A method for the production and characterization of fractionated libraries from Chinese herbal formulas

High-throughput screening (HTS) for pharmaceutical leads requires sufficient number of samples with vast chemical diversity. In this paper, we proposed Chinese herbal formulas as an attractive source for HTS and introduced a strategy for the production of high-quality fractionated libraries. An offline two-dimension liquid chromatography protocol was developed to separate medium- and low-polar extract (MLPE) of Chinese herbal formulas, which implemented the production of semi-purified mixture libraries. HPLC coupled with diode-array detector (DAD) and mass spectrum (MS) analysis was performed to obtain MS and UV spectrum of library components. The detected components were characterized by retention, molecular weight and UV absorbance assisted by WiseProcessor, a customer-developed software to automatically process analytical data. Based on the current understanding in pathophysiology and pharmacology, multiple cell-based bioassays were performed to screening the library samples. Through validation and dereplication process, bioactive compounds could be identified rapidly. The combination of off-line two-dimension liquid chromatography separation, HPLC-DAD-MS analysis and computer-aided data processing is reliable and efficient for the utilization of Chinese herbal formulas as valuable sources for HTS. As a demonstration, a library sample set was generated from Qi-Xue-Bing-Zhi Formula, an efficient Chinese herbal formula to treat atherosclerosis. Several bioactive compounds were quickly identified from this library through the screening and dereplication process.     

Construction and application of a mass spectral and retention time index database generated from plant GC/EI-TOF-MS metabolite profiles

The non-supervised construction of a mass spectral and retention time index data base (MS/RI library) from a set of plant metabolic profiles covering major organs of potato (Solanum tuberosum), tobacco (Nicotiana tabaccum), and Arabidopsis thaliana, was demonstrated. Typically 300-500 mass spectral components with a signal to noise ratio > or =75 were obtained from GC/EI-time-of-flight (TOF)-MS metabolite profiles of methoxyaminated and trimethylsilylated extracts. Profiles from non-sample controls contained approximately 100 mass spectral components. A MS/RI library of 6205 mass spectral components was accumulated and applied to automated identification of the model compounds galactonic acid, a primary metabolite, and 3-caffeoylquinic acid, a secondary metabolite. Neither MS nor RI alone were sufficient for unequivocal identification of unknown mass spectral components. However library searches with single bait mass spectra of the respective reference substance allowed clear identification by mass spectral match and RI window. Moreover, the hit lists of mass spectral searches were demonstrated to comprise candidate components of highly similar chemical nature. The search for the model compound galactonic acid allowed identification of gluconic and gulonic acid among the top scoring mass spectral components. Equally successful was the exemplary search for 3-caffeoylquinic acid, which led to the identification of quinic acid and of the positional isomers, 4-caffeoylquinic acid, 5-caffeoylquinic acid among other still non-identified conjugates of caffeic and quinic acid. All identifications were verified by co-analysis of reference substances. Finally we applied hierarchical clustering to a complete set of pair-wise mass spectral comparisons of unknown components and reference substances with known chemical structure. We demonstrated that the resulting clustering tree depicted the chemical nature of the reference substances and that most of the nearest neighbours represented either identical components, as judged by co-elution, or conformational isomers exhibiting differential retention behaviour. Unknown components could be classified automatically by grouping with the respective branches and sub-branches of the clustering tree.     

Sensitive assay for laboratory evolution of hydroxylases toward aromatic and heterocyclic compounds

Powerful directed evolution methods have been developed for tailoring proteins to our needs in industrial applications. Here, the authors report a medium-throughput assay system designed for screening mutant libraries of oxygenases capable of inserting a hydroxyl group into a C-H bond of aromatic or O-heterocyclic compounds and for exploring the substrate profile of oxygenases. The assay system is based on 4-aminoantipyrine (4-AAP), a colorimetric phenol detection reagent. By using 2 detection wavelengths (509 nm and 600 nm), the authors achieved a linear response from 50 to 800 microM phenol and standard deviations below 11% in 96-well plate assays. The monooxygenase P450 BM-3 and its F87A mutant were used as a model system for medium-throughput assay development, identification of novel substrates (e.g., phenoxytoluene, phenylallyether, and coumarone), and discovery of P450 BM-3 F87A mutants with 8-fold improvement in 3-phenoxytoluene hydroxylation activity. This activity increase was achieved by screening a saturation mutagenesis library of amino acid position Y51 using the 4-AAP protocol in the 96-well format.     

An automated screening method for drugs and toxic compounds in human serum and urine using liquid chromatography–tandem mass spectrometry

A fully automated screening using liquid chromatography–mass spectrometric method applying data-dependent acquisition was developed to identify toxicologically relevant substances in serum and urine. A library including more than 405 spectra of about 365 compounds (main drugs and important metabolites) was established. An easy to use program was created to automate and accelerate library search. Drugs were identified based on their relative retention times, molecular ions and fragment ions. Limits of detection were tested with 100 of the 365 compounds the majority of these were lower than 100 μg/l (67%). The developed LC–MS–MS system seems to be a valuable alternative to other general unknown screening methods allowing fast and specific identification of drugs in serum and urine samples.     

Optimized protocols for the isolation of specific protein-binding peptides or peptoids from combinatorial libraries displayed on beads

Many methods have been published by which combinatorial libraries may be screened for compounds capable of manipulating the function(s) of a target protein. One of the simplest approaches is to identify compounds in a library that bind the protein of interest, since these binding events usually occur on functionally important surfaces of the protein. These protein-binding compounds could also be of utility as protein capture agents in the construction of protein-detecting microarrays or related analytical devices and as reagents for the affinity purification of proteins from complex mixtures. In this article, we provide optimized methods for screening libraries of molecules displayed on the beads on which they were synthesized. This is a particularly convenient format for library screening for laboratories with limited budgets and modest robotics capabilities.     

Identification of protein-binding peptides by direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of peptide beads selected from the screening of one bead-one peptide combinatorial libraries

A fast and inexpensive strategy for the identification of peptide ligands by direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of peptide beads screened from one bead-one peptide combinatorial libraries is herein described. Streptavidin was used as the model protein. A combinatorial library of 6561 peptides was synthesized on ChemMatrix resin by the divide-couple-recombine method. 4-Hydroxymethylbenzoic acid was used as the linker and five residues of Gly were incorporated at the C termini to increase the final peptide molecular weight. Positive control peptides with the HPQ motif and negative control peptides without the HPQ motif evidenced that the linker and the five residues of Gly have neither impaired the specific binding nor facilitated unspecific binding. After screening the library, positive beads were isolated and washed with 8M guanidine hydrochloride. The beads were sliced into two or four pieces, deposited onto the stainless steel MALDI sample plate, and treated with ammonia vapor to release the peptides. In addition, 26 beads picked at random from the library were subjected to the same treatment. All samples were analyzed by MALDI-TOF-MS and the peptides were unambiguously identified with very good reproducibility between the bead pieces, thus evidencing the good homogeneity of the bead. All sequences obtained from the screening contained HPQ.     

Cigarette smoke irreversibly modifies glutathione in airway epithelial cells

In patients with chronic obstructive pulmonary disease (COPD), an imbalance between oxidants and antioxidants is acknowledged to result in disease development and progression. Cigarette smoke (CS) is known to deplete total glutathione (GSH + GSSG) in the airways. We hypothesized that components in the gaseous phase of CS may irreversibly react with GSH to form GSH derivatives that cannot be reduced (GSX), thereby causing this depletion. To understand this phenomenon, we investigated the effect of CS on GSH metabolism and identified the actual GSX compounds. CS and H(2)O(2) (control) deplete reduced GSH in solution [Delta = -54.1 +/- 1.7 microM (P < 0.01) and -39.8 +/- 0.9 microM (P < 0.01), respectively]. However, a significant decrease of GSH + GSSG was observed after CS (Delta = -75.1 +/- 7.6 microM, P < 0.01), but not after H(2)O(2). Exposure of A549 cells and primary bronchial epithelial cells to CS decreased free sulfhydryl (-SH) groups (Delta = -64.2 +/- 14.6 microM/mg protein, P < 0.05) and irreversibly modified GSH + GSSG (Delta = -17.7 +/- 1.9 microM, P < 0.01) compared with nonexposed cells or H(2)O(2) control. Mass spectrometry (MS) showed that GSH was modified to glutathione-aldehyde derivatives. Further MS identification showed that GSH was bound to acrolein and crotonaldehyde and another, yet to be identified, structure. Our data show that CS does not oxidize GSH to GSSG but, rather, reacts to nonreducible glutathione-aldehyde derivatives, thereby depleting the total available GSH pool.     

基于活性和基因从海洋微生物中筛选烯二炔类抗生素

【目的】高质量的海洋微生物菌种库及其天然产物库是新药开发的重要来源。在本研究中我们通过筛选新的烯二炔类抗生素来对已经构建的海洋微生物天然产物库进行质量评价。【方法】首先我们根据烯二炔类抗生素能引起DNA断裂的活性构建了活性筛选模型,并对我们的天然产物库进行筛选;其次根据合成烯二炔核心结构的独特且保守的重复I型聚酮合成酶设计了引物,通过PCR扩增的方法对海洋微生物库进行序列筛选。【结果】通过活性筛选从我们的海洋微生物天然产物库中获得一个阳性的发酵产物。对该阳性菌(LS481)的系统发育学分析表明该菌属于能产生烯二炔类化合物—Dynemicin的Micromonospora chersina,对其发酵产物TLC分析证明该菌确实产生Dynemicin类化合物。通过基因筛选得到了2个具备合成烯二炔核心结构聚酮合成酶的菌株,16S rRNA基因分析显示其中一个很可能为灰色链霉菌(MS098),另外一株菌则同Streptomyces vinaceus NBRC 13425T和Streptomyces cirratus NRRLB-3250T最相近。【结论】我们的活性筛选模型能够有效获得烯二炔类物质,结合基因筛选能够进一步获得可能产生烯二炔物质的菌株。初筛结果也再一次验证了我们海洋微生物天然产物库的质量较好。     

High-throughput fluorescence assay for small-molecule inhibitors of autophagins/Atg4

Autophagy is an evolutionarily conserved process for catabolizing damaged proteins and organelles in a lysosome-dependent manner. Dysregulation of autophagy may cause various diseases, such as cancer and neurodegeneration. However, the relevance of autophagy to diseases remains controversial because of the limited availability of chemical modulators. Herein, the authors developed a fluorescence-based assay for measuring activity of the autophagy protease, autophagin-1(Atg4B). The assay employs a novel reporter substrate of Atg4B composed of a natural substrate (LC3B) fused to an assayable enzyme (PLA(2)) that becomes active upon cleavage by this cysteine protease. A high-throughput screening (HTS) assay was validated with excellent Z' factor (>0.7), remaining robust for more than 5 h and suitable for screening of large chemical libraries. The HTS assay was validated by performing pilot screens with 2 small collections of compounds enriched in bioactive molecules (n = 1280 for Lopac? and 2000 for Spectrum? library), yielding confirmed hit rates of 0.23% and 0.70%, respectively. As counterscreens, PLA(2) and caspase-3 assays were employed to eliminate nonspecific inhibitors. In conclusion, the LC3B-PLA(2) reporter assay provides a platform for compound library screening for identification and characterization of Atg4B-specific inhibitors that may be useful as tools for interrogating the role of autophagy in disease models.     

Determination and identification of estrogenic compounds generated with biosynthetic enzymes using hyphenated screening assays,high resolution mass spectrometry and off-line NMR

This paper describes the determination and identification of active and inactive estrogenic compounds produced by biosynthetic methods. A hyphenated screening assay towards the human estrogen receptor ligand binding domain (hER)α and hERβ integrating target–ligand interactions and liquid chromatography–high resolution mass spectrometry was used. With this approach, information on both biologic activity and structure identity of compounds produced by bacterial mutants of cytochrome P450s was obtained in parallel. Initial structure identification was achieved by high resolution MS/MS, while for full structure determination, P450 incubations were scaled up and the produced entities were purified using preparative liquid chromatography with automated fraction collection. NMR spectroscopy was performed on all fractions for 3D structure analysis; this included 1D-¹H, 2D-COSY, 2D-NOESY, and ¹H-¹³C-HSQC experiments. This multidimensional screening approach enabled the detection of low abundant biotransformation products which were not suitable for detection in either one of its single components. In total, the analytical scale biosynthesis produced over 85 compounds from 6 different starting templates. Inter- and intra-day variation of the biochemical signals in the dual receptor affinity detection system was less than 5%. The multi-target screening approach combined with full structure characterization based on high resolution MS(/MS) and NMR spectroscopy demonstrated in this paper can generally be applied to e.g. metabolism studies and compound-library screening.     

Comparative proteomics using 2-D gel electrophoresis and mass spectrometry as tools to dissect stimulons and regulons in bacteria with sequenced or partially sequenced genomes

We propose two-dimensional gel electrophoresis (2-DE) and mass spectrometry to define the protein components of regulons and stimulons in bacteria, including those organisms where genome sequencing is still in progress. The basic 2-DE protocol allows high resolution and reproducibility and enables the direct comparison of hundreds or even thousands of proteins simultaneously. To identify proteins that comprise stimulons and regulons, peptide mass fingerprint (PMF) with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF-MS) analysis is the first option and, if results from this tool are insufficient, complementary data obtained with electrospray ionization tandem-MS (ESI-MS/MS) may permit successful protein identification. ESI-MS/MS and MALDI-TOF-MS provide complementary data sets, and so a more comprehensive coverage of a proteome can be obtained using both techniques with the same sample, especially when few sequenced proteins of a particular organism exist or genome sequencing is still in progress.     

Experimental validation of a fragment library for lead discovery using SPR biosensor technology

A new fragment library for lead discovery has been designed and experimentally validated for use in surface plasmon resonance (SPR) biosensor-based screening. The 930 compounds in the library were selected from 4.6 million commercially available compounds using a series of physicochemical and medicinal chemistry filters. They were screened against 3 prototypical drug targets: HIV-1 protease, thrombin and carbonic anhydrase, and a nontarget: human serum albumin. Compound solubility was not a problem under the conditions used for screening. The high sensitivity of the sensor surfaces allowed the detection of interactions for 35% to 97% of the fragments, depending on the target protein. None of the fragments was promiscuous (i.e., interacted with a stoichiometry ≥5:1 with all 4 proteins), and only 2 compounds dissociated slowly from all 4 proteins. The use of several targets proved valuable since several compounds would have been disqualified from the library on the grounds of promiscuity if fewer target proteins had been used. The experimental procedure allowed an efficient evaluation and exploration of the new fragment library and confirmed that the new library is suitable for SPR biosensor-based screening.     

A combined A431 cell membrane chromatography and online high performance liquid chromatography/mass spectrometry method for screening compounds from total alkaloid of Radix Caulophylli acting on the human EGFR

We have developed an online analytical method that combines A431 cell membrane chromatography (A431/CMC) with high performance liquid chromatography and mass spectrometry (LC/MS) for identifying active components from Radix Caulophylli acting on human EGFR. Retention fractions on A431/CMC model were captured onto an enrichment column and the components were directly analyzed by combining a 10-port column switcher with an LC/MS system for separation and preliminary identification. Using Sorafenib tosylate as a positive control, taspine and caulophine from Radix Caulophylli were identified as the active molecules which could act on the EGFR. This A431/CMC-online-LC/MS method can be applied for screening active components acting on EGFR from traditional Chinese medicines exemplified by Radix Caulophylli and will be of great utility in drug discovery using natural medicinal herbs as a source of novel compounds.     

Optimization of RNA Isolation from Brittle Leaf Disease Affected Date Palm Leaves and Construction of a Subtractive cDNA Library

A simple and efficient method was described here for the isolation of high-quality RNA from date palm leaves affected with Brittle Leaf Disease (BLD) and containing high amount of phenolic compounds. The procedure was based on the use of a non-ionic detergent Nonidet-P40 (NP-40), Polyvinylpyrrolidone (PVP), and beta-mercaptoethanol in the extraction buffer in order to isolate cytoplasmic RNA and to prevent the oxidation of phenolic compounds. This method allowed the isolation of intact RNA, suitable for cDNA synthesis and library construction. Differential screening of the subtractive cDNA library from affected leaf RNA led to the identification of some BLD-induced genes.     

Physico-chemical and biological analysis of true combinatorial libraries

Combinatorial libraries offer new sources of compounds for the research of pharmacological agents such as receptor ligands, enzyme inhibitors or substrates and antibody-binding epitopes. The present review stresses the main roles played by both physico-chemical analysis, particularly when complex mixture of compounds are synthesized as libraries, and biological analysis from which active compounds are identified. After a brief discussion of semantic problems related to the designation of the product mixtures, the physico-chemical analysis of mixtures is reviewed with special emphasis on mass spectrometric techniques. These methods are able both to give a representative view of a library composition and to identify single critical compounds in large libraries. Then the biological screening of such combinatorial libraries is critically discussed with respect to the power and limitations of the methods used for the identification of the active components. Special attention is given to the complex process of library deconvolution. It is pointed out that while combinatorial techniques have evolved towards sophisticated high-tech methods, simple and robust biochemical tests should be used to deconvolute. From a large panel of published examples, a set of trends are identified which should help investigators to choose the most appropriate assay for the discovery of new entities.     

Proteomic analysis of maternal serum in down syndrome: identification of novel protein biomarkers

Down syndrome (DS) is the most prevalent chromosomal disorder, accounting for significant morbidity and mortality. Definitive diagnosis requires invasive amniocentesis, and current maternal serum-based testing requires a false-positive rate of about 5% to detect 85% of affected pregnancies. We have performed a comprehensive proteomic analysis to identify potential serum biomarkers to detect DS. First- and second-trimester maternal serum samples of DS and gestational age-matched controls were analyzed using multiple, complementary proteomic approaches, including fluorescence 2-dimensional gel electrophoresis (2D-DIGE), 2-dimensional liquid chromatography-chromatofocusing (2D-CF), multidimensional protein identification technology (MudPIT; LC/LC-MS/MS), and MALDI-TOF-MS peptide profiling. In total, 28 and 26 proteins were differentially present in first- and second-trimester samples, respectively. Of these, 19 were specific for the first trimester and 16 for the second trimester, and 10 were differentially present in both trimesters. Analysis of MALDI-TOF-MS peptide profiles with pattern-recognition software also discriminated between DS and controls in both trimesters, with an average recognition capability approaching 96%. A majority of the biomarkers identified are serum glycoproteins that may play a role in cellular differentiation and growth of fetus. Further characterization and quantification of these markers in a larger cohort of subjects may provide the basis for new tests for improved DS screening.     

Support vector machines in HTS data mining: Type I MetAPs inhibition study

This article reports a successful application of support vector machines (SVMs) in mining high-throughput screening (HTS) data of a type I methionine aminopeptidases (MetAPs) inhibition study. A library with 43,736 small organic molecules was used in the study, and 1355 compounds in the library with 40% or higher inhibition activity were considered as active. The data set was randomly split into a training set and a test set (3:1 ratio). The authors were able to rank compounds in the test set using their decision values predicted by SVM models that were built on the training set. They defined a novel score PT50, the percentage of the test set needed to be screened to recover 50% of the actives, to measure the performance of the models. With carefully selected parameters, SVM models increased the hit rates significantly, and 50% of the active compounds could be recovered by screening just 7% of the test set. The authors found that the size of the training set played a significant role in the performance of the models. A training set with 10,000 member compounds is likely the minimum size required to build a model with reasonable predictive power.     

Impact of mass spectrometry on combinatorial chemistry

In the past few years, the emergence of combinatorial chemistry has drawn increasing attention and a great deal of analytical research has been centered around this new methodology. These new methods capable of producing vast numbers of samples, which are in many cases highly complex, demand fast and reliable analytical techniques able to provide high quality information concerning sample compositions. Mass spectrometry (MS) is the method of choice to face these analytical challenges. In particular, the introduction of electrospray ionization (ESI and matrix assisted laser desorption/ionization (MALDI)) have been the driving forces for many of the recent innovations, not only within the fields of the biosciences, but also in combinatorial chemistry. These ionization techniques are extremely versatile for the characterization of both single compound collections and compound mixture collections. The high-throughput capabilities, as well as many possible couplings with separation techniques (HPLC, CE) have been thus facilitated. However, mass spectrometry is not only limited to use as an instrument for synthesis control, but also plays an increasing role in the identification of active compounds from complex libraries. Recently, new initiatives for library analysis and screening have arisen from the application of the latest developments in mass spectrometry, Fourier transform ion cyclotron resonance (FTICR).     

An ultraefficient affinity-based high-throughout screening process: application to bacterial cell wall biosynthesis enzyme MurF

The authors describe the discovery of a new class of inhibitors to an essential Streptococcus pneumoniae cell wall biosyn-thesis enzyme, MurF, by a novel affinity screening method. The strategy involved screening very large mixtures of diverse small organic molecules against the protein target on the basis of equilibrium binding, followed by iterative ultrafiltration steps and ligand identification by mass spectrometry. Hits from any affinity-based screening method often can be relatively nonselective ligands, sometimes referred to as "nuisance" or "promiscuous" compounds. Ligands selective in their binding affinity for the MurF target were readily identified through electronic subtraction of an empirically determined subset of promiscuous compounds in the library without subsequent selectivity panels. The complete strategy for discovery and identification of novel specific ligands can be applied to all soluble protein targets and a wide variety of ligand libraries.