Alkylation of rabbit muscle creatine kinase surface methionine residues inhibits enzyme activity in vitro.

Creatine kinase (CK) catalyzes the formation of phosphocreatine from adenosine triphosphate (ATP) and creatine. The highly reactive free cysteine residue in the active site of the enzyme (Cys) is considered essential for the enzymatic activity. In previous studies we demonstrated that Cys is targeted by the alkylating chemical warfare agent sulfur mustard (SM) yielding a thioether with a hydroxyethylthioethyl (HETE)-moiety.

Kinetic and structural insights into the binding of histone deacetylase 1 and 2 (HDAC1, 2) inhibitors.

The structure-activity and structure-kinetic relationships of a series of novel and selective ortho-aminoanilide inhibitors of histone deacetylases (HDACs) 1 and 2 are described. Different kinetic and thermodynamic selectivity profiles were obtained by varying the moiety occupying an 11Å channel leading to the Zn(2+) catalytic pocket of HDACs 1 and 2, two paralogs with a high degree of structural similarity. The design of these novel inhibitors was informed by two ligand-bound crystal structures of truncated hHDAC2.

Unexpected active-site flexibility in the structure of human neutrophil elastase in complex with a new dihydropyrimidone inhibitor.

Human neutrophil elastase (HNE), a trypsin-type serine protease, is of pivotal importance in the onset and progression of chronic obstructive pulmonary disease (COPD). COPD encompasses a group of slowly progressive respiratory disorders and is a major medical problem and the fifth leading cause of death worldwide. HNE is a major target for the development of compounds that inhibit the progression of long-term lung function decline in COPD patients.

Macrocyclic statine-based inhibitors of BACE-1.

Minimal sequence requirements for binding of substrate-derived statine peptides to the aspartyl enzyme were established on the basis of the X-ray cocrystal structure of the hydroxyethylene-octapeptide OM00-3 in complexation with BACE-1. With this information to hand, macrocyclic compounds that conformationally restrict and preorganize the peptide backbone for an entropically favoured binding to the enzyme active site cleft were designed. By means of a side chain-to-side chain ring closure between two aspartyl residues in the P2 and P3' positions through phenylene-1,3-dimethanamine, a 23-membered ring structure was obtained; this structure retained an extended conformation of the peptide backbone, including the transition state analogue statine for tight interactions with the two aspartyl residues of the active centre.

Development of a simple and robust assay to screen for inhibitors of sphingosine kinases.

Sphingosine kinases (SPHKs) catalyze the formation of the bioactive sphingolipid metabolite sphingosine 1-phosphate (S1P), which plays important roles in a wide variety of intra- and extracellular functions. Conventionally, SPHK activity has been determined using radioisotope thin layer chromatography (TLC) and autoradiography to detect the product S1P. Here we describe the development of a simple and robust in vitro SPHK assay in 384-well format with no requirement for any separation steps such as extraction and TLC.

Cinnabaramides A-G: analogues of lactacystin and salinosporamide from a terrestrial streptomycete.

The cinnabaramides A-G (1-7) were isolated from a terrestrial strain of Streptomyces as potent and selective inhibitors of the human 20S proteasome. Their chemical and biological properties resemble those of salinosporamide A, a recently identified lead compound from an obligate marine actinomycete, which is currently under development as an anticancer agent. Cinnabaramides F and G (6, 7) combine essential structural features of salinosporamide A and lactacystin and show about equal potency in vitro, with IC50 values in the 1 nM range.

Targeted proteomics in biomarker validation: detection and quantification of proteins using a multi-dimensional peptide separation strategy.

Authentic biomarkers, distilling the essence of a complex, functionally significant process in a mammalian system into a precise, physicochemical measurement have been implicated as a tool of increasing importance for drug discovery and development. However, even in spite of recent technological advances, validating a new biomarker candidate, where generation of suitable antibodies is required, is still a long-lasting task. Methods to accelerate initial validation by MS approaches have been suggested, but all methods described so far are associated with serious drawbacks, finally leading to non-generic methods of detection and quantification.

Crystal structure of full length topoisomerase I from Thermotoga maritima.

DNA topoisomerases are a family of enzymes altering the topology of DNA by concerted breakage and rejoining of the phosphodiester backbone of DNA. Bacterial and archeal type IA topoisomerases, including topoisomerase I, topoisomerase III, and reverse gyrase, are crucial in regulation of DNA supercoiling and maintenance of genetic stability. The crystal structure of full length topoisomerase I from Thermotoga maritima was determined at 1.

Discovery of the novel antithrombotic agent 5-chloro-N-({(5S)-2-oxo-3- [4-(3-oxomorpholin-4-yl)phenyl]-1,3-oxazolidin-5-yl}methyl)thiophene- 2-carboxamide (BAY 59-7939): an oral, direct factor Xa inhibitor.

Despite recent progress in antithrombotic therapy, there is still an unmet medical need for safe and orally available anticoagulants. The coagulation enzyme Factor Xa (FXa) is a particularly promising target, and recent efforts in this field have focused on the identification of small-molecule inhibitors with good oral bioavailability. We identified oxazolidinone derivatives as a new class of potent FXa inhibitors.

A versatile high-throughput screen for inhibitors of lipid kinase activity: development of an immobilized phospholipid plate assay for phosphoinositide 3-kinase gamma.

The family of phosphoinositide 3-kinases (PI3K) regulates fundamental cellular responses such as proliferation, apoptosis, motility, and adhesion. In particular, the PI3K gamma isoform plays a critical role in the control of cell migration. Despite the attractiveness of PI3-kinases as drug targets, drug discovery efforts have been hampered by the lack of appropriate lipid kinase assay formats suitable for high-throughput screening.

Development of a simple homogeneous assay to screen for inhibitors of N-acetylglucosamine-6-sulfotransferases.

L-selectin, a leukocyte adhesion molecule, plays a central role in lymphocyte homing to secondary lymphoid tissue and to certain sites of inflammation. Carbohydrate sulfation was implicated in this process, when it was demonstrated that carbohydrate sulfotransferase-mediated sulfation of N-acetylglucosamine (GlcNAc) within sialyl Lewis X of cognate endothelial ligands for L-selectin was an essential modification for L-selectin binding. The recently identified GlcNAc-6-sulfotransferases GlcNAc6ST-1 and -2, which facilitate GlcNAc sulfation by catalyzing the transfer of a sulfonyl group from 3(')-phosphoadenosine 5(')-phosphosulfate (PAPS) to the 6-hydroxy group of the acceptor GlcNAc moiety, contribute to the biosynthesis of the 6-sulfosialyl Lewis X motif.

The Interleukin-4-Receptor: From Recognition Mechanism to Pharmacological Target Structure.

Organic synthesis of hormone derivatives is an established route to yield pharmacologically active agents. Until recently this has only been feasible for small organic compounds, but nowadays it is also possible to produce antagonists for larger protein hormones. In particular, the interleukin-4-receptor was a well-suited target for this approach since it plays a pivotal role in the release and progression of allergic diseases.

Crystal structure of the interleukin-4/receptor alpha chain complex reveals a mosaic binding interface.

Interleukin-4 (IL-4) is a principal regulatory cytokine during an immune response and a crucial determinant for allergy and asthma. IL-4 binds with high affinity and specificity to the ectodomain of the IL-4 receptor alpha chain (IL4-BP). Subsequently, this intermediate complex recruits the common gamma chain (gamma c), thereby initiating transmembrane signaling.

Crystals of a 1:1 complex between human interleukin-4 and the extracellular domain of its receptor alpha chain.

The specific high-affinity binding of interleukin-4 (IL-4) to its receptor alpha chain is the crucial primary event during IL-4 signalling. Single crystals, suitable for high resolution diffraction studies, have been obtained from a complex between IL-4 and the ectodomain of the receptor alpha chain, also called IL-4-binding protein (IL-4BP). The orthorhombic crystals are in spacegroup P2(1)2(1)2(1) with cell constants a = 5.

Cloning, sequencing, crystallization and X-ray structure of glutathione S-transferase-III from Zea mays var. mutin: a leading enzyme in detoxification of maize herbicides.

Glutathione S-transferases (GSTs) are enzymes that inactivate toxic compounds by conjugation with glutathione and are involved in resistance towards drugs, antibiotics, insecticides and herbicides. Their ability to confer herbicide tolerance in plants provides a tool to control weeds in a wide variety of agronomic crops. GST-III was prepared from Zea mays var.

Plant glutathione S-transferases and herbicide detoxification.

Glutathione S-transferases (GSTs) are a family of multifunctional enzymes involved in the metabolism of xenobiotics and reactive endogenous compounds. The interest in plant GSTs may be attributed to their agronomic value, since it has been demonstrated that glutathione conjugation for a variety of herbicides is the major resistance and selectivity factor in plants. The structure of the Arabidopsis thaliana isoenzyme, the first plant GST whose structure has been solved, may serve as a model system for the understanding of herbicide selectivity in crops.

Three-dimensional structure of glutathione S-transferase from Arabidopsis thaliana at 2.2 A resolution: structural characterization of herbicide-conjugating plant glutathione S-transferases and a novel active site architecture.

Glutathione S-transferases (GST) are a family of multifunctional enzymes involved in the metabolization of a broad variety of xenobiotics and reactive endogenous compounds. The interest in plant glutathione S-transferases may be attributed to their agronomic value, since it has been demonstrated that glutathione conjugation for a variety of herbicides is the major resistance and selectivity factor in plants. The three-dimensional structure of glutathione S-transferase from the plant Arabidopsis thaliana has been solved by multiple isomorphous replacement and multiwavelength anomalous dispersion techniques at 3 A resolution and refined to a final crystallographic R-factor of 17.

Crystal structure of the catalytic subunit of human protein phosphatase 1 and its complex with tungstate.

Protein phosphatase 1 (PP1) is a serine/threonine protein phosphatase that is essential in regulating diverse cellular processes. Here we report the crystal structure of the catalytic subunit of human PP1 gamma 1 and its complex with tungstate at 2.5 A resolution.

Mutations that stabilize folding intermediates of phage P22 tailspike protein: folding in vivo and in vitro, stability, and structural context.

The folding of the trimeric phage P22 tailspike protein is affected by single amino acid substitutions designated temperature-sensitive folding (tsf) mutations. Their phenotypes are alleviated by two repeatedly isolated global suppressor (su) mutations (su V331A and su A334V) and by two additional substitutions (su V331G and su A334I), accessible through site-directed mutagenesis. We investigated the influence of the suppressor mutations on tailspike refolding in vitro, on its maturation at high expression levels in vivo, and on the rates of thermal unfolding of the native protein.

The metzincins--topological and sequential relations between the astacins, adamalysins, serralysins, and matrixins (collagenases) define a superfamily of zinc-peptidases.

The three-dimensional structures of the zinc endopeptidases human neutrophil collagenase, adamalysin II from rattle snake venom, alkaline proteinase from Pseudomonas aeruginosa, and astacin from crayfish are topologically similar, with respect to a five-stranded beta-sheet and three alpha-helices arranged in typical sequential order. The four proteins exhibit the characteristic consensus motif HEXXHXXGXXH, whose three histidine residues are involved in binding of the catalytically essential zinc ion. Moreover, they all share a conserved methionine residue beneath the active site metal as part of a superimposable "Met-turn.

X-ray structures of human neutrophil collagenase complexed with peptide hydroxamate and peptide thiol inhibitors. Implications for substrate binding and rational drug design.

Matrix metalloproteinases (MMPs) are a family of zinc endopeptidases involved in tissue remodeling. They have also been implicated in various disease processes including tumour invasion and joint destruction and are therefore attractive targets for inhibitor design. For rational drug design, information of inhibitor binding at the atomic level is essential.

Refined crystal structure of porcine class Pi glutathione S-transferase (pGST P1-1) at 2.1 A resolution.

The crystal structure of class Pi glutathione S-transferase from porcine lung (pGST P1-1) in complex with glutathione sulphonate has been refined at 2.11 A resolution, to a crystallographic R-factor of 16.5% for 21, 165 unique reflections.