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).     

Combinatorial chemistry, automation and molecular diversity: new trends in the pharmaceutical industry

Combinatorial chemistry has emerged as a set of novel strategies for the synthesis of large sets of compounds (combinatorial libraries) for biological evaluation. Within a few years combinatorial chemistry has undergone a series of changes in trends, which are closely related to two important factors in libraries: numbers and quality. While the number of compounds in a library may be easily expressed, it is a lot more difficult to indicate the degree of quality of a library. This degree of quality can be split into two aspects: purity and diversity. The changing trends in combinatorial chemistry with respect to the strategies, the technologies, the libraries themselves (numbers and purity aspects) and the molecular diversity are outlined in this paper.     

A sandwiched microarray platform for benchtop cell-based high throughput screening

The emergence of combinatorial chemistries and the increased discovery of natural compounds have led to the production of expansive libraries of drug candidates and vast numbers of compounds with potentially interesting biological activities. Despite broad interest in high throughput screening (HTS) across varied fields of biological research, there has not been an increase in accessible HTS technologies. Here, we present a simple microarray sandwich system suitable for screening chemical libraries in cell-based assays at the benchtop. The microarray platform delivers chemical compounds to isolated cell cultures by 'sandwiching' chemical-laden arrayed posts with cell-seeded microwells. In this way, an array of sealed cell-based assays was generated without cross-contamination between neighbouring assays. After chemical exposure, cell viability was analyzed by fluorescence detection of cell viability assays on a per microwell basis using a standard microarray scanner. We demonstrate the efficacy of the system by generating four hits from toxicology screens towards MCF-7 human breast cancer cells. Three of the hits were identified in a combinatorial screen of a library of natural compounds in combination with verapamil, a P-glycoprotein inhibitor. A fourth hit, 9-methoxy-camptothecin, was identified by screening the natural compound library in the absence of verapamil. The method developed here miniaturizes existing HTS systems and enables the screening of a wide array of individual or combinatorial libraries in a reproducible and scalable manner. We anticipate broad application of such a system as it is amenable to combinatorial drug screening in a simple, robust and portable platform.     

Separation methods for anthraquinone related anti-cancer drugs

The quinoid anthracycline-related anti-cancer agents represent an important group of anti-tumour drugs with a wide spectrum of activity. We review here some of the separation techniques used for the analysis of anthracyclines and related compounds. In this review we have covered a range of compounds from the early anthracycline antibiotics such as doxorubicin to the more recent anthracenediones and anthrapyrazoles such as mitoxantrone and losoxantrone, respectively. We also include novel compounds such as AQ4N and C1311, both awaiting clinical trial. Separations of the anthraquinone related anti-cancer agents are predominantly by HPLC. These separation techniques have been used for a variety of applications including drug stability, protein binding and therapeutic drug monitoring as well as detailed pharmacokinetic and metabolic studies. Pharmacokinetics, and therefore drug analysis, plays a central role in both the development of new agents and also leads to a better understanding of clinically established agents in this class. Sample preparation and extraction methods including solid-phase and liquid-liquid extraction have also been highlighted. Many anthraquinone related compounds are highly coloured and fluoresce. They are suitable for a range of detection methods including UV-Vis, electrochemical and fluorescence. The methods described are used for sometimes complex separations that are needed for the evaluation of such compounds in biological samples.     

Discovery of new antitumoral and antibacterial drugs from brazilian plant extracts using high throughput screening

Plants have played a significant role in the treatment of cancer and infectious diseases for the last four decades. The discovery and introduction to market of paclitaxel, the vinca alkaloids, etoposide, and many antibacterial drugs support drug discovery programs based on natural products. Natural products have been rediscovered as important tools for drug development despite advances in combinatorial chemistry, due to the complex molecular structures able to interact with mammalian cell targets. The Brazilian flora, the most diverse in the world, has become an interesting spot to prospect for new chemical leads or hits due to its species diversity and associated chemical richness. Screening programs have been established in Brazil as a strategy to identify potentially active substances. High throughput screening techniques allow for the analysis of large numbers of extracts in a relatively short period of time, and can be considered one of the most efficient ways of finding new leads from natural products. An updated review of the current status of the biological screening program is presented and recent results from new antitumoral and antibacterial chemical leads are discussed.     

Methods of screening combinatorial libraries using immobilized or restrained receptors

The screening of combinatorial libraries for compounds with high affinity toward drug receptors is currently a major center of attention. We describe methods recently developed for library screening that involve "constrained" receptors (either immobilized onto a surface or restrained to a compartment by some physical means). These include affinity selection chromatography, ultrafiltration assays, the scintillation proximity assay, a variety of interfacial optical techniques (surface plasmon resonance and its relatives, among others), the quartz crystal microbalance, the jet ring cell, and new interferometric assays using porous silicon to immobilize the receptor. We note some trends in assay development involving assays of membrane-bound complexes, and the coupling of two analytical methods to expand the assay resolution.     

Analyses of compound libraries obtained by high-throughput parallel synthesis: strategy of quality control by high-performance liquid chromatography, mass spectrometry and nuclear magnetic resonance techniques

The growing interest in combinatorial chemistry has led to a new source of compounds from which a large number of leads has emerged over recent years. Parallel synthesis, in particular, allows a quick production of a wide number of individual compounds. A rapid analytical control is needed to determine their quality. A strategy using automated, fast reversed-phase C18 high-performance liquid chromatography with diode-array detection (LC-DAD-MS) followed by atmospheric pressure chemical ionisation mass spectrometry (APCI-MS) and NMR has been developed for their characterisation and purity control. Complementary NMR analyses are done on selected compounds to provide a better structural characterisation of the expected compounds and their potential side-products. Validated libraries are then registered in ISIS databases using automated procedures.     

Solid-phase microextraction for the analysis of biological samples

Solid-phase microextraction (SPME) has been introduced for the extraction of organic compounds from environmental samples. This relatively new extraction technique has now also gained a lot of interest in a broad field of analysis including food, biological and pharmaceutical samples. SPME has a number of advantages such as simplicity, low cost, compatibility with analytical systems, automation and the solvent-free extraction. The last few years, SPME has been combined with liquid chromatography and capillary electrophoresis, besides the generally used coupling to gas chromatography, and has been applied to various biological samples such as, e.g., urine, plasma and hair. The objective of the present paper is a survey of the application of SPME for the analysis of biological samples. Papers about the analysis of biologically active compounds are categorised and reviewed. The impact of SPME on various analytical fields (toxicological, forensic, clinical, biochemical, pharmaceutical, and natural products) is illustrated. The main features of SPME and its modes are briefly described and important aspects about its application for the determination of pharmaceuticals, drugs of abuse and compounds of clinical and toxicological interest are discussed. SPME is compared with other sample pretreatment techniques. The potential of SPME and its main advantages are demonstrated. Special attention is paid to new trends in applications of SPME in bioanalysis.     

The use of combinatorial topographical libraries for the screening of enhanced osteogenic expression and mineralization

Nano- and microstructured surfaces are known to impact on the binding and differentiation of cells, but the detailed basic understanding of the underlying regulatory mechanisms is still scarce, which impedes the rational design of smart biomaterials. Towards a comprehensive analysis of the interplay between topographical parameters such as feature design and lateral and vertical dimensions we here report on a combinatorial screening approach, BioSurface Structure Array (BSSA) of test squares each with a distinct topography. Using such BSSA libraries of 504 topographically distinct surface structures, we have identified combinations of size, gap and height of structures which enhance mineralization as well as the expression of osteogenic markers of a preosteoblastic murine cell line. This generic BSSA screening platform is a versatile technology for the systematic identification of surfaces with specific biological properties, and it may for example be useful for optimizing the design of biomaterials for regulating cellular behaviour.     

Luminescent ultramicroanalysis for the simultaneous determination of biological and chemical substances: the methodology and the ways for solution]

The next revolution in in-vitro bioaffinity assays will be associated with the minimization of the liquid phase of an experiment and with the extremely rapid detection of a large number of samples. The ultra detection unit is one of the essential elements of such devices. The fluorescence detection systems are most promising. The fluorescence detection limit of europium ions is as low as 10(-15) M in the complex with beta-diketone. The authors' approach to designing new highly effective chelates is to synthesize a stable chelate wherein several molecules of beta-diketones are properly oriented and chemically fixed. The molecular mechanic calculation of three dimensional structure of different Eu chelates should be used in optimizing the strategy of chemical synthesis of new compounds. New chelates can be efficiently used for the development of a new approach to measuring distances equal to over 10 nm in the biological systems. Pesticides also represent challenging targets because the levels of compounds are to be detects in minor concentrations, thus the time-resolution fluorescence assay may have some advantages due to its high sensitivity. DNA-based microbiochips are the most promising area in the practical use of a new chelate. A novel europium chelate is a key compound for the development of new methods for studying biological and chemical objects, as well as new diagnostic systems based on a basically new instrumental base that is characterized by extremely high miniaturization.     

Findings on T cell specificity revealed by synthetic combinatorial libraries

Combinatorial libraries and in particular positional scanning synthetic combinatorial libraries (PS-SCL) allow the study of T cell specificity. This is a systematic and unbiased approach that does not require any previous knowledge about the clones to be studied, neither their specificity nor they major histocompatibility complex (MHC) restriction. Two different types of T cell clone ligands can be identified: (1) peptides that do not necessarily correspond to proteins described in the databases, and (2) peptides that are fragments of natural proteins. In this paper, relevant examples of the application of PS-SCL and the deconvolution strategies followed to identify T cell epitopes for clones of known and unknown specificity will be reviewed. Also, important issues like the immunogenicity of such T cell ligands will be discussed.     

Isoprostanes, biomarkers of lipid peroxidation in humans. Part 2: Quantification methods

The isoprostanes are a new class of natural products produced in vivo by a non-enzymatic free-radical-induced peroxidation of polyunsaturated fatty acids. The quantification of these compounds represents a reliable and useful index of lipid peroxidation and oxidant stress in vivo. Then, a large amount of works has been done in the field of isoprostane analysis, but till now, no standardized method seems to emerge. Indeed, described methodologies differ either in the sample preparation steps or in the detection techniques or both. Extraction and purification procedures are often critical and time-consuming, requiring successive chromatographic steps and these procedures lead to a substantial loose of target compounds. Moreover, two main analytical approaches have been adopted for IsoP measurement: immunological methods or mass spectrometry. Some discussion about the methodology used for measurement of isoprostane is important. This review will aim to present and compare different methods developed nowadays for extraction, purification and analysis of F(2)-iPs in various biological samples.     

Chromatographic and capillary electrophoretic methods for the analysis of nicotinic acid and its metabolites

Methods for the assay of nicotinic acid (NiAc) and its metabolites in biological fluids using high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) are reviewed. Most of the references cited in this review concern HPLC methods. A few CE methods that have been recently reported are also included. As these compounds are relatively polar and have a wide range of physico-chemical properties, the sample pre-treatment or clean-up process prior to analysis is included. Most HPLC methods using an isocratic elution system allow determination of a single or few metabolites, but gradient HPLC methods enable simultaneous determination of five to eight compounds. Simultaneous determination of NiAc including many metabolites in a single run can be achieved by CE. We also discuss the pharmacokinetics of NiAc and some of its metabolites.     

X-ray imaging optimization of 3D tissue engineering scaffolds via combinatorial fabrication methods

We have developed a combinatorial method for determining optimum tissue scaffold composition for several X-ray imaging techniques. X-ray radiography and X-ray microcomputed tomography enable non-invasive imaging of implants in vivo and in vitro. However, highly porous polymeric scaffolds do not always possess sufficient X-ray contrast and are therefore difficult to image with X-ray-based techniques. Incorporation of high radiocontrast atoms, such as iodine, into the polymer structure improves X-ray radiopacity but also affects physicochemical properties and material performance. Thus, we have developed a combinatorial library approach to efficiently determine the minimum amount of contrast agent necessary for X-ray-based imaging. The combinatorial approach is demonstrated in a polymer blend scaffold system where X-ray imaging of poly(desaminotyrosyl-tyrosine ethyl ester carbonate) (pDTEc) scaffolds is improved through a controlled composition variation with an iodinated-pDTEc analog (pI(2)DTEc). The results show that pDTEc scaffolds must include at least 9%, 16%, 38% or 46% pI(2)DTEc (by mass) to enable effective imaging by microradiography, dental radiography, dental radiography through 0.75cm of muscle tissue or microcomputed tomography, respectively. Only two scaffold libraries were required to determine these minimum pI(2)DTEc percentages required for X-ray imaging, which demonstrates the efficiency of this new combinatorial approach for optimizing scaffold formulations.     

Computer analysis of toxicological data bases: mutagenicity of aromatic amines in Salmonella tester strains

There is an increasing problem in the world of toxicological evaluation in that, while test results of new compounds are appearing regularly, traditional methods of analysis of such data are cumbersome and slow. The new computer program CASE (computer automated structure evaluator) was designed to handle just such problems. It analyzes molecules and their associated biological activity on the basis of structural fragments found and identified by the program as being important for the activity based on statistical tests of significance. The program was used to examine mutagenicity in Salmonella typhimurium strains TA98 and TA100 (with S9 activation) of approximately 80-100 aromatic amines. The resulting structural features were then used in a predictive fashion to test the expected mutagenic properties of a smaller set of about 20 compounds.     

Application of evaporative light scattering detection to the characterization of combinatorial and parallel synthesis libraries for pharmaceutical drug discovery

The advent of combinatorial and parallel synthesis methodologies in drug discovery have necessitated the development of analytical techniques which permit high throughput quantitative analysis of mixtures of small organic molecules. High pressure liquid chromatography with evaporative light scattering detection has become the major tool for this task. In this article we briefly review the theory of evaporative light scattering detection and the design of commercial instruments, as well as discuss the operational constraints imposed by the exigency of analyzing en masse the product libraries generated by these new drug discovery methods. The application of evaporative light scattering detection to library analysis is illustrated using examples from our library synthesis program. Complemented by ultraviolet absorbance detection for purity assessment and mass spectrometry for product identification, evaporative light scattering detection is the only technique affording sufficient accuracy and sensitivity for high throughput library analysis.     

Isolation of antibodies which neutralise the activity of early pregnancy factor

Early pregnancy factor (EPF) is a secreted protein with growth regulatory and immunomodulatory properties. It functions as an autocrine growth factor for tumour cells and as an autocrine or paracrine growth factor for regenerating normal cells. Anti-EPF antibodies have demonstrable anti-tumour activity and, as a result, hybridomas which produce such antibodies are unstable. In this study, the phage display antibody techniques have been investigated as a means of producing recombinant anti-EPF antibodies. Mice were immunised with synthetic peptides which correspond to the N or C terminal regions of EPF, and their splenic tissue was used to make combinatorial antibody libraries. The Fab repertoire was displayed on the surface of phage and panned over recombinant EPF. Reactive Fabs were identified by ELISA and their binding was characterised by BIAcore analysis and functional studies. Three libraries with a size of greater than 5x10(7)cfu were constructed and a total of 26 unique Fabs with specific reactivity against EPF were identified. Three Fabs were purified and of these one demonstrated strong EPF neutralising activity, one had intermediate activity and the other was not neutralising. Phage display has provided the means of circumventing the problems of anti-EPF hybridoma development and has resulted in the production of antibodies with potential applications in the diagnosis of pregnancy and the diagnosis and therapy of cancer.     

Functional characterization of the NS2B/NS3 protease complex from seven viruses belonging to different groups inside the genus Flavivirus

The genus Flavivirus, family Flaviviridae, comprises more than 70 viruses. Many of them cause severe, potentially fatal, human diseases. Human vaccines are available for only three viruses and no effective antiviral drug is available. In order to limit the consequences of infections with flaviviruses, a promising approach consists in developing specific compounds that target the virus-encoded NS2B/NS3 protease complex, which is crucial for the viral polyprotein processing. In order to develop such compounds active as antiviral drugs against several flaviviruses, identification of biochemical properties shared by proteases from different viruses is essential.

In this work, the functional similarity between the proteases from seven flaviviruses belonging to different major groups was addressed by characterizing their enzymatic properties. For each virus, a catalytically active recombinant protease was designed and expressed as a hexahistidine-tagged protein. Chromogenic and fluorogenic substrates were used to identify optimal conditions for proteolysis. Our study identified important physico-chemical properties shared by all the seven proteases we studied (high pH value requirement for optimal activity, inhibition of substrate processing by salt). However, it also evidenced slight differences in biochemical properties of the flaviviral proteases, which could sustain heterogeneous sensitivity to future inhibitors.     

Recombinant antibodies to small analytes and prospects for deriving them from synthetic combinatorial libraries

During the past 4 years, several laboratories have developed new methods of cloning antibody combining site genes from hybridomas or B‐lymphocytes, and functionally expressing them in bacteria, yeast, mammalian cells or plants. At least three research groups have also constructed ‘antibody display’ libraries with vastly diverse sequence permutations of combining site genes in bacteriophage. These semi‐synthetic combinatorial libraries present an antibody repertoire orders of magnitude greater than that accessed by conventional hybridoma technology. They may yield antibodies not obtainable through conventional immunization. We have cloned the immunoglobulin genes from a hybridoma specific for the phenylurea herbicide, diuron, into a phage display vector. The cloned antibody fragments (Fabs) were as sensitive as the parent monoclonal antibody for detecting free diuron in competition enzyme immunoassays. We also derived diuron hapten‐specific clones from synthetic combinatorial phage libraries, but only a few weakly recognized free diuron. The relative merits of deriving Fabs from synthetic phage display libraries, and the steps in engineering new specificities and other properties into recombinant antibodies, are discussed.     

The applications of FISH in tumor pathology

The current FISH technology was greatly improved during the past 10 years. A large number of cosmids and yeast (YACs), bacterial (BACs), phage P1 derived (PACs) artificial chromosomes have been rapidly mapped and are useful as probes. In parallel, methods were established to specifically "paint" entire chromosomes or chromosome segments. Using these chromosome libraries as probes, complex rearrangements and marker chromosomes can be identified irrespective of their banding pattern. Ripetitive DNA probes specific for each chromosome centromere (alpha satellite sequences), are also available and may be used to identify specific aneuploidies. The use of sensitive digital imaging systems on the basis of "colour" rather than morphology increased the improvement of new FISH techniques. In particular, colour karyotyping results in the differential colour display of all human chromosomes. Another recent development of FISH technology is comparative genome hybridization (CGH), a genome-scanning technique that allows to identify and map chromosomal and subchromosomal gains and losses. FISH techniques may be used to investigate chromosome abnormalities not only on metaphasic chromosomes but also on interphasic nuclei. Any given tissue or cell source, such as sections of frozen tumors, imprinted cells, cultured cells, paraffin-embedded sections may be hybridized. The interphasic FISH may be extremely informative in tumor pathology even if the results are dependent on a good technical quality and adequate controls.