Combinatorial Chemistry & High Throughput Screening - Volume 10, Issue 6, 2007

Instrumentation-based HTS and μHTS is being improved by introducing nanolitre devices for dispensing fluids with extraordinary precision and biosensor-chip based multimode ultra-high sensitive readout protocols both of which are compatible with high density microwell and multiplate formats. A wide range of catalytic and affinity-based sensing elements together with variable fluorescence detection technologies have been implemented successfully into HTS which shifted the focus from observing macroscopic intermolecular events to the nanoscale, supramolecular and even single molecular level. In this way, HTS assays for drug discovery have developed information-rich, multiparameter readout systems for diverse molecular and supramolecular data that can include internal quality controls such as real-time feedback, self-diagnosis and adaptive supervising. These capabilities can reduce false-positive or false-negative rates caused by non-specific binding, degradation, precipitation, evaporation, poor solubility, or viscosity related sub-optimal concentration. Some of these emerging concepts and challenges for HTS are discussed in this issue of Combinatorial Chemistry & High Throughput Screening, which is Part 2 of a special issue commemorating the beginning of supramolecular chemistry in 1967. In the tutorial review by Achyuthan and Whitten opening this issue, some central principles of statistical data analysis in the development, optimization, validation and inter-laboratory comparability and traceability of nanoscale operated HTS as well as in vitro diagnostic (IVD) assays are reconsidered or even redefined. Since the availability of statistical and chemometric data analysis tools today does not always ensure knowledge of their boundaries, this tutorial should be useful to researchers in the pharmaceutical and combinatorial chemist communities. Recent trends, pitfalls and developments in the use of optical biosensors for different HTS protocols, especially for direct label free detection of reagents, beads and cells, are elaborated in the paper by Sanchez Rojas et al. These considerations are also discussed in the paper by Bertucci et al., which presents more detailed applications of optical biosensors in HTS of highly specific drug-protein interactions and determination of ADMET parameters of drug candidates. In addition, the paper by Langner et al., discusses the application of new concept of fluorescence probes and other supramolecular aggregate based biosensors for high-throughput assessment of membrane permeability coefficients and membrane adsorption of drugs and lead compounds. In the two research papers by Kruszewski et al., the binding affinities of a series of hydroxycamptothecins, anticancer drugs candidates, to human serum albumin and dimyristoylphosphatidylcholine liposomes were determined using innovative highthroughput steady-state fluorescence anisotropy measurements and factorial analysis of fluorescence excitation spectra. Similarly, Fadagar-Cosma et al. described the combinatorial synthesis and fluorescence quantum yields of a series of second generation asymmetrical meso-tetraphenylporphyrins as potential photosensitizers in photodynamic therapy. The fluorescence sensing approach was applied in the mini series of research papers by Liu et al., Wang et al., and Jin et al., for the study of molecular recognition and selective supramolecular binding processes between aliphatic oligopeptides and tryptophan-modified permethylated-β-cyclodextrins, fluoroquinolone-type antibiotic norfloxacin and p-sulfonated-calix[4]arene, and copper(II) cations and thiacalix[4]arene coated CdSe/ZnS quantum dots, respectively. These papers illustrate the impact of newly developed supramolecular materials, devices and procedures on peptidomics, proteomics, solubility enhancing drug encapsulation, and highly sensitive determination of inorganic impurities in drugs and pharmaceuticals, thus improving drug safety........

This paper reviews the several different factors that must be considered during the development of assays for high throughput screening (HTS) or in vitro diagnostic (IVD) applications. The reader is introduced to the terminology used in assay development as well as the statistical approaches for evaluating the data. The review is intended to serve as a tutorial to biotechnology, pharmaceutical and clinical professionals, the academic researcher, as well as a guide for established investigators of HTS and IVD. This review is not a comprehensive treatise in its scope or content, but is meant to introduce the reader to key concepts of assay development. Elementary mathematical and statistical tools for designing robust assays and data management are described. While certain design concepts overlap HTS and IVDs, others are more pertinent to one or the other topic. An overview of the regulatory requirements for IVDs is included in the context of the United States Food and Drug Administration. Quality concepts and high content screening are also briefly described. The review does not focus upon any particular assay technology nor does it provide detailed laboratory procedures on specific assays. The references cited are not exhaustive, but meant to steer the reader toward a general status report of the various technologies discussed. The information presented in this review is not intended to replace the judgment of the experienced laboratory scientist. However, this review should assist the scientific professional in executing well designed assays and being aware of design considerations.

Optical biosensors have been commercially available since the early 1990s, and have been used extensively in many areas of research in the life sciences. Optical biosensors developed for drug analysis generally exploit the high selectivity of the antigen-antibody and drug-protein interaction. Optical biosensors can be made based on optical diffraction or electro-chemiluminescence. High throughput screening, (HTS) which includes automated preparation of a large number of samples and then screening of their properties in multi-well plates, improves the efficiency of research in many scientific areas, e.g., catalyst screening, food processing, chemical synthesis, drug discovery, absorption, distribution, metabolism, and excretion and toxicological and cell based screening. The three most common detection techniques used in HTS are UV-VIS absorbance, fluorescence and luminescence. In this review, we summarize some recent trends and developments in the construction of optical chemical biosensors used in high throughput screening of drugs. Also, we have included environmental, biological and other medical applications of biosensors.

Specific molecular interactions provide a fundamental mechanism for selectivity in every aspect of biological structure and function. The ability to measure quantitatively such interaction properties across a wide range of affinity, size, and purity is a growing need. A short review on the use of the optical biosensor techniques is presented, focused on its application for determining the absorption and distribution parameters of drugs and lead compounds. The basic biosensor technology principles are described together with some immobilization methods commonly used for the preparation of selective and specific biosensor surfaces for assays. Some relevant research topics in the field of small molecule recognition phenomena are presented as examples, including binding to plasma proteins, and binding to lipid membranes, in the frame of ADME (absorption, distribution, metabolism and excretion) parameter determinations. These applications demonstrate the applicability of such techniques to the study of low mass compounds and illustrates their potential for the screening of libraries of compounds with regard to their binding to target bio-molecules as part of drug development.

High throughout screening is an approach based on the concept which assumes that when sufficiently large library of compounds are tested, the chance of discovering a new active compound is increased. In order to meet this expectation, proper testing criteria need to be devised. Those criteria should be related to the fate of a compound in the organism to have any predictive power. Not long ago, the main criteria were based exclusively on parameters defined by the maximum activity (QSAR). In this system, the activity criteria have not been included therefore the compound ability to reach the target is not accounted for. Considering that, the construction of yet another set of parameters has been initiated (QSPR). The parameters are in fact semi-empirical numbers which need to be tested on real, physical models. Whereas the activity tests are straightforward, the pharmacokinetic ones are difficult and controversial. One such parameter describes the critical property of an active compound, namely its ability to cross biological membranes. This review describes new concepts in the determination of the permeability coefficient with the help of methods which are based on liposome biosensors. Two methods using fluorescence probes incorporated in the lipid bilayer of liposome are described in detail and compared to other currently available techniques.

Two tryptophan-modified permethylated β-cyclodextrins, 6I-L-Trp-6I-deoxy- 2I,3I-di-O-methyl-hexakis(2IIVII ,3II-VII,6II-VII-tri-O-methyl)-β-cyclodextrin (3) and 6I-D-Trp- 6I-deoxy-2I,3I-di-O-methyl-hexakis(2II-VII,3II-VII,6II-VII-tri-Omethyl)- β-cyclodextrin (4), were synthesized, and their binding behaviors were investigated with the aliphatic oligopeptides, Leu-Gly, Gly-Leu, Gly-Pro, Glu-Glu, and Gly-Gly. Fluorescence spectrophotometric studies indicated that 3 and 4 can act as efficient fluorescence sensors for aliphatic oligopeptides. Due to their intermolecular co-inclusion binding mode with substrates, 3 and 4 not only afforded high binding constants of up to 103??104 M-1 for guest oligopeptides but also good molecular selectivities of up to ca. 7 for Gly-Gly/Leu-Gly and Glu-Glu/Gly-Gly pairs.

Camptothecins (CPTs) are fluorescent compounds exhibiting anticancer activity. They can exist in two forms, a lactone and a carboxylate. In neutral and base solution, lactone forms hydrolyse and convert into carboxylates. Only the lactone forms of CPTs are biologically active. Because of strong affinity of the carboxylate form of the parent drug camptothecin to human serum albumin (HSA), this protein promotes the deactivation of this compound. On the other hand, the lactone forms of camptothecins do not hydrolyse and are stabilized when bound to membranes. The following three hydroxycamptothecins, 10-hydroxycamptothecin (10-OH-CPT), 7-ethyl-10-hydroxy-camptothecin (SN-38) and 7-tertbutyldimethylsil- 10-hydroxycamptothecin (DB-67) were studied. Factor analysis of a set of fluorescence excitation spectra recorded during lactone hydrolysis facilitated the high-throughput determination of the deactivation rates of camptothecin and each hydroxycamptothecin in phosphate buffered saline. The fluorescence spectra of hydroxycamptothecins diluted in HSA solution or suspended in DMPC liposomes were recorded, and the association constants of these drugs to membranes and plasma proteins were calculated. Among the analysed agents, DB-67 exhibited the most desirable properties including low affinity of the carboxylate form for albumin and high affinity of its lactone form for model membranes.

Porphyrins play a major role as active photosensitizers in noninvasive optical photodynamic therapy (PDT). In a modular approach, this paper presents a short review of the recent developments of porphyrin structures and materials with improved photosensitizing properties and then presents the synthesis and characterization of a series of new second generation asymmetrical meso-tetraphenylporphyrins varied by substituent in the meta positions of the phenyl rings with either -OH or -OCH3 groups, whereas in the para positions only with -OCH3 groups. The new series of differentially functionalized porphyrins were obtained by a combinatorial multicomponent synthesis (Adler-Longo method) by simultaneously using two different aldehydes: 3,4-dimethoxybenzaldehyde and 3-hydroxybenzaldehyde. The porphyrins were isolated, purified and characterized by HPLC, TLC, UV-vis, fluorescence, MS, 1H-NMR, and 13C-NMR analysis, accompanied by DEPT 135 experiments. Because of the fact that the medium in cancerous tissues is often more acidic than in normal tissues, the capacity of these porphyrins to exist simultaneously in aggregated and protonated forms was also investigated, in tetrahydrofuran (THF) and acid THF-water systems, underlying the changes in the photophysical behaviour. The relative fluorescence quantum yields (Φf ) were calculated in comparison with meso-tetraphenylporphyrin (TPP), and the values between 0.14-0.26 were found to be promising for further trials. The series of asymmetrically substituted tetraphenylporphyrins, as the new class of supramolecular materials, are suitable for further functionalization in order to improve their photophysical properties, and they could represent interesting potential PDT photosensitizers.

Highly fluorescent water-soluble CdSe/ZnS (core/shell) quantum dots (QDs) as a fluorescent Cu2+ ion probe were synthesized using thiacalix[4]arene carboxylic acid (TCC) as a surface coating agent. Hydrophobic trioctylphosphine oxide (TOPO) capped CdSe/ZnS QDs were overcoated with TCC in tetrahydrofuran at room temperature, and deprotonation of the carboxyl groups of TCC resulted in the formation of water-soluble QDs. The surface structure of the QDs was characterized by using transmission electron microscopy (TEM) and fluorescence correlation spectroscopy (FCS). TEM images showed that TCC-coated QDs were monodispersed with the particle size (core-shell moiety) of approximately 5 nm. Hydrodynamic diameter of the TCC-coated QDs was determined to be 8.9 nm by FCS, showing that the thickness of the surface organic layer of the QDs was approximately 2 nm. These results indicate that the surface layer of TCC-coated QDs forms a bilayer structure consisting of TOPO and TCC molecules. TCC-coated CdSe/ZnS QDs were highly fluorescent (quantum yield, 0.21) compared to the QDs surface-modified with mercaptoacetic acid and mercaptoundecanoic acid. Fluorescence of the TCC-coated QDs was effectively quenched by Cu2+ ions even in the presence of other transition metal ions such as Cd2+, Zn2+, Co2+, Fe2+, and Fe3+ ions in the same solution. The Stern-Volmer plot for the fluorescence quenching by Cu2+ ions showed a linear relationship up to 30 μM of Cu2+ ions. The ion selectivity of TCC-coated QDs was determined by measurements of fluorescence responses towards biologically important transition metal ions (50 μM) including Fe2+, Fe3+, Co2+ > Zn2+, Cd2+. The fluorescence of TCC-coated QDs was almost insensitive to other biologically important ions such as Na+, K+, Mg2+, and Ca2+, suggesting that TCC-coated QDs can be used as a fluorescent Cu2+ ion probe for biological samples. A possible quenching mechanism by Cu2+ ions was also discussed on the basis of a Langmuir- type adsorption isotherm.

The complexation of norfloxacin (NFLX) by p-sulfonated calix[4]arene (SC4A) in aqueous solution has been studied by fluorescence spectroscopy and 1H NMR spectroscopy. A 1:1 stoichiometry and a 8086 L mol-1 stability constant of the NFLX-SC4A complex was obtained by spectrofluorometric titrations. The equimolar solid state inclusion complex of NFLX-SC4A was prepared by the co-precipitation method and then characterized by various techniques, including differential scanning calorimetry (DSC), X-ray powder diffractometry (XRD), Fourier-transform infrared analysis (FT-IR) and scanning electron microscopy (SEM). The experimental results of these chemical property screenings confirmed that NFLX and SC4A can form a stable host-guest complex in the solid state, and SC4A appears to function as a complexing and solubilizing agent for NFLX.

Camptothecin (CPT) and its hydroxycamptothecin analogs are fluorescent compounds exhibiting strong anticancer properties. They exist in two forms: active lactone and inactive carboxylate. The deactivation proceeds via hydrolysis in neutral and base solutions. A serious limitation to the clinical application of CPT is the strong affinity of its carboxylate form to human serum albumin (HSA) which destabilizes its active lactone form. However, binding to membranes in blood improves the stability of the lactone form of CPT and its analogs. A high-throughput screening assay based on the steady-state fluorescence anisotropy method was used to determine the protein- and membrane-binding properties of 10-hydroxycamptothecin (10-OH-CPT), 7-ethyl-10-hydroxycamptothecin (SN-38) and 7-tertbutyldimethylsil- 10-hydroxycamptothecin (DB-67). The relative affinities of hydroxycamptothecins to HSA and model membranes in the form of DMPC liposomes were determined, and DB-67 exhibited the most desirable properties including the highest affinity to membranes in its lactone form and low affinity to HSA in its carboxylate form.