Modern drug development in preclinical pharmaceutics is based on two fundamental processes: library synthesis and screening. Usually, synthesis and screening are separated processes that need a compound storage facility as an interface. Unfortunately, compound storage is a complex process and can lead to compound degradation. For this reason, the chemical identities of found screening hits have to be reanalyzed after the biological screening to be confident. Here, we present a robotic facility that combines solid-phase one-bead one-compound synthesis and screening in a unified workflow using miniaturized formats for on-demand library synthesis. The technical proof of concept was achieved by an exemplary run that combines the solid-phase synthesis of a hydroxamate library and the subsequent biological screening for different histone deacetylase activities using a homogeneous fluorescence assay. Compound storage and related processing steps were thus omitted in this way. Technical concepts for all necessary steps and their robotic solutions for the integration of both synthesis and screening processes in one overall workflow are shown in detail. As a result, different enzyme selective histone deacetylase inhibitors were identified using the integrated workflow.

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. These drawbacks are eliminated by the dual parameter competition assay reported in this study. The assay involves a new fluorescent inhibitor probe that shows an increase in both, fluorescence anisotropy and fluorescence lifetime upon binding to the enzyme. The assay is well suited for high-throughput screening.

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. Here, we show that HC-toxin inhibits the growth of both established NB cell lines and primary cultures with and without amplified MYCN stronger than retinoids (RAs) and other HDACIs (MS-275, n-butyric acid, suberoylanilide hydroxamic acid, trichostatin A, valproic acid). Nanomolar dosages suppress E2F-1, N-myc, Skp2, Mad2 and survivin proteins, found at high levels in high-risk NBs, more efficiently than both RAs and other HDACIs. The level of hypophosphorylated active retinoblastoma (RB) tumor suppressor protein is increased most effectively. HC-toxin's epoxy group is essential for inhibiting HDACs and promoting anti-NB activity. Without this functional group, those cellular effects are not observed. In conclusion, the anti-NB activity of HC-toxin is superior to that of RAs and that of all other HDACIs tested.

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. Therefore HDAC inhibitors are efficient anti-proliferative agents in both in vitro and in vivo pre-clinical models of cancer, making them promising anticancer therapeutics. In the present paper, we present the results of a medium-throughput screening programme aiming at the identification of novel HDAC inhibitors using HDAH (HDAC-like amidohydrolase) from Bordetella or Alcaligenes strain FB188 as a model enzyme. Within a library of 3719 compounds, several new classes of HDAC inhibitor were identified. Among these hit compounds, there were also potent inhibitors of eukaryotic HDACs, as demonstrated by an increase in histone H4 acetylation, accompanied by a decrease in tumour cell metabolism in both SHEP neuroblastoma and T24 bladder carcinoma cells. In conclusion, screening of a compound library using FB188 HDAH as model enzyme identified several promising new lead structures for further development.

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. HDAC inhibitors have shown significant activity against a variety of hematological and solid tumors at doses that are well tolerated by patients, both in monotherapy as well as in combination therapy with other drugs. This paper reviews the most recent developments in HDAC inhibitor design, particularly in the context of anticancer therapy, and other possible pharmaceutical applications.

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 2.3A X-ray crystal structure. The Fourier map displayed clear electron density for the inhibitor. The central part of the inhibitor binds in an improved melagatran-like mode, while the structure identifies a d-tyrosine as P1 residue which forms a charged hydrogen bond with Asp 189 of thrombin. This is the first crystal structure of a thrombin-inhibitor complex, where an uncharged inhibitor residue makes hydrogen bonds within the thrombin S1 pocket. Additionally, novel favourable intermolecular hydrogen bonds of the inhibitor with the thrombin backbone become possible due to the d-configuration of the P1 residue. Two flanking voluminous side chains increase the strength of the subjacent hydrogen bonding system by shielding it from the bulk solvent.

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. Herein, we present the first nonisotopic competition binding assay for HDACs. The assay principle has been demonstrated using the well-established HDAC homolog FB188 histone deacetylase-like amidohydrolase from Bordetella/Alcaligenes species FB188. The assay is based on a new fluorescent HDAC inhibitor that shows fluorescence resonance energy transfer with tryptophans upon binding to the enzyme. In a competition situation with other HDAC inhibitors the displacement of the fluorescent inhibitor is accompanied by a decrease of fluorescence resonance energy transfer. The assay is well suited to kinetic studies of inhibitor binding and to HDAC inhibitor identification, e.g., in the context of high-throughput inhibitor screening in drug discovery.

Histone deacetylases (HDACs) catalyze the deacetylation of ε-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. Different HDAC subtypes can be ranked according to their substrate selectivity: HDAH > HDAC8 > HDAC1 > HDAC3 > HDAC6. HDAC1, HDAC3, and HDAC6 exhibit a similar specificity profile, whereas both HDAC8 and HDAH have rather distinct profiles. Furthermore, it was shown that second-site modification (e.g., phosphorylation) of substrate sequences as well as corepressor binding can modulate the selectivity of enzymatic substrate conversion.

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. Here, the first structure of an enzyme complex with a nonhydroxamate HDAC inhibitor is presented. The structure of the trifluoromethyl ketone inhibitor 9,9,9-trifluoro-8-oxo-N-phenylnonanamide in complex with bacterial FB188 HDAH (histone deacetylase-like amidohydrolase from Bordetella/Alcaligenes strain FB188) has been determined. HDAH reveals high sequential and functional homology to human class 2 HDACs and a high structural homology to human class 1 HDACs. Comparison with the structure of HDAH in complex with SAHA reveals that the two inhibitors superimpose well. However, significant differences in binding to the active site of HDAH were observed. In the presented structure the O atom of the trifluoromethyl ketone moiety is within binding distance of the Zn atom of the enzyme and the F atoms participate in interactions with the enzyme, thereby involving more amino acids in enzyme-inhibitor binding.

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. In this study, the conformational stability of a well-established histone deacetylase homolog with high structural similarity (histone deacetylase-like amidohydrolase from Bordetella/Alcaligenes species FB188) was investigated using denaturation titrations and stopped-flow kinetics. Based on the results of these complementary approaches, we conclude that the interconversion of native histone deacetylase-like amidohydrolase into its denatured form involves several intermediates possessing different enzyme activities and conformational structures. The refolding kinetics has shown to be strongly dependent on Zn(2+) and to a lesser extent on K(+), which underlines their importance not only for catalytic function but also for maintaining the correct conformational structure of the enzyme. Two main unfolding processes of histone deacetylase-like amidohydrolase were differentiated. The unfolding occurring at submolar concentrations of the denaturant guanidine hydrochloride was not affected by inhibitor binding, whereas the unfolding at higher concentrations of guanidine hydrochloride was strongly affected. It was shown that the known inhibitors suberoylanilide hydroxamic acid and cyclopentylpropionyl hydroxamate are capable of stabilizing the conformational structure of histone deacetylase-like amidrohydrolase. Judging from the free energies of unfolding, the protein stability was increased by 9.4 and 5.4 kJ.mol(-1) upon binding of suberoylanilide hydroxamic acid and cyclopentylpropionyl hydroxamate, respectively.

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. This paper reviews the most recent insights into the diverse biological roles of HDACs as well as the evolution of this important protein family.

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. In the present paper, we studied in more detail the esterase activity of HDACs, focussing on the HDAH (histone deacetylase-like amidohydrolase) from Bordetella/Alcaligenes strain FB188. This enzyme was classified as a class 2 HDAC based on sequence comparison as well as functional data. Using chromogenic and fluorogenic ester substrates we show that HDACs such as FB188 HDAH indeed have esterase activity that is comparable with those of known esterases. Similar results were obtained for human HDAC1, 3 and 8. Standard HDAC inhibitors were able to block both activities with similar IC(50) values. Interestingly, HDAC inhibitors such as suberoylanilide hydroxamic acid (SAHA) also showed inhibitory activity against porcine liver esterase and Pseudomonas fluorescens lipase. The esterase and the amidohydrolase activity of FB188 HDAH both appear to have the same substrate specificity concerning the acyl moiety. Interestingly, a Y312F mutation in the active site of HDAH obstructed amidohydrolase activity but significantly improved esterase activity, indicating subtle differences in the mechanism of both catalytic activities. Our results suggest that, in principle, HDACs may have other biological roles besides acting as protein deacetylases. Furthermore, data on HDAC inhibitors affecting known esterases indicate that these molecules, which are currently among the most promising drug candidates in cancer therapy, may have a broader target profile requiring further exploration.

Miniature labs: A microstructured silicon wafer was used as a microreactor array for single-bead synthesis on a nanovolume scale. Products were prepared by combinatorial synthesis and spatially encoded by their position in the array. Three-component Biginelli condensations were carried out at 100 °C for 14 h and therefore required the development of a specially sealed reactor device.

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. Here, we compare in more detail representatives of class 1 HDACs and FB188 HDAH as a close bacterial homologue of class 2 HDAC6, in regard of substrate and inhibitor specificity. Structure comparison is used to identify candidate regions responsible for observed specificity differences. Knowledge of these structural elements expedite studies on the biochemical role of different HDAC subtypes as well as the development of highly selective HDAC inhibitors as antitumor agents.

Thrombin: A series of thrombin inhibitors 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. The identified lead compounds exhibited thrombin inhibitory constants in the lower nanomolar range. They are by far the most selective synthetic thrombin inhibitors, with selectivities of >100,000-fold toward other proteases such as Factor Xa, Factor XIIa, urokinase, plasmin, and Plasma kallikrein. Furthermore, these compounds exhibit a favorable profile, comprising nontoxicity, high metabolic stability, low serum protein binding, good solubility, high anticoagulant activity, and a slow and exclusively renal elimination from the circulation in a rat model. Finally, x-ray crystallographic analysis of a thrombin inhibitor complex revealed a binding mode with a neutral moiety in the S1 pocket of thrombin.Histone-Deacetylases HDAC: Human HDAC8 was manufactured as active enzyme with Escherichia coli as effective expression system and purified over an anion exchanger. A fluorogenic HDAC assay with easy to be synthesized substrates was developed. Due to its high sensitivity the assay is feasible for miniaturized assay-formats (microtiterplate MTP). This Assay was optimized by new substrate developments regarding conversion and quantity of necessary substrate, so that a high throughput screening (HTS) suited instrument is available for the search of inhibitors. Different synthetic substrates served to probe the requirements of the proteins regarding structural and stereochemical demands of the substrate and in particular to experience possible structural motives for inhibitors and to find out more over the substrate specificity of the HDAC`s and the processes in their active center. Idications for a two-stage reaction mechanism were found. All preceding tasks can be considered as pre-working for a development of selective, specific inhibitors. This project was begun with few small series of molecules from the class of the peptides, phenylendiamines and pyranone-derivatives, which are to be considered as basis to the further development of non-hydroxamate-inhibitors.

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. The identified lead compounds exhibited thrombin inhibitory constants in the lower nanomolar range. They are by far the most selective synthetic thrombin inhibitors, with selectivities of >100,000-fold toward other proteases such as Factor Xa, Factor XIIa, urokinase, plasmin, and Plasma kallikrein. Furthermore, these compounds exhibit a favorable profile, comprising nontoxicity, high metabolic stability, low serum protein binding, good solubility, high anticoagulant activity, and a slow and exclusively renal elimination from the circulation in a rat model. Finally, x-ray crystallographic analysis of a thrombin-inhibitor complex revealed a binding mode with a neutral moiety in the S1 pocket of thrombin.