Non-isotopic dual parameter competition assay suitable for high-throughput screening of histone deacetylases.

Histone deacetylases reside among the most important and novel target classes in oncology. Selective lead structures are intensively developed to improve efficacy and reduce adverse effects. The common assays used so far to identify new lead structures suffer from many false positive hits due to auto-fluorescence of compounds or triggering undesired signal transduction pathways.

Identification of novel small-molecule histone deacetylase inhibitors by medium-throughput screening using a fluorigenic assay.

HDACs (histone deacetylases) are considered to be among the most important enzymes that regulate gene expression in eukaryotic cells. In general, increased levels of histone acetylation are associated with increased transcriptional activity, whereas decreased levels are linked to repression of gene expression. HDACs associate with a number of cellular oncogenes and tumour-suppressor genes, leading to an aberrant recruitment of HDAC activity, which results in changes of gene expression, impaired differentiation and excessive proliferation of tumour cells.

Anti-neuroblastoma activity of Helminthosporium carbonum (HC)-toxin is superior to that of other differentiating compounds in vitro.

Treatment of high-risk neuroblastoma (NB) is difficult. Novel therapeutics improving survival rates are urgently required. We have previously shown that the histone deacetylase inhibitor (HDACI) Helminthosporium carbonum (HC)-toxin induces differentiation of neuroblastoma (NB) cells.

Structure of a novel thrombin inhibitor with an uncharged D-amino acid as P1 residue.

Thrombin, the ultimate proteinase of the coagulation cascade, is an attractive target for the treatment of a variety of cardiovascular diseases. Previously, a series of novel thrombin inhibitors, discovered by employing a powerful and new computer-assisted multiparameter optimization process (CADDIS), have been synthesized. We have now crystallized the complex of human alpha-thrombin with the most potent of these inhibitors, 8-5 (K(i)=3 nM), and have determined its 2.

Inhibitor-mediated stabilization of the conformational structure of a histone deacetylase-like amidohydrolase.

Histone deacetylases are major regulators of eukaryotic gene expression. Not unexpectedly, histone deacetylases are among the most promising targets in cancer therapy. However, despite huge efforts in histone deacetylase inhibitor design, very little is known about the impact of histone deacetylase inhibitors on enzyme stability.

Factors affecting the substrate specificity of histone deacetylases.

Histone deacetylases (HDACs) catalyze the deacetylation of epsilon-acetyl-lysine residues within the N-terminal tail of core histones and thereby mediate changes in the chromatin structure and regulate gene expression in eukaryotic cells. So far, surprisingly little is known about the substrate specificities of different HDACs. Here, we prepared a library of fluorogenic tripeptidic substrates of the general format Ac-P(-2)-P(-1)-Lys(Ac)-MCA (P(-1), P(-2)=all amino acids except cysteine) and measured their HDAC-dependent conversion in a standard fluorogenic HDAC assay.

Complex structure of a bacterial class 2 histone deacetylase homologue with a trifluoromethylketone inhibitor.

Histone deacetylases (HDACs) have emerged as attractive targets in anticancer drug development. To date, a number of HDAC inhibitors have been developed and most of them are hydroxamic acid derivatives, typified by suberoylanilide hydroxamic acid (SAHA). Not surprisingly, structural information that can greatly enhance the design of novel HDAC inhibitors is so far only available for hydroxamic acids in complex with HDAC or HDAC-like enzymes.

Histone deacetylases--an important class of cellular regulators with a variety of functions.

The elucidation of mechanisms of chromatin remodeling, particular transcriptional activation, and repression by histone acetylation and deacetylation has shed light on the role of histone deacetylases (HDAC) as a new kind of therapeutic target for human cancer treatment. HDACs, in general, act as components of large corepressor complexes that prevent the transcription of several tumor suppression genes. In addition, they appear to be also involved in the deacetylation of nonhistone proteins.

Histone deacetylase inhibitors--turning epigenic mechanisms of gene regulation into tools of therapeutic intervention in malignant and other diseases.

Histone deacetylase inhibitors reside among the most promising targeted anticancer agents that are potent inducers of growth arrest, differentiation, and/or apoptotic cell death of transformed cells. In October 2006, the US Food and Drug Administration approved the first drug of this new class, vorinostat (1, Zolinza, Merck). Several histone deacetylase (HDAC) inhibitors more are in clinical trials.

Histone deacetylase inhibitor assay based on fluorescence resonance energy transfer.

Histone deacetylases (HDACs) are important enzymes for the transcriptional regulation of gene expression in eukaryotic cells. Furthermore, in recent years HDACs occupied a major position as key targets for chemotherapeutic intervention in malignant diseases. However, progress in the development of these new chemotherapeutics is largely dependent on the existence of bioassays well-suited to inhibitor screening.

An active site tyrosine residue is essential for amidohydrolase but not for esterase activity of a class 2 histone deacetylase-like bacterial enzyme.

HDACs (histone deacetylases) are considered to be among the most important enzymes that regulate gene expression in eukaryotic cells acting through deacetylation of epsilon-acetyl-lysine residues within the N-terminal tail of core histones. In addition, both eukaryotic HDACs as well as their bacterial counterparts were reported to also act on non-histone targets. However, we are still far from a comprehensive understanding of the biological activities of this ancient class of enzymes.

Substrate and inhibitor specificity of class 1 and class 2 histone deacetylases.

Histone deacetylases (HDACs) are key enzymes in the transcriptional regulation of gene expression in eukaryotic cells. In recent years HDACs have attracted considerable attention as promising new targets in anticancer therapy. Currently, different histone deacetylase subtypes are divided into four groups denoted as classes 1-4.

Thrombin inhibitors identified by computer-assisted multiparameter design.

Here, we present a series of thrombin inhibitors that were generated by using powerful computer-assisted multiparameter optimization process. The process was organized in design cycles, starting with a set of randomly chosen molecules. Each cycle combined combinatorial synthesis, multiparameter characterization of compounds in a variety of bioassays, and algorithmic processing of the data to devise a set of compounds to be synthesized in the next cycle.

Members of the histone deacetylase superfamily differ in substrate specificity towards small synthetic substrates.

Histone deacetylases (HDACs) are important enzymes for the transcriptional regulation of gene expression in eukaryotic cells. Deacetylation of epsilon-acetyl-lysine residues within the N-terminal tail of core histones mediates changes in both histone-DNA and histone-non-histone protein interactions. However, surprisingly little is known about the substrate specificities of different HDACs.

A new amidohydrolase from Bordetella or Alcaligenes strain FB188 with similarities to histone deacetylases.

The full-length gene encoding the histone deacetylase (HDAC)-like amidohydrolase (HDAH) from Bordetella or Alcaligenes (Bordetella/Alcaligenes) strain FB188 (DSM 11172) was cloned using degenerate primer PCR combined with inverse-PCR techniques and ultimately expressed in Escherichia coli. The expressed enzyme was biochemically characterized and found to be similar to the native enzyme for all properties examined. Nucleotide sequence analysis revealed an open reading frame of 1,110 bp which encodes a polypeptide with a theoretical molecular mass of 39 kDa.

Directed evolution towards protease-resistant hirudin variants.

Hirudin, a thrombin-specific inhibitor, is efficiently digested and inactivated by proteases with pepsin- and chymotrypsin-like specificity. Using a combination of phage display selection and high-throughput screening methods, several variants of recombinant hirudin were generated. Only very few variants comprising amino acid substitutions in the amino-terminal domain (residues 1-5) and in the carboxyl-terminal tail (residues 49, 50, and/or 56, 57, 62-64) were identified that showed thrombin inhibition activities similar to those of the wild-type polypeptide.

Improved fluorogenic histone deacetylase assay for high-throughput-screening applications.

Histone deacetylases (HDACs) are key targets for chemotherapeutic intervention in malignant diseases. In this paper, a highly sensitive, nonisotopic, homogeneous assay for high-throughput screening of HDAC inhibitors is presented. The assay is based on a new fluorogenic peptidic substrate of HDACs comprising an epsilon-acetylated lysyl moiety and an adjacent 4-methylcoumarin-7-amide moiety at the C terminus of the peptide chain.

Genetic algorithm for the design of molecules with desired properties.

The design of molecules with desired properties is still a challenge because of the largely unpredictable end results. Computational methods can be used to assist and speed up this process. In particular, genetic algorithms have proved to be powerful tools with a wide range of applications, e.

A fluorogenic histone deacetylase assay well suited for high-throughput activity screening.

Histone deacetylases (HDACs) are important enzymes for the transcriptional regulation of gene expression in eukaryotic cells. Recent findings suggest that HDACs could be key targets for chemotherapeutic intervention in malignant diseases. A convenient and sensitive fluorogenic assay for HDAC activity would therefore expedite studies of HDAC in transcriptional regulation and in vitro screening for drug discovery.