Combinatorial Chemistry & High Throughput Screening - Volume 14, Issue 4, 2011

Optimizing cell-material interactions is critical for maximizing regeneration in tissue engineering. Combinatorial and high-throughput (CHT) methods can be used to systematically screen tissue scaffolds to identify optimal biomaterial properties. Previous CHT platforms in tissue engineering have involved a two-dimensional (2D) cell culture format where cells were cultured on material surfaces. However, these platforms are inadequate to predict cellular response in a three-dimensional (3D) tissue scaffold. We have developed a simple CHT platform to screen cell-material interactions in 3D culture format that can be applied to screen hydrogel scaffolds. Herein we provide detailed instructions on a method to prepare gradients in elastic modulus of photopolymerizable hydrogels.

The procaine effect on human erythrocytes was investigated by atomic force microscopy (AFM) at three procaine concentrations, about 5 x 10-7 M, 5 x 10-5 M and 5 x 10-4 M. The changes in surface morphology of erythrocyte membrane bring direct evidence on the procaine effect on the cell membrane at micro- and nanometer scale. AFM images of the control erythrocytes (without procaine) showed a well defined concave (donut) shape of cells. The structure of control erythrocytes membrane is featured by closely packed nanometer size intra-membranous particles. After the incubation of the fresh blood with increasing procaine concentrations, a progressive increase in both concave depth and surface roughness of erythrocyte membrane was observed. The particles (granules) of the membrane surface increased progressively with increasing procaine concentrations. The changes in the surface morphology of erythrocyte membrane can be associated with the enlargement of surface granules, due to the aggregation of membranous particles within the cell surface, and the domain structure formation induced by procaine. A large number of moderate elevations from 25 nm to almost 40 nm in lateral size were found to be rather uniformly distributed on the surface of whole erythrocytes at low and medium procaine concentrations, respectively. At the highest procaine concentration, the granules of about 80 nm to almost 90 nm lateral size were found to form rows rather well separated. These data are in substantial agreement with the published results obtained on membrane models in the presence of procaine.

Steroid hormone receptors represent a major target in drug discovery. As ligand inducible transcription factors, their activity can be modulated by small lipophilic molecules. Here we describe two panels of potent and selective luciferase reporter cell lines based on cells with low endogenous steroid receptor activity (U2OS). The panels contain reporter cell lines for estrogen receptors α and β, androgen, glucocorticoid, mineralocorticoid, and progesterone receptors. In the first panel, the activation of either synthetic, steroid response elements containing promoter or viral promoter is mediated by full-length steroid receptors. The second panel is based on the expression of the chimeric receptor, which was created by the replacement of the N-terminal part of the molecule by Gal4 DBD and that binds to multiple UAS sites in the reporter promoter. Both panels were extensively characterized by profiling 28 ligands in dose response manner in agonist and antagonist mode. We have analyzed and compared the responses to tested ligands from both panels and concluded that in general both systems generated similar qualitative response in terms of potency, efficacy, partial agonism/antagonism, mixed agonistic/antagonistic profiles and the rank of potencies was well conserved between both panels. However, we have also identified some artifacts introduced by the Gal4/LBD reporter assays in contrast to their full-length receptor reporter counterparts. Keeping in mind the advantages and drawbacks of each reporter format, these cell lines represent powerful and selective tools for profiling large compound libraries (HTS) and for detailed study of mechanisms by which compounds exert their biological effects.

Spatially addressable combinatorial libraries were synthesized by solution phase chemistry and screened for binding to human serum albumin. Members of arylidene diamide libraries were among the best hits found, having submicromolar binding affinities. The results were analyzed by the frequency with which particular substituents appeared among the most potent compounds. After immobilization of the ligands either through the oxazolone or the amine substituent, characterization by surface plasmon resonance showed that ibuprofen affected the binding kinetics, but phenylbutazone did not. It is therefore likely that these compounds bind to Site 2 in sub domain IIIA of human serum albumin (HSA).

Molecular topology can be considered an application of graph theory in which the molecular structure is characterized through a set of graph-theoretical descriptors called topological indices. Molecular topology has found applications in many different fields, particularly in biology, chemistry, and pharmacology. The first topological index was introduced by H. Wiener in 1947 [1]. Although its very first application was the prediction of the boiling points of the alkanes, the Wiener index has demonstrated since then a predictive capability far beyond that. Along with the Wiener index, in this paper we focus on a few pioneering topological indices, just to illustrate the connection between physicochemical properties and molecular connectivity.

Prime concerns with modern developments are attributed to high level undetected but important biological substances or even toxicants cycled often among individual and populations; which in turn agonizes environmental monitoring, trace-gas detection, water treatment facilities, in vivo detection in biological fluids and other accomplishments. For the detection of such analytes, several analytical devices combined with biological component have been designed with a physiochemical detector component. Here, we essentially focus on drug-based potentiometric membrane sensors known as ion selective electrodes (ISEs). The functionality of ion-selective membrane is quite intricate, challenging, and our undertsanding is yet to be thrived with more interventions. ISEs have applied explications to enormous variety of analytical inquires as well as informative tools for probing host-guest chemistry. However, expansion of ISEs based applications is aimed to improve the system performance, acquiring enhanced understanding of their response mechanism, and finding new chemical or physical configurations mainly for human welfare. The major strength of ISEs is the precised analytical information, assured by using the ion-selective membrane electrodes used successfully for both in vitro and in vivo assays of pharmaceutical products as well as in clinical analyses. In this review, we attempt to provide a brief prologue to the applicability and advantages of potentiometric sensors in the analysis of pharmaceutically active compounds emphasizing their employment at molecular level for in situ selection of biologically important analytes.

The section on patent review will be focused in the areas of interest to the readers of CCHTS. The search was conducted using the following key words: combinatorial chemistry, high throughput screening, drug repurposing, chemical library, high content screening, drug discovery and natural products. All patents highlighted here are identified by the patent number issued either by the World Intellectual Property Organization or by a regional patent office. DRUG REPOSITIONING The paradigm for finding new uses for known or failed drugs has generated new investments and interest in pharmaceutical sector. Drug repositioning or repurposing includes finding different therapeutic indications for drugs with established or even unknown pharmacological mechanism. Both marketed drugs as well as compounds with history of failure during clinical development are candidates for repositioning. Recent patent activity has focused on the experimental methods or theoretical mathematical modeling for selecting drugs for drug repurposing. WO2010141592A2: Chemical fragment screening and assembly utilizing common chemistry for NMR probe introduction and fragment linkage, (Sem, Daniel, S., Marquette University, USA) patent describes a potentially powerful tool to overcome the bottleneck in fragment-based drug design and extends the approach to tailoring known marked drugs for repurposing or increasing potency or reducing side-effects of targeted treatments with existing drugs. The fragment based approach requires identification of short molecular fragments that bind to adjacent sites of a target. One of the fragments, an active scaffold is labeled with 13C-methyl group. The challenges associated with linking two short fragments for drug design is approached using NMR-based fragment assembly. NMR is used at an early stage to detect any transfer of magnetization (NOE) between the labeled scaffold and test fragments, which is detectable only if the fragments bind to a target within 5 Angstrom distance, followed by chemical tethering of the fragments to sites where NOEs were detected. Any substantial increase in the binding affinity of tethered compounds selects the fragments and also precludes the time and cost associated with chemical synthesis of linked fragments. In a related set of patent applications, the uptake of isotope-labeled substrates/precursors in a biological system is accompanied with administration of known or unknown drug (s) or test compound(s). In the patent WO05051434A1: Method for high-throughput screening of compounds and combinations of compounds for discovery and quantification of actions, particularly unanticipated therapeutic or toxic actions, in biological systems (Hellerstein, Marc, K, University of California), the changes in patterns or content of isotopically labeled target molecules is determined in cells, tissues or whole animals through expected or unanticipated metabolic pathways and the rates are compared between labeled systems left untreated with drugs/compounds. An extension of the same principle in related patents WO06017812A1 and WO2007041611A2 focus on quantifying the effect of drugs/compounds on the dynamics of assembly or disassembly of isotopically labeled subunits of cytoskeletal system like the microtubules, amyloid plaques or plasma membrane disruptions. In the following set of patents, methods involving data mining information on diseases, pathways and known drugs forms the basis for the development of various approaches and models to drug repositioning. The patent WO2009068659A2: Novel disease treatment by predicting drug association (Cohen, Daniel; Chumkov, Ilya, Pharnet, France), describes a methodology based on mining of public database to select a disease and build a dynamic model of the disease and the molecular pathways. This is followed by in silico screening of drugs approved for other diseases that encompass all target pathways directly or indirectly implicated in the model. The in silico selected drugs either alone or in combination(s), are then tested in available biological model of the disease, to identify candidates for the treatment of the selected disease. Using this in silico approach, some compounds were proven to have the required biological activity against the targets selected in the patent. The strategy was shown to be effective The patent WO2009027843A2: Techniques for purposing a new compound and for repurposing a drug, (Zoref, Tali, Eilam and Agur, Zvia, Optimata ltd , Israel) describes a computer modeling based approach for repurposing and is based on information and disease models available for an approved drug or for compound(s) that failed in clinical development. Based on information on the drug's pre-clinical and clinical trials, modified pharmacokinetic and pharmacodynamic mathematical models are reconstructed and the model is adjusted based upon information about new patient populations or new indications. A new treatment protocol is suggested to salvage the failed drug or a new way to use an approved drug. HIGH THROUGHPUT SCREENING In patent WO2009078876A1: Assay method for group transfer reactions (Lowery, Robert et al., Bellbrook Labs, LLC, USA), a generic methodology is described for high throughput screening of catalytic activities generating the donor-products in group-transfer reactions of enzymes like methyltransferases, sulfotransferases, kinases, glycosyltransferases, uridine glucuronide transferase, UDP- glucuronosyltransferases, acetyl transferases, glutathione transferases, and ADPribosyltransferases. The methodology is based on generating an antibody which binds with high specificity to the cleaved donor product in a reaction, donor-X + acceptor -» donor-product + acceptor -X. In a methyltransferase reaction, the antibody that specifically binds to S-adenosylhomocysteine with high affinity is used at the detection step in which the enzymatically generated donor product displaces the tracer-labeled donor-product from its complex with the specific antibody, as in fluorescence polarization immunoassay. Because the donor product is the same for all enzymes that catalyze a given type of group transfer reaction, the same detection reagents can be used for all the members within a family of group transfer enzymes and with any acceptor substrate. The assay products can be detected using homogenous fluorescence or chemiluminescence methods.....