Arylaminoethyl amides as inhibitors of the cysteine protease cathepsin K-investigating P1' substituents

Modeling, synthesis and in vitro activities of a series of arylaminoethyl amide based inhibitors of the cysteine protease cathepsin K are described.     

Synthesis and SAR of arylaminoethyl amides as noncovalent inhibitors of cathepsin S: P3 cyclic ethers

The synthesis and structure-activity relationship of a series of arylaminoethyl amide cathepsin S inhibitors are reported. Optimization of P3 and P2 groups to improve overall physicochemical properties resulted in significant improvements in oral bioavailability over early lead compounds. An X-ray structure of compound 37 bound to the active site of cathepsin S is also reported.     

Screening for in vitro metabolites of Abelmoschus manihot extract in intestinal bacteria by ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry

Abelmoschus manihot has drawn much attention recently due to its potential beneficial health effects after oral administration. However, the metabolic fate of A. manihot in intestinal flora is not well understood. In this paper, we describe a strategy using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF MS) with automated data analysis software (MetaboLynx?) for fast analysis of the metabolic profile of flavonoids from A. manihot in intestinal flora. The human and rat incubated samples collected 72 h in the anaerobic incubator were analyzed by UPLC-Q-TOF MS within 10 min. A total of 14 metabolites were identified in human and rat incubated solution compared with blank samples. The results indicated that hydrolysis, hydroxylation and acetylation were the major metabolic pathways of flavonoids in A. manihot extract in vitro. MS(E) was used for simultaneous acquisition of precursor ion information and fragment ion data at high and low collision energy in one analytical run, which facilitated the fast structural characterization of metabolites. This work demonstrated the potential of the UPLC-Q-TOF MS approach using Metabolynx for fast and automated identification of metabolites of natural product in intestinal flora.     

Design and synthesis of arylaminoethyl amides as noncovalent inhibitors of cathepsin S. Part 1

A series of Nalpha-acyl-alpha-amino acid-(arylaminoethyl)amides were found to be potent and noncovalent cathepsin S inhibitors. Compound 20 possessed high cathepsin S affinity (Ki=3.3 nM) and showed excellent selectivity over cathepsin K, L, F, and V. Molecular modeling, design, synthesis, and in vitro activity are described.     

Chemical derivatization and mass spectral libraries in metabolic profiling by GC/MS and LC/MS/MS

An overview is presented of gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS), the two major hyphenated techniques employed in metabolic profiling that complement direct 'fingerprinting' methods such as atmospheric pressure ionization (API) quadrupole time-of-flight MS, API Fourier transform MS, and NMR. In GC/MS, the analytes are normally derivatized prior to analysis in order to reduce their polarity and facilitate chromatographic separation. The electron ionization mass spectra obtained are reproducible and suitable for library matching, mass spectral collections being readily available. In LC/MS, derivatization and library matching are at an early stage of development and mini-reviews are provided. Chemical derivatization can dramatically increase the sensitivity and specificity of LC/MS methods for less polar compounds and provides additional structural information. The potential of derivatization for metabolic profiling in LC/MS is demonstrated by the enhanced analysis of plant extracts, including the potential to measure volatile acids such as formic acid, difficult to achieve by GC/MS. The important role of mass spectral library creation and usage in these techniques is discussed and illustrated by examples.     

Primary amides as selective inhibitors of cathepsin K

The nitrile warhead used in a series of cathepsin K inhibitors can be replaced by a less electrophilic primary amide. The accompanying loss of potency can be partially recovered by introducing a substituent alpha to the amide. The potency gain resulting from this addition is not achieved with the nitrile derivatives due to a different geometry of the cysteine adduct in the enzyme active site. This study led to the identification of the primary amide 2g, which is an inhibitory substrate, with an IC(50) of 10 nM against cathepsin K and excellent selectivity versus the other cathepsins.     

Clarification of pathway-specific inhibition by Fourier transform ion cyclotron resonance/mass spectrometry-based metabolic phenotyping studies

We have developed a metabolic profiling scheme based on direct-infusion Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR/MS). The scheme consists of: (1) reproducible data collection under optimized FT-ICR/MS analytical conditions; (2) automatic mass-error correction and multivariate analyses for metabolome characterization using a newly developed metabolomics tool (DMASS software); (3) identification of marker metabolite candidates by searching a species-metabolite relationship database, KNApSAcK; and (4) structural analyses by an MS/MS method. The scheme was applied to metabolic phenotyping of Arabidopsis (Arabidopsis thaliana) seedlings treated with different herbicidal chemical classes for pathway-specific inhibitions. Arabidopsis extracts were directly infused into an electrospray ionization source on an FT-ICR/MS system. Acquired metabolomics data were comprised of mass-to-charge ratio values with ion intensity information subjected to principal component analysis, and metabolic phenotypes from the herbicide treatments were clearly differentiated from those of the herbicide-free treatment. From each herbicide treatment, candidate metabolites representing such metabolic phenotypes were found through the KNApSAcK database search. The database search and MS/MS analyses suggested dose-dependent accumulation patterns of specific metabolites including several flavonoid glycosides. The metabolic phenotyping scheme on the basis of FT-ICR/MS coupled with the DMASS program is discussed as a general tool for high throughput metabolic phenotyping studies.     

Synthesis and evaluation of arylaminoethyl amides as noncovalent inhibitors of cathepsin S. Part 3: heterocyclic P3

A series of N(alpha)-2-benzoxazolyl-alpha-amino acid-(arylaminoethyl)amides were identified as potent, selective, and noncovalent inhibitors of cathepsin S. Structure-activity relationships including strategies for modulating the selectivities among cathepsins S, K, and L, and in vivo pharmacokinetics are discussed. A X-ray structure of compound 3 bound to the active site of cathepsin S is also reported.     

Benzodioxocin-3-ones and N-acyl-3-amino-3-buten-2-ones: novel classes of cathepsin K cysteine protease inhibitors.

The design, synthesis, enzymologic, and protein mass spectrometric characterization of benzodioxocin-3-one and N-acyl-3-amino-3-buten-2-one inhibitors of the cysteine protease cathepsin K are described. The benzodioxocin-3-one ring system is chemically unstable giving rise to a mixture of N-acyl-3-amino-3-buten-2-one and hemiketals. This mixture of N-acyl-3-amino-3-buten-2-one and hemiketals potently inhibits recombinant, human cathepsin K (IC50 = 36 nM) by a time-independent, irreversible mechanism. Formation of a covalent adduct between cathepsin K and inhibitor has been confirmed by mass spectrometry.     

Unique scanning capabilities of a new hybrid linear ion trap mass spectrometer (Q TRAP) used for high sensitivity proteomics applications

The unique scanning capabilities of a hybrid linear ion trap (Q TRAP) mass spectrometer are described with an emphasis on proteomics applications. The combination of the very selective triple quadrupole based tandem mass spectrometry (MS/MS) scans with the very sensitive ion trap product ion scans allows rapid identification of peptides at low concentrations derived from post-translationally modified proteins on chromatographic time scales. The Q TRAP instrument also offers the opportunity to conduct a variety of ion processing steps prior to performing a mass scan. For example, the enhancement of the multiple-charge ion contents of the ion trap can be performed resulting in a survey mass spectrum dominated by double- and triple-charge peptides. This facilitates the identification of relevant biological species in both separated and unseparated peptide mixtures for further MS/MS experiments.     

Identification of a potent and selective non-basic cathepsin K inhibitor

Based on our previous study with trifluoroethylamine as a P2-P3 amide isostere of cathepsin K inhibitor, further optimization led to identification of compound 22 (L-873724) as a potent and selective non-basic cathepsin K inhibitor. This compound showed excellent pharmacokinetics and efficacy in an ovariectomized (OVX) rhesus monkey model. The volumes of distribution close to unity were consistent with this compound not being lysosomotropic, which is a characteristic of basic cathepsin K inhibitors.     

Identification of p21Cip1 binding proteins by gel electrophoresis and capillary liquid chromatography microelectrospray tandem mass spectrometry

Proteins bound to a glutathione-S-transferase-p21Cip1 affinity column were separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified using tandem mass spectrometry. Capillary liquid chromatography coupled to microelectrospray tandem mass spectrometry (capLC-microESI MS/MS) in an ion trap allowed identification of the proteins present in the gel bands. Of eleven bands analyzed, fifty-three proteins were identified. More than one hundred tryptic peptides were detected on-line, automatically fragmented and used for protein characterization in databases. Samples were also analyzed by off-line nanospray and matrix-assisted laser desorption/ionization mass spectrometry. CapLC-microESI MS/MS was the most efficient technique for the analysis of these protein mixtures.     

Nearline acquisition and processing of liquid chromatography-tandem mass spectrometry data

Liquid chromatography–mass spectrometry (LC–MS) is a commonly used analytical platform for non-targeted metabolite profiling experiments. Although data acquisition, processing and statistical analyses are almost routine in such experiments, further annotation and subsequent identification of chemical compounds are not. For identification, tandem mass spectra provide valuable information towards the structure of chemical compounds. These are typically acquired online, in data-dependent mode, or offline, using handcrafted acquisition methods and manually extracted from raw data. Here, we present several methods to fast-track and improve both the acquisition and processing of LC–MS/MS data. Our nearly online (nearline) data-dependent tandem MS strategy creates a minimal set of LC–MS/MS acquisition methods for relevant features revealed by a preceding non-targeted profiling experiment. Using different filtering criteria, such as intensity or ion type, the acquisition of irrelevant spectra is minimized. Afterwards, LC–MS/MS raw data are processed with feature detection and grouping algorithms. The extracted tandem mass spectra can be used for both library search and de-novo identification methods. The algorithms are implemented in the R package MetShot and support the export to Bruker, Agilent or Waters QTOF instruments and the vendor-independent TraML standard. We evaluate the performance of our workflow on a Bruker micrOTOF-Q by comparison of automatically acquired and extracted tandem mass spectra obtained from a mixture of natural product standards against manually extracted reference spectra. Using Arabidopsis thaliana wild-type and biosynthetic gene knockout plants, we characterize the metabolic products of a biosynthetic pathway and demonstrate the integration of our approach into a typical non-targeted metabolite profiling workflow.     

Advanced mass spectrometric methods for the rapid and quantitative characterization of proteomes

Progress is reviewed towards the development of a global strategy that aims to extend the sensitivity, dynamic range, comprehensiveness and throughput of proteomic measurements based upon the use of high performance separations and mass spectrometry. The approach uses high accuracy mass measurements from Fourier transform ion cyclotron resonance mass spectrometry (FTICR) to validate peptide 'accurate mass tags' (AMTs) produced by global protein enzymatic digestions for a specific organism, tissue or cell type from 'potential mass tags' tentatively identified using conventional tandem mass spectrometry (MS/MS). This provides the basis for subsequent measurements without the need for MS/ MS. High resolution capillary liquid chromatography separations combined with high sensitivity, and high resolution accurate FTICR measurements are shown to be capable of characterizing peptide mixtures of more than 10(5) components. The strategy has been initially demonstrated using the microorganisms Saccharomyces cerevisiae and Deinococcus radiodurans. Advantages of the approach include the high confidence of protein identification, its broad proteome coverage, high sensitivity, and the capability for stableisotope labeling methods for precise relative protein abundance measurements.Abbreviations: LC, liquid chromatography; FTICR, Fourier transform ion cyclotron resonance; AMT, accurate mass tag; PMT, potential mass tag; MMA, mass measurement accuracy; MS, mass spectrometry; MS/MS, tandem mass spectrometry; ppm, parts per million.     

Metabolome searcher: a high throughput tool for metabolite identification and metabolic pathway mapping directly from mass spectrometry and using genome restriction

Background

Mass spectrometric analysis of microbial metabolism provides a long list of possible compounds. Restricting the identification of the possible compounds to those produced by the specific organism would benefit the identification process. Currently, identification of mass spectrometry (MS) data is commonly done using empirically derived compound databases. Unfortunately, most databases contain relatively few compounds, leaving long lists of unidentified molecules. Incorporating genome-encoded metabolism enables MS output identification that may not be included in databases. Using an organism’s genome as a database restricts metabolite identification to only those compounds that the organism can produce.

Results

To address the challenge of metabolomic analysis from MS data, a web-based application to directly search genome-constructed metabolic databases was developed. The user query returns a genome-restricted list of possible compound identifications along with the putative metabolic pathways based on the name, formula, SMILES structure, and the compound mass as defined by the user. Multiple queries can be done simultaneously by submitting a text file created by the user or obtained from the MS analysis software. The user can also provide parameters specific to the experiment’s MS analysis conditions, such as mass deviation, adducts, and detection mode during the query so as to provide additional levels of evidence to produce the tentative identification. The query results are provided as an HTML page and downloadable text file of possible compounds that are restricted to a specific genome. Hyperlinks provided in the HTML file connect the user to the curated metabolic databases housed in ProCyc, a Pathway Tools platform, as well as the KEGG Pathway database for visualization and metabolic pathway analysis.

Conclusions

Metabolome Searcher, a web-based tool, facilitates putative compound identification of MS output based on genome-restricted metabolic capability. This enables researchers to rapidly extend the possible identifications of large data sets for metabolites that are not in compound databases. Putative compound names with their associated metabolic pathways from metabolomics data sets are returned to the user for additional biological interpretation and visualization. This novel approach enables compound identification by restricting the possible masses to those encoded in the genome.     

C-terminal sequencing by mass spectrometry: application to gelatine-derived proline-rich peptides

Protonated peptides derived from proline‐rich proteins (PRP) are often difficult to sequence by standard collision‐induced dissociation (CID) mass spectrometry (MS) due to preferential amide bond cleavage N‐terminal to proline. In connection with bovine spongiform encephalopathy regulations, proteolytic products derived from the PRP collagen have been suggested as markers for contamination of animal feedstuffs with processed animal protein (Fernandez Ocaña, M. et al., Analyst 2004, 129, 111–115). Herein, we report the identification of these marker peptides using the strategy of C‐terminal sequencing by CID MS from their sodium and lithium adducts. Upon fragmentation a new cationized peptide was produced that is one C‐terminal amino acid shorter in length. This dissociation pathway allowed for the facile identification of the C‐terminal residue by matrix‐assisted laser desorption/ionization tandem time‐of‐flight mass spectrometry. Each newly formed cationized peptide was further fragmented by up to seven stages of electrospray ionization ion trap MS. Proline‐rich C‐terminal sequence tags were established which permitted successful database identification of collagen alpha type I proteins.     

Formation of a new oxidative metabolite from kobophenol A by human intestinal bacterium Klebsiella pneumoniae

During our studies on the metabolism of kobophenol A (1) in rats, we had isolated, purified, and identified the main new oxidative metabolite of 1, koboquinone A (2) from rats' feces. To elucidate the metabolic pathway of 1 in rats, we conducted the in vitro metabolic experiments of 1 by human intestinal bacteria and found that Klebsiella pneumoniae produced appreciable amounts of 2. This was verified by means of high performance liquid chromatography mass/mass spectrometry (HPLC/MS/MS) analysis.     

Metabolic profiling of human saliva before and after induced physiological stress by ultra-high performance liquid chromatography–ion mobility–mass spectrometry

A method has been developed for metabolite profiling of the salivary metabolome based on protein precipitation and ultra-high performance liquid chromatography coupled with ion mobility-mass spectrometry (UHPLC–IM–MS). The developed method requires 0.5 mL of human saliva, which is easily obtainable by passive drool. Standard protocols have been established for the collection, storage and pre-treatment of saliva. The use of UHPLC allows rapid global metabolic profiling for biomarker discovery with a cycle time of 15 min. Mass spectrometry imparts the ability to analyse a diverse number of species reproducibly over a wide dynamic range, which is essential for profiling of biofluids. The combination of UHPLC with IM–MS provides an added dimension enabling complex metabolic samples to be separated on the basis of retention time, ion mobility and mass-to-charge ratio in a single chromatographic run. The developed method has been applied to targeted metabolite identification and untargeted metabolite profiling of saliva samples collected before and after exercise-induced physiological stress. δ-Valerolactam has been identified as a potential biomarker on the basis of retention time, MS/MS spectrum and ion mobility drift time.     

Analysis of carbohydrate heterogeneity in a glycoprotein using liquid chromatography/mass spectrometry and liquid chromatography with tandem mass spectrometry

High-performance liquid chromatography with on-line electrospray ionization mass spectrometry (ESI-LC/MS) was investigated for the analysis of carbohydrate heterogeneity using RNase B as a model glycoprotein. Oligosaccharides released from RNase B with endoglycosidase H were reduced and separated on a graphitized carbon column (GCC). GCC-HPLC/MS in the positive-ion mode was successful in the identification of one Man5GlcNAc, three Man6GlcNAc, three Man7GlcNAc, three Man8GlcNAc, one Man9GlcNAc, and an oligosaccharide having six hexose units (Hex) and two N-acetylhexosamine units (HexNAc). The branch structures of the three Man7GlcNAc isomers were determined by liquid chromatography with tandem mass spectrometry (LC/MS/MS). LC/MS/MS analysis was shown to be useful for the detection and identification of a trace amount of Hex6HexNAc2 alditol as a hybrid-type oligosaccharide. Its structure was confirmed by the combination of LC/MS with enzymatic digestion using beta-galactosidase and N-acetyl-beta-glucosaminidase. The relative quantities of high-mannose-type oligosaccharides in RNase B detected by ESI-LC/MS are in reasonable agreement with those by UV, high-pH anion-exchange chromatography with pulsed amperometric detection, fluorophore-assisted carbohydrate electrophoresis. Our results indicate that LC/MS and LC/MS/MS can be utilized to elucidate the distribution of oligosaccharides and their structures, which differ in molecular weight, sugar sequence, and branch structure.     

Strategic proteome analysis of Candida magnoliae with an unsequenced genome

Erythritol is a noncariogenic, low calorie sweetener. It is safe for people with diabetes and obese people. Candida magnoliae is an industrially important organism because of its ability to produce erythritol as a major product. The genome of C. magnoliae has not been sequenced yet, limiting the available proteome database. Therefore, systematic approaches were employed to construct the proteome map of C. magnoliae. Proteomic analysis with systematic approaches is based on two-dimensional electrophoresis, matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), tandem mass spectrometry (MS/MS) and database interrogation. First, 24 spots were analyzed using peptide mass fingerprinting along with MALDI-TOF MS with high mass accuracy. Only four spots were reliably identified as carbonyl reductase and its isoforms. The reason for low sequence coverage seemed to be that these identification strategies were based on the presence of the protein database obtained from the publicly accessible genome database and the availability of cross-species protein identification. MS/MS (MS/MS ion search and de novo sequencing) in combination with similarity searches allowed successful identification of 39 spots. Several proteins including transaldolase identified by MS/MS ion searches were further confirmed by partial sequences from the expressed sequence tag database. In this study, 51 protein spots were analyzed and then potentially identified. The identified proteins were involved in glycolysis, stress response, other essential metabolisms and cell structures.