (NAD+-dependent histone deacetylases) should enable an in depth study of the molecular biology underpinning these targets and how they are deregulated in diseases such as cancer and neurodegeneration. Herein, we present the discovery of structurally novel SIRT2 inhibitors. Hit molecule 8 was discovered through the chemical synthesis and biological characterization of a small-molecule compound library based around the 10,11-dihydro-5H-dibenz[b,f]azepine scaffold. In vitro screening assays revealed compound 8 to have an IC50 of 18 μM against SIRT2 and to exhibit more than 30-fold selectivity compared to SIRT1. Cellular assays, performed on MCF-7 cells, confirmed the in vitro selectivity and showed hit 8 to have antiproliferative activity at a concentration of 30 μM. Computational studies were performed to predict the SIRT2 binding mode and to rationalise the observed selectivity.

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

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.