Ralf Heinke

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.

Virtual screening and biological characterization of novel histone arginine methyltransferase PRMT1 inhibitors.

Lysine and arginine methyltransferases participate in the posttranslational modification of histones and regulate key cellular functions. Protein arginine methyltransferase 1 (PRMT1) has been identified as an essential component of mixed lineage leukemia (MLL) oncogenic complexes, revealing its potential as a novel therapeutic target in human cancer. The first potent arginine methyltransferase inhibitors were recently discovered by random‐ and target‐based screening approaches. Herein we report virtual and biological screening for novel inhibitors of PRMT1. Structure‐based virtual screening (VS) of the Chembridge database composed of 328 000 molecules was performed with a combination of ligand‐ and target‐based in silico approaches. Nine inhibitors were identified from the top‐scored docking solutions; these were experimentally tested using human PRMT1 and an antibody‐based assay with a time‐resolved fluorescence readout. Among several aromatic amines, an aliphatic amine and an amide were also found to be active in the micromolar range.

Acyl derivatives of p-aminosulfonamides and dapsone as new inhibitors of the arginine methyltransferase hPRMT1.

Arginine methylation is an epigenetic modification that receives increasing interest as it plays an important role in several diseases. This is especially true for hormone-dependent cancer, seeing that histone methylation by arginine methyltransferase I (PRMT1) is involved in the activation of sexual hormone receptors. Therefore, PRMT inhibitors are potential drugs and interesting tools for cell biology. A dapsone derivative called allantodapsone previously identified by our group served as a lead structure for inhibitor synthesis. Acylated derivatives of p-aminobenzenesulfonamides and the antilepra drug dapsone were identified as new inhibitors of PRMT1 by in vitro testing. The bis-chloroacetyl amide of dapsone selectively inhibited human PRMT1 in the low micromolar region and was selective for PRMT1 as compared to the arginine methyltransferase CARM1 and the lysine methyltransferase Set7/9. It showed anticancer activity on MCF7a and LNCaP cells and blocked androgen dependent transcription specifically in a reporter gene system. Likewise, a transcriptional block was also demonstrated in LNCaP cells using quantitative RT-PCR on the mRNA of androgen dependent genes.

Targeting epigenetic modifiers: Inhibitors of histone methyltransferases

The term epigenetics is defined as inheritable changes that influence the outcome of a phenotype without changes in the genome. Epigenetics is based upon DNA methylation and posttranslational histone modifications. While there is much known about reversible acetylation as a posttranslational modification, research on histone methylation is still emerging, especially with regard to drug discovery. As aberrant epigenetic modifications have been linked to many diseases, inhibitors of histone methylation are very much in demand. This article gives an outline on the different histone methyltransferases, their involvement in disease, the available inhibitors and their potential as drugs.

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.

Computer- and structure-based lead design for epigenetic targets

The term epigenetics is defined as inheritable changes that influence the outcome of a phenotype without changes in the genome. Epigenetics is based upon DNA methylation and posttranslational histone modifications. While there is much known about reversible acetylation as a posttranslational modification, research on reversible histone methylation is still emerging, especially with regard to drug discovery. As aberrant epigenetic modifications have been linked to many diseases, inhibitors of histone modifying enzymes are very much in demand. This article will summarize the progress on small molecule epigenetic inhibitors identified by structure- and computer-based approaches.

Evaluation of the performance of 3D virtual screening programs: docking vs. structure-based pharmacophore

Virtual screening (VS) techniques are well established methods in the modern drug discovery process and an almost unmanageable number of different 3D VS techniques are available today. Beside protein-ligand docking approaches, pharmacophore search methods are applied to screen large compound libraries to identify potential hits. In recent years, structure-based pharmacophores became more and more popular for virtual screening when 3D information about the target protein is available. In the present work we have analyzed and compared the performance of two docking programs (GOLD and ParaDocks) and the pharmacophore-based procedure within the program LigandScout. For our evaluation study we choose the Astex Diverse Set including 85 protein-ligand structures and decoy sets derived from the NCI and the Derwent WDI database. Beside the reproducibility of the experimentally derived structures, a special focus was given to the discrimination between known actives and inactives. For this purpose we have analyzed in a retrospective way several real life examples from our laboratory. from 4th German Conference on Chemoinformatics Goslar, Germany. 9–11 November 2008