Development of an analytical platform for the affinity screening of natural extracts by SEC-MS towards PPARα and PPARγ receptors.

BACKGROUND: Peroxisome proliferator-activated receptors (PPARs) belong to the superfamily of nuclear receptors and represent the targets for the therapeutical treatment of type 2 diabetes, dyslipidemia and hyperglycemia associated with metabolic syndrome. Some medicinal plants have been traditionally used to treat this kind of metabolic diseases. Today only few drugs targeting PPARs have been approved and for this reason, the rapid identification of novel ligands and/or chemical scaffolds starting from natural extracts would benefit of a selective affinity ligand fishing assay. RESULTS: In this paper we describe the development of a new ligand fishing assay based on size exclusion chromatography (SEC) coupled to LC-MS for the analysis of complex samples such as botanical extracts. The known PPARα and PPARγ ligands, WY-14643 and rosiglitazone respectively, were used for system development and evaluation. The system has found application on an Allium lusitanicum methanolic extract, containing saponins, a class of chemical compounds which have attracted interest as PPARs ligands because of their hypolipidemic and insulin-like properties. SIGNIFICANCE: A new SEC-AS-MS method has been developed for the affinity screening of PPARα and PPARγ ligands. The system proved to be highly specific and will be used to improve the throughput for the identification of new selective metabolites from natural souces targeting PPARα and PPARγ.

Development of an integrated chromatographic system for ω-transaminase-IMER characterization useful for flow-chemistry applications.

An integrated chromatographic system was developed to rapidly investigate the biocatalytic properties of ω-transaminases useful for the synthesis of chiral amines. ATA-117, an (R)-selective ω-transaminase was selected as a proof of concept. The enzyme was purified and covalently immobilized on an epoxy monolithic silica support to create an immobilized enzyme reactor (IMER). Reactor efficiency was evaluated in the conversion of a model substrate. The IMER was coupled through a switching valve to an achiral analytical column for separation and quantitation of the transamination products. The best conditions of the transaminase-catalyzed bioconversion were optimized by a design of experiments (DoE) approach. The production of (R)-1-(4-methoxyphenyl)propan-2-amine and (R)-1-methyl-3-phenylpropylamine, intermediates for the synthesis of the bronchodilator formoterol and the antihypertensive dilevalol respectively, was achieved in the presence of different amino donors. The enantiomeric excess (ee) was determined off-line by developing a derivatization procedure using Nα-(2,4-dinitro-5-fluorophenyl)-L-alaninamide reagent. The most satisfactory conversion yields were 60% for (R)-1-(4-methoxyphenyl)propan-2-amine and 29% for (R)-1-methyl-3-phenylpropylamine, using isopropylamine as amino donor. The enantiomeric excess of the reactions were 84%R and 99%R, respectively.

Application of a rapid HILIC-UV method for synthesis optimization and stability studies of immunogenic neo-glycoconjugates.

Proteins and glycoproteins with therapeutic activity are susceptible to environmental factors, which can cause their degradation and the loss of their activity. Thus, the maintenance of their stability during the production process is a critical factor. In this work, a simple and rapid hydrophilic interaction liquid chromatography HILIC-UV method was validated in terms of accuracy, precision, linearity, LOD, LOQ and specificity and applied to the investigation of the stability of intact proteins and their neo-glycoconjugates with antigenic activity against tuberculosis. The method proved to be suitable for the estimation of the degradation of the proteins under critical conditions (i.e. freeze-thaw cycles) and for the monitoring of their coupling reaction with saccharidic moieties, without the need of sample preparation. In addition, the chromatographic analysis allowed to calculate the yields of the protein glycosylation reaction.

Resveratrol and its metabolites bind to PPARs.

Resveratrol, a modulator of several signaling proteins, can exert off-target effects involving the peroxisome proliferator-activated receptor (PPAR) transcription factors. However, evidence for the direct interaction between this polyphenol and PPARs is lacking. Here, we addressed the hypothesis that resveratrol and its metabolites control aspects of PPAR transcriptional activity through direct interaction with PPARs. Bioaffinity chromatographic studies with the immobilized ligand-binding domains (LBDs) of PPARγ and PPARα and isothermal titration calorimetry allowed the binding affinities of resveratrol, resveratrol 3-O-glucuronide, resveratrol 4-O-glucuronide, and resveratrol 3-O-sulfate to both PPAR-LBDs to be determined. Interaction of resveratrol, resveratrol 3-O-glucuronide, and resveratrol 4-O-glucuronide with PPARγ-LBD occurred with binding affinities of 1.4, 1.1, and 0.8 µM, respectively, although only resveratrol bound to the PPARα-LBD with a binding affinity of 2.7 µM. Subsequently, X-ray crystallographic studies were carried out to characterize resveratrol binding to the PPARγ-LBD at the molecular level. The electron density map from the crystal structure of the complex between PPARγ-LBD and resveratrol revealed the presence of one molecule of resveratrol bound to the LBD of PPARγ, with the ligand occupying a position close to that of other known PPARγ ligands. Transactivation assays were also performed in HepG2 cells, with the results showing that resveratrol was not a PPAR agonist but instead was able to displace rosiglitazone from PPARγ and Wy-14643 from PPARα with IC50 values of (27.4±1.8) µM and (31.7±2.5) µM, respectively. We propose that resveratrol acts as a PPAR antagonist through its direct interaction with PPARγ and PPARα.

Isolation and characterization of bioactive compounds from plant resources: the role of analysis in the ethnopharmacological approach.

The phytochemical research based on ethnopharmacology is considered an effective approach in the discovery of novel chemicals entities with potential as drug leads. Plants/plant extracts/decoctions, used by folklore traditions for treating several diseases, represent a source of chemical entities but no information are available on their nature. Starting from this viewpoint, the aim of this review is to address natural-products chemists to the choice of the best methodologies, which include the combination of extraction/sample preparation tools and analytical techniques, for isolating and characterizing bioactive secondary metabolites from plants, as potential lead compounds in the drug discovery process. The work is distributed according to the different steps involved in the ethnopharmacological approach (extraction, sample preparation, biological screening, etc.), discussing the analytical techniques employed for the isolation and identification of compound/s responsible for the biological activity claimed in the traditional use (separation, spectroscopic, hyphenated techniques, etc.). Particular emphasis will be on herbal medicines applications and developments achieved from 2010 up to date.

Open tubular columns containing the immobilized ligand binding domain of peroxisome proliferator-activated receptors α and γ for dual agonists characterization by frontal affinity chromatography with mass spectrometry detection.

The peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptor superfamily. In the last years novel PPARs ligands have been identified and these include PPARα/γ dual agonists. To rapidly identify novel PPARs dual ligands, a robust binding assay amenable to high-throughput screening toward PPAR isoforms would be desirable. In this work we describe a parallel assay based on the principles of frontal affinity chromatography coupled to mass spectrometry (FAC-MS) that can be used to characterize dual agonists. For this purpose the ligand binding domain of PPARα receptor was immobilized onto the surface of open tubular capillaries to create new PPAR-alpha-OT columns to be used in parallel with PPAR-gamma-OT columns. The two biochromatographic systems were used in both ranking and Kd experiments toward new ureidofibrate-like dual agonists for subtype selectivity ratio determination. In order to validate the system, the Kd values determined by frontal analysis chromatography were compared to the affinity constants obtained by ITC experiments. The results of this study strongly demonstrate the specific nature of the interaction of the ligands with the two immobilized receptor subtypes.

New monolithic chromatographic supports for macromolecules immobilization: challenges and opportunities.

This mini-review reports on some recent advances in the field of immobilized protein employing both silica and polymer-based monoliths as supports, and their application in affinity chromatography and immobilized enzyme reactors (IMERs) developments. The major emphasis is put on some interesting challenges and opportunities related to the development of new monolithic affinity supports based on biofriendly sol-gel inorganic monoliths with entrapped proteins and on organic monolithic supports with improved hydrophilicity for IMERs development in proteomic studies. The ease of preparation of monoliths and the multitude of functionalization techniques, make monoliths interesting for an increasing number of biochemical and medical applications.

Frontal affinity chromatography with MS detection of the ligand binding domain of PPARγ receptor: ligand affinity screening and stereoselective ligand-macromolecule interaction.

In this study we report the development of new chromatographic tools for binding studies based on the gamma isoform ligand binding domain (LBD) of peroxisome proliferator-activated receptor (PPARγ) belonging to the nuclear receptor superfamily of ligand-activated transcription factors. PPARγ subtype plays important roles in the functions of adipocytes, muscles, and macrophages with a direct impact on type 2 diabetes, dyslipidemia, atherosclerosis, and cardiovascular disease. In order to set up a suitable immobilization chemistry, the LBD of PPARγ receptor was first covalently immobilized onto the surface of aminopropyl silica particles to create a PPARγ-Silica column for zonal elution experiments and then onto the surface of open tubular (OT) capillaries to create PPARγ-OT capillaries following different immobilization conditions. The capillaries were used in frontal affinity chromatography coupled to mass spectrometry (FAC-MS) experiments to determine the relative binding affinities of a series of chiral fibrates. The relative affinity orders obtained for these derivatives were consistent with the EC(50) values reported in literature. The optimized PPARγ-OT capillary was validated by determining the K(d) values of two selected compounds. Known the role of stereoselectivity in the binding of chiral fibrates, for the first time a detailed study was carried out by analysing two enantioselective couples on the LBD-PPARγ capillary by FAC and a characteristic two-stairs frontal profile was derived as the result of the two saturation events. All the obtained data indicate that the immobilized form of PPARγ-LBD retained the ability to specifically bind ligands.

Immobilized trypsin on epoxy organic monoliths with modulated hydrophilicity: novel bioreactors useful for protein analysis by liquid chromatography coupled to tandem mass spectrometry.

The development of epoxy organic monoliths with modulated hydrophilicity for the preparation of novel trypsin-based microreactors is reported. Porous polymer monoliths have been prepared using methacrylate chemistry triggered by γ-ray irradiation. In situ polymerization has been optimized and extended to medium and high polymer densities using glycidyl methacrylate (GMA) as reactive monomer as well as to the hydrophilic nature of the co-monomers (glyceryl monomethacrylate, GlyMA and acrylamide, AMD). Enzyme immobilization was smoothly achieved by passing a buffered trypsin solution through the columns kept at room temperature. The activities of the immobilized enzyme were characterized by the apparent Michaelis constant (K(m)) and the apparent maximum velocity (V(max)) of the reaction using a non chromogenic, low-molecular mass substrate N-α-benzoyl-l-arginine ethyl ester (BAEE). For the kinetic constants determination a new off-line chromatographic procedure was developed on purpose. The most efficient IMERs were obtained by immobilizing trypsin on monolithic skeleton prepared with hydrophilic monomers (GlyMA and AMD). One of the most promising bioreactor was applied to the digestion of model proteins with different molecular weight and complexity such as human serum albumin (HSA), ß-casein and ribonuclease B (RNase B), and the produced peptides were analyzed by liquid chromatography-mass spectrometry. Using a digestion time of only 25 min the proteins were recognized by the database with satisfactory sequence coverage, which was 78.22, 49.76 and 80.68% for HSA, ß-casein and RNase B, respectively.

Frontal affinity chromatography in characterizing immobilized receptors.

The state-of-the-art in frontal affinity chromatography (FAC) applied to receptor of pharmaceutical interest is here reported. This review will first discuss the principles of FAC for ligand characterization (K(d) determination) and for screening studies, and will examine the different strategies that have been followed for the immobilization of a broad range of receptors (cytosolic and membrane receptors). Several reported applications will then be presented demonstrating that FAC is an interesting tool enabling convenient and efficient screening in the identification of new potential ligands. Moreover new applications of FAC including dual binding site assay, receptor subtype characterization, and multi-receptor binding experiments will be underlined.

Penicillin G acylase as chiral selector in LC and CE: exploring the origins of enantioselectivity.

The review examines the most recent achievement of immobilized Penicillin G acylase (PGA) as chiral stationary phases (PGA-CSP) for the separation of acidic enantiomers. Particular attention is paid to the influence of structural variations of a large number of analytes on retention and enantioselectivity with a special emphasis on advances in the elucidation of the chiral recognition mechanism. Docking calculations and molecular dynamic simulations are discussed to rationalize the origins of enantioselective behaviour. The review also touches the chiral behaviour of PGA in partial filling CE. The CE results, obtained using PGA in free solution, suggest that the immobilization procedure used in the development of PGA-CSPs did not alter the enantioselective properties of the enzyme.

Determination of glycine and threonine in topical dermatological preparations.

In the present study, a single HPLC method was developed for the determination of glycine and threonine in cicatrizants. Two different preparations of a cream and an ointment, and the corresponding bandages, onto which the formulations were applied, were studied. The method involved matrix solubilisation with dichloromethane, liquid-liquid isolation of gly and thr with aqueous 1N NaOH, and derivatization with phenylisothiocyanate. Reversed-phase HPLC separation was carried out by gradient elution with 20mM aqueous NaClO4 and acetonitrile (from 90% to 30% aqueous NaClO4 in 10 min) on a LiChrospher 100 RP-18 cartridge (125 mm x 4.6 mm). Analytes were determined with a UV detector set at 245 nm. Quantitation was accomplished by internal standardization with methionine. Linearity was studied in the range 60-120% of the concentrations expected for gly and thr (viz. for gly from 200 to 400 microgml(-1), and for thr from 100 to 200 microgml(-1)). In reference aqueous samples, linear correlation (r) was better than 0.99 for gly and thr, while in spiked matrix samples r ranged from 0.97 to 0.98. Recoveries were in the 95-105% interval, and precision (CV%, N=6) was better than 5% for both analytes either in cream, ointment or bandages. The method was successfully used for the quality control of topical dermatological preparations.

Development of an integrated chromatographic system for on-line digestion and characterization of phosphorylated proteins.

The development of an integrated chromatographic system for complete phosphoprotein analysis is described. The digestion of phosphoproteins with trypsin- or pronase-based monolithic bioreactors is carried out on-line with selective enrichment on a TiO(2) trap and separation of the produced phosphopeptides by reversed-phase liquid chromatography-multiple mass spectrometry (RPLC/MS(n)). A detailed study on the selective extraction of peptides with different degrees of phosphorylation on TiO(2) cartridges is discussed. This analytical strategy has been optimized using beta-casein as a standard phosphoprotein, and then applied to the identification of phosphorylation sites in insulin-like grow factor-binding protein 1 (IGFBP-1) isolated from amniotic fluid.

Development and integration of an immunoaffinity monolithic disk for the on-line solid-phase extraction and HPLC determination with fluorescence detection of aflatoxin B1 in aqueous solutions.

The development and characterization of an anti-aflatoxin B1 (anti-AFB1) immunoaffinity monolithic disk is reported. Polyclonal anti-AFB1 was covalently immobilized in batch on an epoxy-activated monolithic Convective Interaction Media (CIM) disk (12 mm x 3 mm i.d.) by a one-step reaction via epoxy groups of the polymer surface. 0.96 mg of antibody were immobilized and the binding capacity of the CIM disk was determined by frontal analysis. The CIM disk was coupled through a switching valve to a reversed-phase column, namely Chromolith Performance RP-18e. A fully automated HPLC method with fluorescence detection for the determination of aflatoxin B1 in aqueous solution was developed. The total analysis time with the integrated system is 46 min and the retention time of AFB1 is approximately 29 min. The binding capacity of the immunoaffinity disk was evaluated in terms of linearity, precision and accuracy of the extraction procedure. The immunoaffinity support was stable after repeated runs.

Optimization of a trypsin-bioreactor coupled with high-performance liquid chromatography-electrospray ionization tandem mass spectrometry for quality control of biotechnological drugs.

The optimization of a silica-based trypsin bioreactor and its use in the quality control of biotechnological drugs like peptides and proteins is described. Five bioreactors based on monolithic material have been prepared, with different amount of bound trypsin. The performances of these bioreactors were compared to the proteolytic activity of a bioreactor based on silica material. The trypsin-based chromatographic columns were coupled on-line with an LC/ESI/MS/MS system for digestion and identification of proteins. First, human serum albumin has been used as test protein to compare the ability of the bioreactors to hydrolyse high-molecular-weight proteins. The best chromatographic material (epoxy monolithic silica) and the optimum amount of enzyme bound (7.13 mg) have been identified to obtain the highest protein recovery and an analytical reproducibility of the whole digestion, separation and identification process. The optimized enzyme-reactor has been used for the on-line digestion of some biotechnological drugs such as somatotropin. Somatotropin for parentheral use has been analyzed, without sample pre-treatment, with both an on-line procedure and the traditional off-line procedure described in the European Pharmacopoeia. It was found that the cleavage efficiency (aminoacidic recovery, %AA) achieved within minutes by the developed protocol is at least comparable or even better than the conventional 4h consuming method.

Effect of hippuric acid on the gaschromatographic retention of S-phenylmercapturic acid.

S-phenylmercapturic acid (PMA) is one specific urinary biomarker of low-level benzene exposure. It is used for biological monitoring of benzene-exposed workers in the petrochemical industry and normally ranges from non-measurable to 10 microg/l levels in non-exposed non-smoking subjects. Benzene-exposure caused by workplace or lifestyle sources is frequently accompanied by toluene exposure, which can cause the occurrence of high levels (from 10 mg/l to more than 2000 mg/l) of hippuric acid (HA) in urine. Both solvents are toxic, and benzene is classified as a human carcinogen. The biological monitoring of benzene and toluene is therefore required for preventive care of exposed workers health. In this study a GC-MS method was adopted for measuring urinary PMA, which involved liquid-liquid extraction (LLE) with ethyl acetate from acidified urine and esterification with 0.5 N hydrochloric acid in methanol. The method evidenced a GC effect in a conventional HP-5 (30 m x 0.25 mm i.d., 0.25 microm film-thickness) methyl-phenylsilicone capillary column produced by HA on PMA. The results demonstrate that HA at concentrations as low as 250 mg/l can delay the elution of PMA and labelled internal standard from the column. The recognition and discussion of this particular GC phase soaking effect may be of help for those who are occupied in the determination of PMA and of urinary acidic metabolites by GC.

Development of a bioreactor based on trypsin immobilized on monolithic support for the on-line digestion and identification of proteins.

The preparation and characterization of a new trypsin-based bioreactor is here described for on-line protein digestion and peptide analysis. Trypsin was immobilized on an epoxy-modified silica monolithic support with a single reaction step and the amount of immobilized enzyme was found to be 66.07 mg (+/-11.75 S.D.)/column (n = 6). The bioreactor was coupled through a switching valve to an analytical column for the on-line digestion, peptide separation and identification of test proteins by ESI-MS-MS. The influence of various parameters (flow rate, temperature, buffer pH and molarity, etc.) on enzymatic activity was investigated by an experimental design and the mostly significant factor was found to be the flow rate. The efficacy of the reported on-line bioreactor for tryptic mapping is reported for somatostatin and myoglobin, selected as model compounds. Tryptic peptide maps obtained by on-line digestion of myoglobin were compared to those obtained by traditional off-line digestion. Sequence coverage obtained with the on-line protocol (21 peptides, 75.16% coverage of myoglobin sequence) was found to be comparable to the one obtained with the off-line protocol (18 peptides, 76.47% coverage). Sensitivity for myoglobin digestion and identification was 0.1 mg/ml. The reproducibily of the peptide maps in terms of retention time was from 1.53 to 4.31%, R.S.D.

Development of a chromatographic bioreactor based on immobilized beta-glucuronidase on monolithic support for the determination of dextromethorphan and dextrorphan in human urine.

We here reported the development and application of an immobilized enzyme reactor (IMER) based on beta-glucuronidase to the on-line determination of urinary molar ratios of dextromethorphan (DOMe)/dextrorphan (DOH) for the assessment of the metabolic activity of CYP2D6, a genetically variable isoform of cytochrome P-450 (CYP). beta-Glucuronidase was immobilized on an HPLC monolithic aminopropyl silica support. Catalytic activity and stability of the chromatographic reactor were evaluated using 8-hydroxyquinoline glucuronide (8-HQG) as substrate. The IMER was coupled through a switching valve to a reversed-phase column (C8) for the simultaneous determination of dextromethorphan and its main metabolite dextrorphan. On purpose a selective reversed-phase ion pair HPLC method coupled with fluorescence detection was developed. Urine samples were first centrifuged to remove insoluble materials and then aliquots of the supernatants were injected into the coupled-column analyser. Linearity, precision and accuracy of the method were established. The method reliability was verified by comparing our data with previous data of a phenotyping study carried out by the Poison Control Centre of Pavia-Clinical Toxicology Division.

Penicillin G acylase-based stationary phases: analytical applications.

A review of Penicillin G Acylase (PGA)-based stationary phases is given, focusing on immobilisation methods, selection of immobilisation material and applications in chiral liquid chromatography. Two immobilization methods, namely "in situ" and "in batch" techniques, are described for the immobilisation of PGA on silica supports. Microparticulate and monolithic silica, both functionalized with aminopropyl- and epoxy-groups, were used in the development of the PGA immobilised enzyme reactor (IMER). The best results, in terms of PGA immobilised amount and enzyme activity, were obtained with the "in situ" immobilisation on epoxy monolithic silica. The use of PGA columns as enzyme reactors for the preparation of 6-APA and for the production of enantiomeric pure drugs in a one-step reaction in described. The review also covers the application of PGA-columns as chiral stationary phases for the separation of acidic enantiomers. An on-line chromatographic system based on the PGA-IMER combined with a switching valve to an analytical column is also described as a highly efficient tool to study the enantioselective hydrolyses properties of PGA. Finally a molecular modelling study is reported with the aim to give more insights into PGA-substrates interactions and to expand the application of these stationary phases as a chiral biocatalysts for pharmaceutical processes.

Evaluation of a monolithic epoxy silica support for penicillin G acylase immobilization.

In this work, a new type of penicillin G acylase (PGA)-based monolithic silica support was developed and evaluated for the chiral separation in HPLC. The preparation procedure consisted of two steps: preparation of an epoxy derivatized monolithic silica column and chemical modification of the epoxide groups with the enzyme chiral selector. The epoxy Silica-Rod column for the immobilization of PGA was prepared with the in situ modification process by using epoxy-silanes and the identification of the species bound to the surface was achieved by solid-state nuclear magnetic resonance. The enzyme was covalently immobilized to the surface of the derivatized monolithic column. The enantioselectivity and the performance of the developed column are discussed and compared to the corresponding experimental data obtained with a PGA-based microparticulate (5 microm) silica column.