Combinatorial Chemistry & High Throughput Screening - Volume 7, Issue 4, 2004

In this review, we discuss a number of computational methods that have been developed or adapted for molecule classification and virtual screening (VS) of compound databases. In particular, we focus on approaches that are complementary to high-throughput screening (HTS). The discussion is limited to VS methods that operate at the small molecular level, which is often called ligand-based VS (LBVS), and does not take into account docking algorithms or other structure-based screening tools. We describe areas that greatly benefit from combining virtual and biological screening and discuss computational methods that are most suitable to contribute to the integration of screening technologies. Relevant approaches range from established methods such as clustering or similarity searching to techniques that have only recently been introduced for LBVS applications such as statistical methods or support vector machines. Finally, we discuss a number of representative applications at the interface between VS and HTS.

Lead identification and optimisation have evolved into multidimensional, multidisciplinary and information-driven processes. Herein, we review the contribution of computational chemistry to these processes. We focus on computational approaches developed for modelling biopharmaceutical properties, including in vitro activity, selectivity, absorption, distribution, metabolism, excretion and toxicity. Whenever possible, successful applications are mentioned.

There is a need for fast testing of drug candidates for properties of pharmacokinetics and pharmacodynamics importance, in particular lipophilicity and acidity. These two parameters can conveniently be estimated by gradient reversed-phase HPLC. Appropriate conventional organic solvent gradient and the new pH gradient HPLC procedures are presented. The chromatographic parameter of lipophilicity, log kw, can be determined from two organic solvent gradient runs instead of 6-8 runs necessary in the standard isocratic (polycratic) approach. The newly introduced pH gradient reversed-phase HPLC consists in a programmed increase during the chromatographic run of the eluting power of the mobile phase with regards to ionizable analytes. The eluting strength of the mobile phase increases due to its increasing (in case of acidic analytes) or decreasing (basic analytes) pH, whereas the content of organic modifier remains constant. It has been theoretically and experimentally demonstrated that the pKa and log kw values can be evaluated based on retention data from a pH gradient run, combined with appropriate data from two organic solvent gradient runs. The gradient HPLCderived log kw parameters correlate well with analogous parameters determined isocratically as well as with reference lipophilicity parameter log P (logarithm of n-octanol / water partition coefficient). Also, the HPLCderived pKa parameters correlate to the literature pKa values ( ww pKa), conventionally determined by titrations in water. The approach described allows rapid and high-throughput assessment of log kw and pKa for large series of drugs candidates, also when the analytes are available in a form of mixture, e.g. produced by combinatorial synthesis.

Stationary phases with specific structural properties for high-throughput liquid chromatographic (LC) techniques are described. Special attention was paid to phases with special structural properties, mainly containing internal functional group (e.g. amide). Such materials are generally called “embedded phases”. There are phases created in amidation process of aminopropylated silica gel, especially phases based on biological compounds, like phospholipids and cholesterol, which are called immobilized artificial membranes (IAM's). The synthesis and applications of polar embedded amide LC stationary phases were also reviewed. Methods of characterization of synthesized packing materials were presented, with general focusing on spectroscopic measurementS like (13C and 29Si CP / MAS NMR and FT-IR), elemental and thermal analysis as well as chromatographic quantitative structure-retention relationships (QSRR) and extended chemometric tests. The potential applications of various dedicated stationary phases in a high-throughput LC screening procedures were also presented.

The performance of artificial neural network (ANN) in predicting the affinity of a series of 65 new azapirone derivatives for rat brain serotonin 5-HT1A receptors based on high-performance liquid chromatography (HPLC) retention data and on non-empirical structural parameters of the compounds' was studied. Affinity of the agents for rat brain 5-HT1A receptors were assessed in vitro and expressed as inhibitor constant values, Ki. The retention parameters determined in 14 HPLC systems along with compounds' structural descriptors from calculation chemistry were considered in ANN analysis. Supervised method of ANN learning with back-propagation strategy was used in ANN calculations. Two models of ANN of similar architecture were designed: the first one for the data based on chromatographic retention data and the second based on structural parameters of the agents. Each ANN model was trained with the data of training set. It was next used to classify the agents from the testing set into two groups: active (Ki ≤ 50 nM) and inactive compounds (Ki ≥ 50 nM). A high prediction performance of both ANN models considered as regards the affinity of new azapirone derivatives for the serotonin 5-HT1A receptors was demonstrated. However, the percent of correctly classified compounds was higher in the case of the ANN processing of the non-empirical structural descriptors of azapirone derivatives. Since the ANN analysis of the retention data and of the structural parameters originating from calculation chemistry allows to segregate drug candidates according to their pharmacological properties that, in consequence, may be of help to limit the number of biological assays in the search for new drugs.

Artificial neural networks (ANNs) have been applied for the quantitative structure-activity relationships (QSAR) studies of antibacterial activity against Escherichia coli, Serratia marcescens, Proteus vulgaris, Klebsiella pneumoniae and Pseudomonas aeruginosa of a large series of new imidazole derivatives. Antibacterial activity against individual bacteria, expressed as logarithm of reciprocal of the minimal inhibitory concentrations, log 1 / MIC, has been related to a number of physicochemical and structural parameters of the imidazole derivatives investigated. Molecular descriptors of agents were obtained by quantum-chemical calculations combined with molecular modelling and from respective structure fragment reference data (e.g., log P). A high correlation resulted between the predicted from ANN model antibacterial activity, log 1 / MICANN, and that from biological experiments, log 1 / MICexp, both for the data used in learning and in the testing sets of imidazoles. Correlation coefficient, R, depending on the type of bacteria and structural subset of analysed imidazole compounds, varies from 0.875 to 0.969. The applicability of ANNs has been demonstrated for the prediction of pharmacological potency of new imidazole derivatives based on their structural descriptors generated exclusively by calculation chemistry.

Multicomponent essential oils Tagetes Minuta and Poleo as well as pure limonene were encapsulated in Tween doped-high methoxylated pectin gels. Optical microscopy reveals that the obtained gels containing limonene consisted in a highly heterogeneous oil-in-water emulsion stabilised by the gelled medium. The influence of limonene encapsulation in pectin gelation kinetics and the gel structural properties were followed by dynamic rheological measurements. An electronic nose device developed in our laboratory was used to follow the flavour release of the three systems in order to discriminate the samples according to the main components released to the headspace. PCA and Neural Network Analysis allowed us to discriminate Tagetes Minuta from Poleo due to the difference in their limonene content. It is remarkable that the fingerprints of encapsulated complex mixtures differ from those obtained for the non-encapsulated oils, showing a preferential release of some components. In the case of limonene, the effect of the encapsulated concentration on the detected odour was also studied.

Performance of potentiometric quasi-array detection system consisted with the seven poly (vinyl chloride) (PVC) based liquid membrane electrodes in the cation-exchange HPLC using acetonitrile - 40 mM phosphoric acid (15 : 85, v / v, pH* 2.35) for assessing of molecular similarity / diversity in a mini-library of beta-adrenergic and beta-blocking chiral drugs was presented. Macrocyclic compounds differing in stability of their conformers as well as in a size, steric hindrance and polarity of its internal cavities, comprising a series of five calix[6]arene derivatives completed with one modified calix[4]resorcinarene, were used as neutral ionophores to compose mentioned set of PVC-based electrodes. The output potentiometric responses were registered in the linear supernerstian range of calibration graph of each electrode, i.e. for a constant injected concentration 2.0 × 10-4 M of investigated drugs, which is related to the amount frequently used at in vitro studies on pharmacological effects of these drugs. The impact of symmetry oriented supramolecular interactions on the responses of developed electrodes were characterised with proposed series of the highly significant quantitative structure-potentiometric response relationships (QSPRRs) combining both threedimensional (3D) molecular descriptors of analysed drugs as well as lipophilicity and volume polarizability of calixarene-type ionophores. The principal components analysis (PCA) and unweighted hierarchical clustering analysis (HCA) were used as the pattern recognition techniques into collected potentiometric database for extraction of the useful information on the molecular and pharmacological similarity / diversity of analysed drugs, thus a high-throughput and consistent identification of therapeutically relevant agonists of beta2- and beta3-adrenoceptors and antagonists of beta1-adrenoceptor was especially achieved. This evidence supports also a hypothesis formulated by results of homology modelling on the subtle significance of an unrecognised supramolecular insertion processes of the chiral drug molecule into the flexible hydrophobic pocket(s) formed by seven helical transmembrane moving domains of beta-adrenoceptors on their final activation, sequestration, down-regulation or blockade.

Boronic esters incorporated into a poly(vinyl chloride) (PVC)-supported liquid membrane electrodes have displayed an anionic ionophore properties enabling their use in the potentiometric high-throughput screening procedures. These compounds belong to the class of ligands in which the anion recognition process can be explained on the concept of Lewis type acid-base interactions. Membranes containing boronic esters showed fairly good sensitivity for maleate (cis-isomer) in comparison to fumarate anions (trans-isomer). The potentiometric selectivity coefficients of proposed electrodes proved that common anions did not interfered with the maleate anion determination. The influence of structure of the three boronic esters ionophores on generation of potentiometric signal by developed liquid membrane electrodes was shortly discussed.

The [Ni{(TAP)(SBn)8}] complex (I), i.e. ({2,3,7,8,12,13,17,18-octakis(benzylthio)-5,10,15,20- tetraazaporphyrin})nickel(II), has been explored as an ionophore for fabrication of the PVC based membrane electrodes used in the screening of thiocyanate anion (SCN-). The membrane having [Ni{(TAP)(SBn)8}] complex (I) as an electroactive material and dioctylphthalate (DOP) as plasticizer in the PVC matrix with the percentage ratio 5 : 158 : 200 (I : DOP : PVC, % w / w) exhibited a linear response in the concentration range 7.0 × 10-6 - 1.0 × 10-1 M of SCN- with a sub-Nernstian slope 32.5 ± 0.2 mV / decade of activity and a fast response time of 10 ± 2 s. The sensor works well in the pH range 3.0 - 9.5 and could be satisfactorily used in presence of 50 % (v / v) methanol, ethanol and acetone, and is selective for SCN- over a large number of anions with slight interference from iodide (I-) and azide (N3-) if present at a level ≥ 1.0 × 10-5 M. Described electrode works well over a period of six months. The sensor can be successfully applied for the screening of SCN- in both aqueous and real samples and also as indicator electrodes in precipitation titrations.

The results of studies on the use of corrole derivatives as a host ligand in the PVC liquid membrane electrodes and their ability for the potentiometric high-throughput discrimination of nitrophenol guests have been presented. The significance of parameters which govern the mechanism of generation of potentiometric signals such as the attachment of substituents in the corrole structure, acidity and lipophilicity of the guests, and pH of the aqueous solutions has been discussed in details. Supramolecular recognition processes between corroles and para-nitrophenol molecules have been confirmed by independent NMR measurements.

Four stationary phases containing different groups such as: C18, C30, alkylamide, and cholesterolic, were presented for simultaneous HPLC analysis of structural isomers of tocopherol. Especially, the influence of stationary phase structure and properties on tuning of the highly selective HPLC separation of β- and γ- tocopherol pair demonstrating, respectively, para- and ortho- arrangement of methyl substituents on the 6- chromanol ring, has been elucidated. It was pointed out that selectivity of each stationary phase has been a result of modulation in the mass transfer and set of unspecific interactions in the tertiary system comprising analyte stationary phase mobile phase. Differences in observed retention and specific selectivity of tocopherols together with the stationary phase structure investigations indicated that a spatial organization changing of chemically bonded ligands as predominantly a solvation consequence. Additional molecular modeling studies preliminary explained some of these complicated supramolecular phenomena which caused that cholesterolic stationary phase offered beneficial performance in screening of tocopherols by HPLC and biomimetic studies of not completely recognized interactions of tocopherol isomers and biological membranes.

Dr. Grzegorz Bazylak is currently an associate professor in the Department of Food Sciences & Drug Chemistry, Faculty of Pharmacy, Ludwik Rydygier Medical University (LRMU) in Bydgoszcz, Poland, where he directs research on HTS of potential hypotensive drugs, construction of genosensors, and use of components of edible transgenic plants in drug formulation. He joined this department after completing his D.Sc. (habilitation) in pharmaceutical sciences at the Faculty of Pharmacy, Medical University of Gdansk, Poland, at May 2003, and finishing his intensive research program on the use of supramolecular effects for enhancement of potentiometric detection in HPLC and capillary electrophoresis systems at the Department of Chemistry, University of Antwerpen, Belgium, in course of 2001-2003 years. In this period he was also the visiting scientist and visiting professor, respectively, at the Structural Chemistry Group and Department of Medicinal Chemistry, University of Antwerpen, Belgium, working on the structure-activity relationships of poly(p-phenylene vinylene) oligomers used in construction of electronic nose systems and enantiomeric HPLC separation of small molecule inhibitors of dipeptydyl peptidase IV. In years 1997-2001 he was an adjunct professor at the Department of General Chemistry, Faculty of Medicine, Medical University of Lodz, Poland, conducting research on development and use of helically distorted chiral selectors for the high-throughput enantioselective HPLC of adrenolytic / adrenomimetic drugs and their candidates as well as implementation of screening chromatographic systems which mimics G-protein coupled receptors. In period of 1991-1997 he administered the biochromatographic laboratory and held position of adjunct professor at the Human Nutrition Division, Hygienics Department, Medical University of Lodz, Poland, and carried out analytical research on separation of iridoid glycosides and chemometrically aided characterization of aminoalcohols as the potential lysosomotropic agents and mitochondrial apoptosis inhibitors. In 1997 he shortly visited Department of Applied Chemistry, University of Nagoya, Japan, and in 1996 he was visiting fellow of Centre of Analytical Chemistry at the Imperial College of Science, Technology and Medicine (South Kensington, London, UK). In 1995 he visited Department of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden, extending his knowledge on nutritional epidemiology and biostatistics. In years 1979 - 1991 he worked as the research scientists and finally adjunct professor at the Department of Bioinorganic and Analytical Chemistry, Institute of Fundamental Food Chemistry, Technical University of Lodz, Poland. In this last university he achieved his M.Sc. (1979) and Ph.D. (1990) degree in, respectively, food chemistry and technical sciences. During this pioneering time his research interest lied in the area of the heterogeneity of gas chromatographic adsorbents, synthesis of biomimetic nickel chelates, application of complexation gas chromatography in separation of pyridine-like analytes, quantitation of acroleine in waste animal fats, and HPLC determination of carcinogenic polycyclic aromatic hydrocarbons in mineral oils as part of his collaboration with cosmetic industry. Until today Dr. Bazylak published over 70 scientific papers in the open literature, give more than 60 conference presentations, and compiled 3 chapters in student teaching handbooks. His scientific activity was honored in 2003 by the individual scientific award from the National Ministry of Health, Warsaw, Poland. He serves now as the permanent member of the Establishers Council of the Polish Supramolecular Chemistry Network Foundation (from 1997), and Institutional Coordinator of Socrates-Erasmus Programes in LRMU (from 2003). In 2000 he served as the Guest Editor of special issue of CC In addition, during the years 1998-2001 he was a member of editorial board of three literary journals in Poland, serving as the reviewer of theatre events, and published also four poetry books (in Polish).