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 (Cys283) is considered essential for the enzymatic activity. In previous studies we demonstrated that Cys283 is targeted by the alkylating chemical warfare agent sulfur mustard (SM) yielding a thioether with a hydroxyethylthioethyl (HETE)-moiety. In the present study, the effect of SM on rabbit muscle CK (rmCK) activity was investigated with special focus on the alkylation of Cys283 and of reactive methionine (Met) residues. For investigation of SM-alkylated amino acids in rmCK, micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry measurements were performed using the Orbitrap technology. The treatment of rmCK with SM resulted in a decrease of enzyme activity. However, this decrease did only weakly correlate to the modification of Cys283 but was conclusive for the formation of Met70-HETE and Met179-HETE. In contrast, the activity of mutants of rmCK produced by side-directed mutagenesis that contained substitutions of the respective Met residues (Met70Ala, Met179Leu, and Met70Ala/Met179Leu) was highly resistant against SM. Our results point to a critical role of the surface exposed Met70 and Met179 residues for CK activity.

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. BRD4884 and BRD7232 possess kinetic selectivity for HDAC1 versus HDAC2. We demonstrate that the binding kinetics of HDAC inhibitors can be tuned for individual isoforms in order to modulate target residence time while retaining functional activity and increased histone H4K12 and H3K9 acetylation in primary mouse neuronal cell culture assays. These chromatin modifiers, with tuned binding kinetic profiles, can be used to define the relation between target engagement requirements and the pharmacodynamic response of HDACs in different disease applications.

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. The conformational preorganization of the inhibitor molecule was verified by NMR structural analysis and was then confirmed by the crystal structure of the BACE-1/inhibitor complex. Detailed insights into the binding mode of this macrocyclic inhibitor explained its moderate binding affinity in cell-free assays (K(i)=2.5 microM) and yielded precious information for possible structural optimization in view of the lack of steric clashes of the macrocycle with the flap domain of the enzyme.

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 1.7A resolution and represents an intact and fully active bacterial topoisomerase I. It reveals the torus-like structure of the conserved transesterification core domain comprising domains I-IV and a tightly associated C-terminal zinc ribbon domain (domain V) packing against domain IV of the core domain. The previously established zinc-independence of the functional activity of T.maritima topoisomerase I is further supported by its crystal structure as no zinc ion is bound to domain V. However, the structural integrity is preserved by the formation of two disulfide bridges between the four Zn-binding cysteine residues. A functional role of domain V in DNA binding and recognition is suggested and discussed in the light of the structure and previous biochemical findings. In addition, implications for bacterial topoisomerases I are provided.

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. The assay is based on (33)P-phosphate transfer from [gamma-(33)P]ATP to sphingosine and subsequent detection of the [(33)P]S1P using AquaBind plates (Asahi Techno Glass, Tokyo, Japan). Enzymatic and inhibition characteristics determined with this assay are in good agreement with previously reported values determined in the conventional TLC assay. K(m) values for D-erythro-sphingosine and ATP were determined to be 17.5 microM and 19.2 microM, respectively. The kinase reaction could be inhibited by ADP and N,N-dimethylsphingosine with a 50% inhibitory concentration of 410 microM and 450 microM, respectively. The established assay format was easily adapted to an automated screening platform and is characterized by a high signal-to-background ratio, small variation, and excellent Z factors.

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. Lead optimization led to the discovery of BAY 59-7939 (5), a highly potent and selective, direct FXa inhibitor with excellent in vivo antithrombotic activity. The X-ray crystal structure of 5 in complex with human FXa clarified the binding mode and the stringent requirements for high affinity. The interaction of the neutral ligand chlorothiophene in the S1 subsite allows for the combination of good oral bioavailability and high potency for nonbasic 5. Compound 5 is currently under clinical development for the prevention and treatment of thromboembolic diseases.

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. The authors report the development of a simple and robust 384-well plate assay that is based on(33) P-phosphate transfer from radiolabeled [gamma(33) P]ATP to phosphatidylinositol immobilized on Maxisorp plates. The established assay format for PI3K gamma was easily adapted to the automated screening platform and was successfully employed for high-throughput screening. Enzymatic and inhibition characteristics of recombinant human PI3K gamma determined with the plate assay are in very good agreement with previously reported values determined in other assay formats. Maximal catalytic activity of PI3K gamma was observed at pH 7.0. The apparent K(m) value for ATP using a 1:1 mixture of phosphatidylinositol and phosphatidylserine was determined to be 7.3 microM (6.0-8.6 microM, 95% confidence interval [CI]). IC(50) values for known PI3-kinase inhibitors were determined to be 1.45 nM (1.17-1.80 nM, 95% CI) for wortmannin and estimated from partial inhibition data to be 1400, 2830, and 21,400 nM for quercetin, LY294002, and staurosporine, respectively. This novel assay approach allows for screening of inhibitors of lipid kinases in high-throughput mode and thereby may facilitate the identification of novel inhibitory structures for drug development.

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. Here, we present the three-dimensional structure of a potent dihydropyrimidone inhibitor (DHPI) non-covalently bound to HNE at a resolution of 2.0 Å. The inhibitor binds to the active site in a unique orientation addressing S1 and S2 subsites of the protease. To facilitate further analysis of this binding mode, we determined the structure of the uncomplexed enzyme at a resolution of 1.86 Å. Detailed comparisons of the HNE:DHPI complex with the uncomplexed HNE structure and published structures of other elastase:inhibitor complexes revealed that binding of DHPI leads to large conformational changes in residues located in the S2 subsite. The rearrangement of residues Asp95-Leu99B creates a deep, well-defined cavity, which is filled by the P2 moiety of the inhibitor molecule to almost perfect shape complementarity. The shape of the S2 subsite in complex with DHPI clearly differs from all other observed HNE structures. The observed structural flexibility of the S2 subsite is a key feature for the understanding of the binding mode of DHPIs in general and the development of new HNE selective inhibitors.

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. The properties and phylogenetic position of the producer organism, the production and isolation of compounds 1-7, their structure elucidation by MS and NMR, and their biological activities are reported. Additionally, an X-ray crystal structure was obtained from cinnabaramide A (1).

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. Moreover, when complex body fluids are used as samples, efficient debulking strategies are crucial to open a window of analytical sensitivity in the ng/mL range, where many diagnostically relevant analytes are present. Here we report the proof-of-principle of a multi-dimensional strategy for accelerated initial validation of biomarker candidates by MS, which promises to be generally applicable, sensitive and quantitative. The method presented employs a combination of electrophoretic and chromatographic steps on the peptide level, followed by MS quantification using isotopically labeled synthetic peptides as internal standards. Our proposed workflow includes up to four dimensions, finally resulting in a desired LOD sufficient to detect and quantify diagnostically relevant analytes from complex samples. Although the current state of the method only represents a starting point for further validation and development, it reveals great potential in biomarker validation.

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. Due to their pivotal role in L-selectin ligand biosynthesis, this enzyme class has recently emerged as an important and relatively unexplored class of potential targets for anti-inflammatory therapy. However, no inhibitors have been reported to date and screening for lead inhibitors has been hampered by the lack of simple assay formats suitable for high-throughput screening. Here, we report the development of a simple homogeneous in vitro sulfotransferase assay using a newly synthesized biotinylated glycoside as a substrate. The assay is based on GlcNAc6ST-2-mediated [35S]sulfate transfer from [35S]PAPS to the biotinylated glycoside and subsequent detection using streptavidin-coated SPA beads. K(m) values with partially purified GlcNAc6ST-2 for PAPS and the biotinylated glycoside were estimated to be 8.4 and 34.5 microM, respectively. The sulfotransferase reaction could be inhibited by 3('),5(')-ADP with an IC(50) of 2.1 microM. The assay can be operated in 384-well format; is characterized by a high signal-to-noise ratio, low variation, and excellent Z factors; and is highly suitable for high-throughput screening.