Natural Products in High Throughput Screening: Automated High-Quality Sample Preparation

At present, compound libraries from combinatorial chemistry are the major source for high throughput screening (HTS) programs in drug discovery. On the other hand, nature has been proven to be an outstanding source for new and innovative drugs. Secondary metabolites from plants, animals, and microorganisms show a striking structural diversity that supplements chemically synthesized compounds or libraries in drug discovery programs. Unfortunately, extracts from natural sources are usually complex mixtures of compounds, often generated in time-consuming and, for the most part, manual processes. Because quality and quantity of the provided samples play a pivotal role in the success of HTS programs, this poses serious problems. In order to make samples of natural origin competitive with synthetic compound libraries, we devised a novel, automated sample preparation procedure based on solid-phase extraction (SPE). By making use of modified Zymark (Hopkinton, MA) RapidTrace? SPE workstations, we developed an easy-to-handle and effective fractionation method that generates high-quality samples from natural origin, fulfilling the requirements for an integration in high throughput drug discovery programs.     

Mass spectrometric analysis in combinatorial chemistry.

The continuing success of the combinatorial approach is heavily reliant on analytical methodologies, which allow for the rapid and accurate characterisation of medicinally relevant molecules from compound libraries. Mass spectrometry has recently been touted as the most suitable tool for a range of combinatorial applications such as structural elucidation and screening. The refinement of conventional methods, developments of techniques such as Fourier transform ion cyclotron resonance and new screening methodologies have allowed the medicinal chemist to tackle the growing analytical challenges posed by combinatorial chemistry.     

Qualitative screening for basic drugs in autopsy liver samples by dual-plate overpressured layer chromatography

An overpressured layer chromatography (OPLC) method was evaluated for broad-scale screening of basic drugs in 5g autopsy liver samples using two parallel OPLC systems. Sample preparation included enzymatic digestion with trypsin and liquid-liquid extraction with butyl chloride. Chromatographic separation was performed as dual-plate analysis, with mobile phases composed of trichloroethylene-methylethylketone-n-butanol-acetic acid-water (17:8:25:6:4, v/v) (OPLC1), and butyl acetate-ethanol (96.1%)-tripropylamine-water (85:9.25:5:0.75, v/v). Identification was based on automated comparison of corrected R(f) values (hR(f)c) and in situ UV spectra with library values by dedicated software. The identification limit was determined for 25 basic drugs in liver ranging from 0.5 to 10mg/kg. The OPLC method proved to be well suited for routine screening analysis of basic drugs in post-mortem samples of varying quality, combining the benefit from moderately high separation power with the ease of disposable plates.     

Solid- and solution-phase synthesis of highly-substituted-pyrrolidine libraries.

Starting from a complex bicyclic beta-lactam scaffold we have demonstrated the possible production of libraries of a new class of drug-like, highly substituted pyrrolidines. The choice of the type of substitution was made by optimizing various synthetic routes. The selection of each compound is the result of a filtration of a large virtual combinatorial chemical space, using simple criteria. The access to these complex pyrrolidines needed only four to six synthetic steps.     

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).     

Chemical biology of dynamic combinatorial libraries

Dynamic combinatorial chemistry (DCC) is a recently introduced supramolecular approach to generate libraries of chemical compounds based on reversible exchange processes. The building elements are spontaneously and reversibly assembled to virtually encompass all possible combinations, allowing for simple one-step generation of complex libraries. The method has been applied to a variety of combinatorial systems, ranging from synthetic models to materials science and drug discovery, and enables the establishment of adaptive processes due to the dynamic interchange of the library constituents and its evolution toward the best fit to the target. In particular, it has the potential to become a useful tool in the direct screening of ligands to a chosen receptor without extensive prior knowledge of the site structure, and several biological systems have been targeted. In the vast field of glycoscience, the concept may find special perspective in response to the highly complex nature of carbohydrate-protein interactions. This chapter summarises studies that have been performed using DCC in biological systems, with special emphasis on glycoscience.     

High-throughput characterization and quality control of small-molecule combinatorial libraries

To fully realize the potential of combinatorial synthesis and high-throughput screening for increasing the efficiency of the drug discovery and development process, issues related to compound purity must be addressed. Impurities, often present after synthesis, can lead to ambiguous screening results and inhibit the development of quality structure-activity relationships. The demand for high-throughput analytical characterization of combinatorial libraries has prompted the development of more rapid methods to keep pace with compound production. Recent progress has focused upon the development of parallel separation methods, multiplexed detector interfaces, and synergistic combinations of different detectors possessing complementary selectivities.     

Discovering novel ligands for macromolecules using X-ray crystallographic screening

The need to decrease the time scale for clinical compound discovery has led to innovations at several stages in the process, including genomics/proteomics for target identification, ultrahigh-throughput screening for lead identification, and structure-based drug design and combinatorial chemistry for lead optimization. A critical juncture in the process is the identification of a proper lead compound, because a poor choice may generate costly difficulties at later stages. Lead compounds are commonly identified from high-throughput screens of large compound libraries, derived from known substrates/inhibitors, or identified in computational prescreeusing X-ray crystal structures. Structural information is often consulted to efficiently optimize leads, but under the current paradigm, such data require preidentification and confirmation of compound binding. Here, we describe a new X-ray crystallography-driven screening technique that combines the steps of lead identification, structural assessment, and optimization. The method is rapid, efficient, and high-throughput, and it results in detailed crystallographic structure information. The utility of the method is demonstrated in the discovery and optimization of a new orally available class of urokinase inhibitors for the treatment of cancer.     

One-bead–one-structure combinatorial libraries

Combinatorial libraries employing the one-bead–one-compound technique are reviewed. Two distinguishing features characterize this technique. First, each compound is identified with a unique solid support, enabling facile segregation of active compounds. Second, the identity of a compound on a positively reacting bead is elucidated only after its biological relevance is established. Direct methods of structure identification (Edman degradation and mass spectroscopy) as well as indirect “coding” methods facilitating the synthesis and screening of nonpeptide libraries are discussed. Nonpeptide and “scaffold” libraries, together with a new approach for the discovery of a pentide binding motif using a “library of libraries,” are also discussed. In addition, the ability to use combinatorial libraries to optimize initially discovered leads is illustrated with examples using peptide libraries. © 1994 John Wiley & Sons, Inc.     

Affinity Selection and Mass Spectrometry-Based Strategies to Identify Lead Compounds in Combinatorial Libraries

The screening of diverse libraries of small molecules created by combinatorial synthetic methods is a recent development which has the potential to accelerate the identification of lead compounds in drug discovery. We have developed a direct and rapid method to identify lead compounds in libraries involving affinity selection and mass spectrometry. In our strategy, the receptor or target molecule of interest is used to isolate the active components from the library physically, followed by direct structural identification of the active compounds bound to the target molecule by mass spectrometry. In a drug design strategy, structurally diverse libraries can be used for the initial identification of lead compounds. Once lead compounds have been identified, libraries containing compounds chemically similar to the lead compound can be generated and used to optimize the binding characteristics. These strategies have also been adopted for more detailed studies of protein–ligand interactions.     

Peptide-binding antibiotics: A solid-phase assay for screening libraries of vancomycin mimics for selective d-Ala-d-Ala binding

Due to recent increases of vancomycin-resistant bacterial infections, research on more effective antibiotics has intensified. As part of such research, screening of combinatorial libraries is a valuable approach for discovering new lead antibiotics. A screening method is presented here that uses a solid state library and a fluorescent label to detect RCO-d-Ala-d-Ala binding in aqueous media.     

Solution-phase library generation: methods and applications in drug discovery

Solution-phase high throughput synthesis has emerged as a powerful method for the rapid generation of chemical libraries. The success of this approach is largely due to the development of novel synthetic methodologies that expedite the preparation of compounds. Several isolation/purification techniques have also been developed to eliminate the time-consuming purification procedures often associated with solution-phase chemistry. These methods are amenable to parallel synthesis and combinatorial strategies and can be fully automated. In addition, the compound libraries generated using solution-phase high throughput synthesis have been used to accelerate both lead identification and lead optimization programs at various companies.     

Pyrazole CCK(1) receptor antagonists. Part 1: Solution-phase library synthesis and determination of Free-Wilson additivity

High throughput screening revealed compound 1 as a potent antagonist of the CCK(1) receptor. Evaluation of the CCK(1) SAR in a series of these diarylpyrazole antagonists was conducted in a matrix synthesis format revealing additive (Free-Wilson) and non-additive SAR. This use of additive QSAR modeling in conjunction with combinatorial libraries represents a unique approach to the evaluation of SAR interactions between the variables of any combinatorial matrix.     

Construction of Chemical Libraries of Volatile Compounds by Combinatorial Synthesis of Homologous Mixtures: Alk-4-en-1-ols,Alk-4-enals and Methyl Alk-4-enoates

A compound library of sixty six linear compounds, eleven representatives of six molecular families: (E)- and (Z)-isomers of alk-4-en-1-ols, alk-4-enals, and methyl alk-4-enoates, was prepared by combinatorial syntheses to allow the creation of a mass spectral database directly usable for their identification in GC/MS analyses. We demonstrate here that compound libraries can be prepared by combinatorial syntheses using long linear synthetic sequences, i. e., eight step in the case of 4-enals. The resulting mixtures of homologues are still perfectly exploitable to deliver the requested information such as clean mass spectra and good gas chromatographic retention indices.     

Diversity-oriented synthesis: exploring the intersections between chemistry and biology

Diversity-oriented synthesis (DOS) is an emerging field involving the synthesis of combinatorial libraries of diverse small molecules for biological screening. Rather than being directed toward a single biological target, DOS libraries can be used to identify new ligands for a variety of targets. Several different strategies for library design have been developed to target the biologically relevant regions of chemical structure space. DOS has provided powerful probes to investigate biological mechanisms and also served as a new driving force for advancing synthetic organic chemistry.     

Novel miniaturized systems in high-throughput screening

High-throughput screening (HTS) using high-density microplates is the primary method for the discovery of novel lead candidate molecules. However, new strategies that eschew 2D microplate technology, including technologies that enable mass screening of targets against large combinatorial libraries, have the potential to greatly increase throughput and decrease unit cost. This review presents an overview of state-of-the-art microplate-based HTS technology and includes a discussion of emerging miniaturized systems for HTS. We focus on new methods of encoding combinatorial libraries that promise throughputs of as many as 100,000 compounds per second.     

FTICR-mass spectrometry for high-resolution analysis in combinatorial chemistry

The diversity of compound collections required for finding lead structures in pharmaceutical research can be provided by means of combinatorial organic chemistry. The resultant enormous number of single compounds but also of compound mixtures represents a challenge for the analyst. With the introduction of Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS or FT-MS), a new and, as yet, not widespread mass spectrometric technique (a means of analysis of such compound libraries with a very high mass resolution) high mass accuracy and high sensitivity has become available. Moreover, in combination with electrospray ionization (ESI), not only high-throughput measurements via flow-injection analysis (FIA) but also coupling with separation techniques such as high-performance liquid chromatography (HPLC) or capillary electrophoresis (CE) is possible. Structural verification by way of decomposing ions (MS(n); n > or = 2) using a variety of different dissociation techniques can be performed by FTICR-MS. This is the first review specifically covering applications of FTICR-MS in the field of combinatorial chemistry.     

Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid phase by using combinatorial chemistry. One of the most well studied classes of peptidomimetics is peptoids. Peptoids are easy to synthesize and have been shown to be proteolysis-resistant and cell-permeable. Over the past decade, many useful protein ligands have been identified through screening of peptoid libraries. However, most of the ligands identified from peptoid libraries do not display high affinity, with rare exceptions. This may be due, in part, to the lack of chiral centers and conformational constraints in peptoid molecules. Recently, we described a new synthetic route to access peptide tertiary amides (PTAs). PTAs are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. With side chains on both α-carbon and main chain nitrogen atoms, the conformation of these molecules are greatly constrained by sterical hindrance and allylic 1,3 strain. (Figure 1) Our study suggests that these PTA molecules are highly structured in solution and can be used to identify protein ligands. We believe that these molecules can be a future source of high-affinity protein ligands. Here we describe the synthetic method combining the power of both split-and-pool and sub-monomer strategies to synthesize a sample one-bead one-compound (OBOC) library of PTAs.     

Genetic optimization of combinatorial libraries

Most agrochemical and pharmaceutical companies have set up high-throughput screening programs which require large numbers of compounds to screen. Combinatorial libraries provide an attractive way to deliver these compounds. A single combinatorial library with four variable positions can yield more than 10(12) potential compounds, if one assumes that about 1000 reagents are available for each position. This is far more than any high-throughput screening facility can afford to screen. We have proposed a method for iterative compound selection from large databases, which identifies the most active compounds by examining only a small fraction of the database. In this article, we describe the extension of this method to the problem of selecting compounds from large combinatorial libraries. Copyright 1998 John Wiley & Sons, Inc.     

Formaldehyde and some fully n-methylated substances in boar seminal fluids. Short communication

On the basis of recent observations it is supposed that seminal fluids may contain--mainly in hydroxymethyl groups--formaldehyde (HCHO) and quaternary ammonium compounds as potential HCHO generators, therefore, preliminary investigations were carried out for the identification of these compounds in pig seminal fluids using OPLC, HPLC and MALDI MS techniques. The fresh pig seminal fluid was frozen in liquid nitrogen, powdered and aliquots (0.25 g) were treated with 0.7 ml ethanolic dimedone solution. The suspension was centrifuged and the clear supernatant was used for analysis by OPLC or after dilution with HPLC or MALDI MS technique. After OPLC separation of formaldemethone the fully N-methylated compounds which are stayed on the start point were separated by OPLC using an other eluent system. It has been established that the HCHO is really a normal component of the pig seminal fluid, as well. It can be isolated and identified in dimedone adduct form. The measurable amount of HCHO depended on the concentration applied of dimedone. According to OPLC and MALDI MS investigations L-carnitine is the main quaternary ammonium compound in pig seminal fluid which can generate a protection of the sperm cells against environmental and other influences. Considerable differences have been found among individuals concerning concentrations of quaternary ammonium compounds in the seminal fluid of pigs.