Alexander-Thomas Hauser

The emerging therapeutic potential of histone methyltransferase and demethylase inhibitors.

Epigenetics is defined as heritable changes to the transcriptome that are independent of changes in the genome. The biochemical modifications that govern epigenetics are DNA methylation and posttranslational histone modifications. Among the histone modifications, acetylation and deacetylation are well characterized, whereas the fields of histone methylation and especially demethylation are still in their infancy. This is particularly true with regard to drug discovery. There is strong evidence that these modifications play an important role in the maintenance of transcription as well as in the development of certain diseases. This article gives an overview of the mechanisms of action of histone methyltransferases and demethylases, their role in the formation of certain diseases, and available inhibitors. Special emphasis is placed on the strategies that led to the first inhibitors which are currently available and the screening approaches that were used in that process.

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.

Targeting Epigenetic Mechanisms: Potential of Natural Products in Cancer Chemoprevention

The term epigenetics is defined as heritable changes in gene expression patterns that occur without changes in DNA sequence. Epigenetic changes according to this definition are achieved by methylation of cytosine bases in the DNA and by histone modifications, such as acetylation, methylation or phosphorylation. These modifications play an important role in regulating gene expression and the existence of an epigenetic code which maintains these modifications even upon cell division has been underlined by many investigations. Targeting the enzymes which catalyze DNA methylation or histone modifications may be a possibility not only for cancer therapy but also for chemoprevention since disruption of epigenetic balance is known to cause diseases such as cancer. In this review, we want to present the key epigenetic targets. We highlight natural products that modulate these epigenetic mechanisms and show their potential for cancer chemoprevention.

Structure-Based Design and Synthesis of Novel Inhibitors Targeting HDAC8 from Schistosoma mansoni for the Treatment of Schistosomiasis

Schistosomiasis is a major neglected parasitic disease that affects more than 265 million people worldwide and for which the control strategy consists of mass treatment with the only available drug, praziquantel. In this study, a series of new benzohydroxamates were prepared as potent inhibitors of Schistosoma mansoni histone deacetylase 8 (smHDAC8). Crystallographic analysis provided insights into the inhibition mode of smHDAC8 activity by these 3-amidobenzohydroxamates. The newly designed inhibitors were evaluated in screens for enzyme inhibitory activity against schistosome and human HDACs. Twenty-seven compounds were found to be active in the nanomolar range, and some of them showed selectivity toward smHDAC8 over the major human HDACs (1 and 6). The active benzohydroxamates were additionally screened for lethality against the schistosome larval stage using a fluorescence-based assay. Four of these showed significant dose-dependent killing of the schistosome larvae and markedly impaired egg laying of adult worm pairs maintained in culture.

Molecular Basis for the Antiparasitic Activity of a Mercaptoacetamide Derivative That Inhibits Histone Deacetylase 8 (HDAC8) from the Human Pathogen Schistosoma mansoni

Schistosomiasis, caused by the parasitic flatworm Schistosoma mansoni and related species, is a tropical disease that affects over 200 million people worldwide. A new approach for targeting eukaryotic parasites is to tackle their dynamic epigenetic machinery that is necessary for the extensive phenotypic changes during the life cycle of the parasite. Recently, we identified S. mansoni histone deacetylase 8 (smHDAC8) as a potential target for antiparasitic therapy. Here, we present results on the investigations of a focused set of HDAC (histone deacetylase) inhibitors on smHDAC8. Besides several active hydroxamates, we identified a thiol-based inhibitor that inhibited smHDAC8 activity in the micromolar range with unexpected selectivity over the human isotype, which has not been observed so far. The crystal structure of smHDAC8 complexed with the thiol derivative revealed that the inhibitor is accommodated in the catalytic pocket, where it interacts with both the catalytic zinc ion and the essential catalytic tyrosine (Y341) residue via its mercaptoacetamide warhead. To our knowledge, this is the first complex crystal structure of any HDAC inhibited by a mercaptoacetamide inhibitor, and therefore, this finding offers a rationale for further improvement. Finally, an ester prodrug of the thiol HDAC inhibitor exhibited antiparasitic activity on cultured schistosomes in a dose-dependent manner.

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.

Carbamate prodrug concept for hydroxamate HDAC inhibitors.

Reversible acetylation of histones and other proteins has emerged over the last 10 years as an important mechanism for cell proliferation and has been identified as a valuable target for anticancer drug design. Acetylation is executed and maintained by the histone acetyltransferases and reversed by their counterparts, histone deacetylases (HDACs). The first HDAC inhibitors have already been approved for therapeutic use, and many additional clinical studies are currently under way. Herein, we describe virtual screening efforts that identified several novel HDAC6 inhibitors with cellular isoform selectivity. In particular, a carbamate-protected hydroxamic acid exhibited improved effects with respect to protein hyperacetylation compared with the parent hydroxamate, possibly because of improved cell permeability. The carbamate structure therefore represents a potential prodrug concept for hydroxamic acidcontaining HDAC inhibitors. HDACs are zinc-dependent amidohydrolases, and 11 human subtypes are known. Among the dozens of nonhistone substrates of HDACs, tubulin has attracted a lot of attention because it is a validated target for established anticancer drugs, such as taxanes, the vinca-alkaloids and their derivatives. Tubulin is deacetylated by a single zinc-dependent HDAC subtype, HDAC6, 7] and by the NAD-dependent histone deacetylase Sirt2. HDAC6-specific inhibitors and nonselective HDAC inhibitors synergize in cytotoxicity with the proteasome inhibitor bortezomib (Velcade), which makes HDAC6 an interesting target for inhibitor development. So far, reports of HDAC6selective inhibitors are limited 11, 12, 13] compared to nonselective or class I-selective inhibitors. Aiming at novel selective HDAC6 inhibitors, we were particularly interested in whether structure-based virtual screening using a HDAC6 homology model could deliver such compounds. We recently reported a homology model for HDAC6 used for docking studies of hydroxamates. The model was generated by exploiting multiple solved crystal structures of related HDACs as templates. Based on a substructure search for hydroxamates and related zinc binding groups, we identified 252 potential compounds by virtually screening the Maybridge compound collection comprising 55 000 molecules. The docking and subsequent visual inspection of the docking poses showed that, among the compounds with the highest docking scores, several hydroxamate and hydrazide derivatives could be identified. Finally, the five top-ranked compounds (1–5) were selected for in vitro testing.

Assays for Histone Deacetylases

Epigenetics is defined as heritable changes in gene expression, i.e. the presentation of a certain phenotype, without alterations to the genetic code. This is a central phenomenon in developmental biology but also for the pathogenesis of a variety of diseases, e.g. for cancer in the case of silencing of tumor suppressor proteins. Reversible histone acetylation is an important player within the epigenetic machinery and histone deacetylases are the best studied epigenetic target for drug discovery. A first drug (vorinostat) is already approved for human cancer treatment. The strategies and protocols for monitoring histone deacetylase (HDAC) activity are an integral part of drug discovery approaches towards new HDAC inhibitors and will be reviewed in this article. The main focus is on biochemical in-vitro assays but cellular hit validation models are also presented.

Screening Assays for Epigenetic Targets Using Native Histones as Substrates

In the past years, a lot of attention has been given to the identification and characterization of selective and potent inhibitors of chromatin-modifying enzymes to better understand their specific role in transcriptional regulation. As aberrant histone methylation is involved in different pathological processes, the search for methyltransferase and demethylase inhibitors has emerged as a crucial issue in current medicinal chemistry research. High-throughput in vitro assays are important tools for the identification of new methyltransferase or demethylase inhibitors. These usually use oligopeptide substrates derived from histone sequences, although in many cases, they are not good substrates for these enzymes. Here, the authors report about the setup and establishment of in vitro assays that use native core histones as substrates, enabling an assay environment that better resembles native conditions. They have applied these substrates for the known formaldehyde dehydrogenase assay for the histone demethylase LSD1 and have established two new antibody-based assays. For LSD1, a heterogeneous assay format was set up, and a homogeneous assay was used for the characterization of the arginine methyltransferase PRMT1. Validation of the system was achieved with reference inhibitors in each case.

Chemical probes: sharpen your epigenetic tools.

A potent and selective inhibitor for the protein lysine methyltransferases G9a and GLP has been discovered. This small molecule serves as a useful probe to decipher the specific role of these enzymes in the development of various diseases.