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Daniel Riester

Publications and source records attributed to Daniel Riester.

10 recordsLinked to original sources
PubMed2007

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. 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.

Alcaligenes↗
PubMed2006

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. 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.

Amino Acid Sequence↗
PubMed2005

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. 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.

Antithrombins↗
PubMed2004

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. Here, we use the epsilon-acyl moieties of epsilon-modified l-lysine in peptidic substrates as a probe to examine the active site cavity of HDACs and HDAC-like enzymes. Measurements were based on a fluorogenic assay with small synthetic substrates. Four different enzyme preparations were used derived from rat, human, and bacterial sources. None of the enzymes was able to utilize substrates with epsilon-acyl moieties larger than acetyl, except rat liver HDAC, which was the only enzyme to convert a substrate containing epsilon-propionyl-l-lysine. All enzymes exhibited a distinct enantioselectivity toward l-lysine-containing substrates except FB188 HDAH which also deacetylated Boc-d-Lys(epsilon-acetyl)-MCA. Moreover, all enzymes also exhibited a distinct specificity for the length of the lysine side chain; acetylated ornithine, which comprises one CH(2) unit less in the side chain, was not a substrate. In line with these results, only acetylcadaverin the metabolic degradation product of lysine but neither acetylputrescine (degradation product of ornithine) nor acetylspermidine strongly inhibited enzyme activity. Boc-l-Lys(epsilon-trifluoroacetyl)-MCA was observed to be a superior substrate for FB188 HDAH, Pseudomonas aeruginosa HDAH (PA3774), and particularly HDAC 8 compared to rat liver HDAC, and is the first suitable (synthetic) substrate for (human-derived) HDAC 8 reported to date. Altogether, the results reveal clear differences in substrate specificity between different HDACs as analyzed in the fluorogenic HDAC assay. Finally, we present the first candidates for HDAC-type-selective substrates that may be useful as biochemical tools to establish the function of particular pathways and to elucidate the role of distinct HDAC subtypes in cellular differentiation and cancer.

Animals↗
PubMed2004

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. Interestingly, peptide sequencing disclosed that the N-terminal methionine is lacking in the mature wild-type enzyme, presumably due to the action of methionyl aminopeptidase. Sequence database searches suggest that the new amidohydrolase belongs to the HDAC superfamily, with the closest homologs being found in the subfamily assigned acetylpolyamine amidohydrolases (APAH). The APAH subfamily comprises enzymes or putative enzymes from such diverse microorganisms as Pseudomonas aeruginosa, Archaeoglobus fulgidus, and the actinomycete Mycoplana ramosa (formerly M. bullata). The FB188 HDAH, however, is only moderately active in catalyzing the deacetylation of acetylpolyamines. In fact, FB188 HDAH exhibits significant activity in standard HDAC assays and is inhibited by known HDAC inhibitors such as trichostatin A and suberoylanilide hydroxamic acid (SAHA). Several lines of evidence indicate that the FB188 HDAH is very similar to class 1 and 2 HDACs and contains a Zn(2+) ion in the active site which contributes significantly to catalytic activity. Initial biotechnological applications demonstrated the extensive substrate spectrum and broad optimum pH range to be excellent criteria for using the new HDAH from Bordetella/Alcaligenes strain FB188 as a biocatalyst in technical biotransformations, e.g., within the scope of human immunodeficiency virus reverse transcriptase inhibitor synthesis.

Amidohydrolases↗
PubMed2003

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. Upon deacetylation of the acetylated lysyl moiety, molecules are recognized as substrates by trypsin, which releases highly fluorescent 7-amino-4-methylcoumarin molecules in a subsequent step of the assay. The fluorescence increase is directly proportional to the amount of deacetylated substrate molecules, i.e., HDAC activity. Validation of an improved version of the assay revealed (i) a significantly lower enzyme consumption, (ii) an increased screening window coefficient, (iii) a good tolerance toward organic solvents, and (iv) a good suitability for a whole range of different HDAC-like enzymes. The novel assay thus will expedite studies of HDAC-like enzymes and in vitro screening for drug discovery.

Acetylation↗
PubMed2003

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. Analysis of protease susceptibility, however, revealed that mutations, which conferred protease resistance, simultaneously diminish thrombin inhibition activity. This is particularly apparent for substitutions in the region of residues 56-64, which forms a large number of electrostatic and hydrophobic interactions with thrombin in the crystal structure of the complex. Unlike wild-type hirudin, the variant comprising Pro(50)- ...-His(56)-Asp(57)- ...-Pro(62)-Pro(63)-His(64) is completely resistant to pepsin and chymotrypsin cleavage; however, this is at the expense of thrombin inhibition activity where there is a 100-fold increase in the IC50 value. The frequent replacement of wild-type amino acids by proline at major protease cleavage sites indicates that at least pepsin- and chymotrypsin-like enzymes may exhibit a (conformational) specificity concerning the P1 and P2 positions. On the basis of these results, proline substitutions appear to be a general strategy to design polypeptides that are not susceptible to digestion by a broader range of different proteases.

Amino Acid Sequence↗
PubMed2003

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. In this study, novel fluorogenic substrates of HDACs were synthesized with an epsilon-acetylated lysyl moiety and an adjacent MCA moiety at the C terminus of the peptide chain. Upon deacetylation of the acetylated lysyl moiety, molecules became substrates for trypsin, which released highly fluorescent AMC molecules in a subsequent step of the assay. The fluorescence increased in direct proportion to the amount of deacetylated substrate molecules, i.e., HDAC activity. The nonisotopic, homogeneous assay is well suited for high-throughput HDAC inhibitor screening.

Acetylation↗
PubMedDate not supplied

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.g. in the field of drug development. Here, we propose a new genetic algorithm that has been tailored to meet the demands of de novo drug design, i.e. efficient optimization based on small training sets that are analyzed in only a small number of design cycles. The efficiency of the design algorithm was demonstrated in the context of several different applications. First, RNA molecules were optimized with respect to folding energy. Second, a spinglass was optimized as a model system for the optimization of multiletter alphabet biopolymers such as peptides. Finally, the feasibility of the computer-assisted molecular design approach was demonstrated for the de novo construction of peptidic thrombin inhibitors using an iterative process of 4 design cycles of computer-guided optimization. Synthesis and experimental fitness determination of only 600 different compounds from a virtual library of more than 10(17) molecules was necessary to achieve this goal.

Algorithms↗