Fragment-based activity space: smaller is better

Fragment-based drug discovery has the potential to supersede traditional high throughput screening based drug discovery for molecular targets amenable to structure determination. This is because the chemical diversity coverage is better accomplished by a fragment collection of reasonable size than by larger HTS collections. Furthermore, fragments have the potential to be efficient target binders with higher probability than more elaborated drug-like compounds. The selection of the fragment screening technique is driven by sensitivity and throughput considerations, and we advocate in the present article the use of high concentration bioassays in conjunction with NMR-based hit confirmation. Subsequent ligand X-ray structure determination of the fragment ligand in complex with the target protein by co-crystallisation or crystal soaking can focus on confirmed binders.     

NMR techniques for characterization of ligand binding: utility for lead generation and optimization in drug discovery.

Over the last ten years, nmr spectroscopy has evolved into an important discipline in drug discovery. Initially, nmr was most useful as a technique to provide structural information regarding protein drug targets and target-ligand interactions. More recently, it has been shown that nmr may be used as an alternative method for identification of small molecule ligands that bind to protein drug targets. High throughput implementation of these experiments to screen small molecule libraries may lead to identification of potent and novel lead compounds. In this review, we will use examples from our own research to illustrate how nmr experiments to characterize ligand binding may be used to both screen for novel compounds during the process of lead generation, as well as provide structural information useful for lead optimization during the latter stages of a discovery program.     

Enthalpy screen of drug candidates

The enthalpic and entropic contributions to the binding affinity of drug candidates have been acknowledged to be important determinants of the quality of a drug molecule. These quantities, usually summarized in the thermodynamic signature, provide a rapid assessment of the forces that drive the binding of a ligand. Having access to the thermodynamic signature in the early stages of the drug discovery process will provide critical information towards the selection of the best drug candidates for development. In this paper, the Enthalpy Screen technique is presented. The enthalpy screen allows fast and accurate determination of the binding enthalpy for hundreds of ligands. As such, it appears to be ideally suited to aid in the ranking of the hundreds of hits that are usually identified after standard high throughput screening.     

Applications of calorimetric methods to drug discovery and the study of protein interactions

Recent studies report the application of isothermal titration calorimetry and differential scanning calorimetry to the study of protein-ligand interactions, allosteric cooperativity and aspects of protein folding. New methods of data analysis compare alternative methods for determining ligand binding enthalpy and analyze potential sources of error in the experimental measurement of other thermodynamic parameters. Several reports examine issues concerning drug design and the correlation of thermodynamic and X-ray structural data. New instruments allow volumetric effects in biochemical systems to be evaluated calorimetrically and to substantially expand the throughput of differential scanning calorimetry measurements in drug discovery and other high-throughput applications.     

Emerging challenges in ligand discovery: new opportunities for chromatographic assay

Ligand discovery initiatives are facing interesting challenges as ever-increasing numbers of proteins are entering screening programs. As an answer to steady pressure to improve performance in drug discovery, ligand discovery can expect to play an expanded role in generating small molecules as probes to help uncover the function of novel proteins. Chromatographic assay formats can offer new entry points into standard interaction characterization (binding and rate constants) as well as powerful, scaleable methods for compound screening. This review presents recent advancements in chromatographic assay technology, with a particular focus on frontal affinity chromatography as a platform technology for interaction analysis.     

Homogeneous Cell- and Bead-Based Assays for High Throughput Screening Using Fluorometric Microvolume Assay Technology

High throughput drug screening has become a critical component of the drug discovery process. The screening of libraries containing hundreds of thousands of compounds has resulted in a requirement for assays and instrumentation that are amenable to nonradioactive formats and that can be miniaturized. Homogeneous assays that minimize upstream automation of the individual assays are also preferable. Fluorometric microvolume assay technology (FMAT) is a fluorescence-based platform for the development of nonradioactive cell- and bead-based assays for HTS. This technology is plate format-independent, and while it was designed specifically for homogeneous ligand binding and immunological assays, it is amenable to any assay utilizing a fluorescent cell or bead. The instrument fits on a standard laboratory bench and consists of a laser scanner that generates a 1 mm(2) digitized image of a 100-μmm deep section of the bottom of a microwell plate. The instrument is directly compatible with a Zymark Twistertrade mark (Zymark Corp., Hopkinton, MA) for robotic loading of the scanner and unattended operation in HTS mode. Fluorescent cells or beads at the bottom of the well are detected as localized areas of concentrated fluorescence using data processing. Unbound flurophore comprising the background signal is ignored, allowing for the development of a wide variety of homogeneous assays. The use of FMAT for peptide ligand binding assays, immunofluorescence, apoptosis and cytotoxicity, and bead-based immunocapture assays is described here, along with a general overview of the instrument and software.     

Emerging challenges in ligand discovery: new opportunities for chromatographic assay

Ligand discovery initiatives are facing interesting challenges as ever-increasing numbers of proteins are entering screening programs. As an answer to steady pressure to improve performance in drug discovery, ligand discovery can expect to play an expanded role in generating small molecules as probes to help uncover the function of novel proteins. Chromatographic assay formats can offer new entry points into standard interaction characterization (binding and rate constants) as well as powerful, scaleable methods for compound screening. This review presents recent advancements in chromatographic assay technology, with a particular focus on frontal affinity chromatography as a platform technology for interaction analysis.     

Methyl group reorientation under ligand binding probed by pseudocontact shifts

Liquid-state NMR spectroscopy is a powerful technique to elucidate binding properties of ligands on proteins. Ligands binding in hydrophobic pockets are often in close proximity to methyl groups and binding can lead to subtle displacements of methyl containing side chains to accommodate the ligand. To establish whether pseudocontact shifts can be used to characterize ligand binding and the effects on methyl groups, the N-terminal domain of HSP90 was tagged with caged lanthanoid NMR probe 5 at three positions and titrated with a ligand. Binding was monitored using the resonances of leucine and valine methyl groups. The pseudocontact shifts (PCS) caused by ytterbium result in enhanced dispersion of the methyl spectrum, allowing more resonances to be observed. The effects of tag attachment on the spectrum and ligand binding are small. Significant changes in PCS were observed upon ligand binding, indicating displacements of several methyl groups. By determining the cross-section of PCS iso-surfaces generated by two or three paramagnetic centers, the new position of a methyl group can be estimated, showing displacements in the range of 1–3 Å for methyl groups in the binding site. The information about such subtle but significant changes may be used to improve docking studies and can find application in fragment-based drug discovery.     

High Throughput Quantitation of cAMP Production Mediated by Activation of Seven Transmembrane Domain Receptors

Impairment of G protein-coupled seven-transmembrane (7 TM) receptor function has been implicated in a variety of different pathologic conditions, suggesting that the discovery of specific antagonists may lead to the development of successful therapeutic agents. The effect of different agents on receptor-ligand interaction is often measured directly in a receptor binding assay; however, this assay format can be time consuming and does not detect agents that interact at sites distal to the native ligand binding site. Cyclic adenosine monophospate (cAMP) represents a ubiquitous second messenger generated in response to ligand binding to many 7 TM receptors. The present study describes the practical adaptation of scintillation proximity methodology, using FlashPlate (NEN Life Sciences, Boston, MA) technology to evaluate cAMP production. The bioassay is based on competition between endogenously produced cAMP and exogenously added radiolabeled [125I]-cAMP. Cyclic AMP capture is mediated through an anti-cAMP antibody onto a microplate well surface. Removal of unbound radioligand is not necessary because only ligand within #20 mm of the plate surface is detected due to the proximity effect. The data indicate that the use of scintillation proximity technology allows accurate and specific evaluation of G protein-coupled receptor function as measured by cAMP production and is suitable for high throughput screening.     

Recent trends and some applications of isothermal titration calorimetry in biotechnology

Isothermal titration calorimeters (ITCs) are thermodynamic instruments used for the determination of enthalpy changes in any physical/chemical reaction. This can be applied in various fields of biotechnology. This review explains ITC applications, especially in bioseparation, drug development and cell metabolism. In liquid chromatography, the separation/purification of specific proteins or polypeptides in a mixture is usually achieved by varying the adsorption affinities of the different proteins/polypeptides for the adsorbent under different mobile-phase conditions and temperatures. Using ITC analysis, the binding mechanism of proteins with adsorbent solid material is derived by elucidating enthalpy and entropy changes, which offer valuable guidelines for designing experimental conditions in chromatographic separation. The binding affinity of a drug with its target is studied by deriving binding enthalpy and binding entropy. To improve the binding affinity, suitable lead compounds for a drug can be identified and their affinity tested by ITC. Recently ITC has also been used in studying cell metabolism. The heat produced by animal cells in culture can be used as a primary indicator of the kinetics of cell metabolism, which provides key information for drug bioactivity and operation parameters for process cell culture.     

基因芯片技术及应用研究进展

采用高速打印或光刻合成技术可在硅片、玻璃或尼龙膜上制造ＤＮＡ微阵列。样品ＤＮＡ／ＲＮＡ通过ＰＣＲ扩增、体外转录等技术掺入荧光标记分子,与微阵列杂交后通过荧光扫描仪器扫描及计算机分析即可获得样品中大量基因序列及表达的信息。该技术可应用于高通量基因表达平行分析、大规模基因发现及序列分析、基因多态性分析和基因组研究等 。     

Weak affinity chromatography as a new approach for fragment screening in drug discovery

Fragment-based drug design (FBDD) is currently being implemented in drug discovery, creating a demand for developing efficient techniques for fragment screening. Due to the intrinsic weak or transient binding of fragments (mM–μM in dissociation constant (K_D)) to targets, methods must be sensitive enough to accurately detect and quantify an interaction. This study presents weak affinity chromatography (WAC) as an alternative tool for screening of small fragments. The technology was demonstrated by screening of a selected 23-compound fragment collection of documented binders, mostly amidines, using trypsin and thrombin as model target protease proteins. WAC was proven to be a sensitive, robust, and reproducible technique that also provides information about affinity of a fragment in the range of 1 mM–10 μM. Furthermore, it has potential for high throughput as was evidenced by analyzing mixtures in the range of 10 substances by WAC–MS. The accessibility and flexibility of the technology were shown as fragment screening can be performed on standard HPLC equipment. The technology can further be miniaturized and adapted to the requirements of affinity ranges of the fragment library. All these features of WAC make it a potential method in drug discovery for fragment screening.     

Large-scale integrated super-computing platform for next generation virtual drug discovery

Traditional drug discovery starts by experimentally screening chemical libraries to find hit compounds that bind to protein targets, modulating their activity. Subsequent rounds of iterative chemical derivitization and rescreening are conducted to enhance the potency, selectivity, and pharmacological properties of hit compounds. Although computational docking of ligands to targets has been used to augment the empirical discovery process, its historical effectiveness has been limited because of the poor correlation of ligand dock scores and experimentally determined binding constants. Recent progress in super-computing, coupled to theoretical insights, allows the calculation of the Gibbs free energy, and therefore accurate binding constants, for usually large ligand-receptor systems. This advance extends the potential of virtual drug discovery. A specific embodiment of the technology, integrating de novo, abstract fragment based drug design, sophisticated molecular simulation, and the ability to calculate thermodynamic binding constants with unprecedented accuracy, are discussed.     

Evaluation of cellular dielectric spectroscopy, a whole-cell, label-free technology for drug discovery on Gi-coupled GPCRs

Cellular dielectric spectroscopy (CDS) is an emerging technology capable of detecting a range of whole-cell responses in a label-free manner. A new CDS-based instrument, CellKey, has been developed that is optimized for G-protein coupled receptor (GPCR) detection and has automated liquid handling in microplate format, thereby making CDS accessible to lead generation/optimization drug discovery. In addition to having sufficient throughput, new assay technologies must pass rigorous standards for assay development, signal window, dynamic range, and reproducibility to effectively support drug discovery SAR studies. Here, the authors evaluated CellKey with 3 different G(i)-coupled GPCRs for suitability in supporting SAR studies. Optimized assay conditions compatible with the precision, reproducibility, and throughput required for routine screening were quickly achieved for each target. Across a 1000-fold range in compound potencies, CellKey results correlated with agonist and antagonist data obtained using classical methods ([(35)S]GTPgammaS binding and cAMP production). For partial agonists, relative efficacy measurements also correlated with GTPgammaS data. CellKey detection of positive allosteric modulators appeared superior to GTPgammaS methodology. Agonist and antagonist activity could be accurately quantified under conditions of low receptor expression. CellKey is a new technology platform that uses label-free detection in a homogeneous assay that is unaffected by color quenching and is easily integrated into existing microtiter-based compound testing and data analysis procedures for drug discovery.     

Exact analysis of competition ligand binding by displacement isothermal titration calorimetry

A rigorous method for the least-squares nonlinear regression analysis of displacement isothermal titration calorimetric data is presented. The method can fit the binding isotherm of a ligand which is competitively inhibited in its binding by another bound ligand to a molecule with n identical and independent binding sites. There are no other assumptions for the method and no approximations. Analysis of previously published data of the strong binding of acarbose to glucoamylase is presented as an example. The regression equations have been programmed for the Origin software supplied with the widely used titration calorimeters from Microcal, Inc., and an Origin Function Definition File with instructions is freely available from the author upon e-mail request.     

Development of surface plasmon resonance biosensor assays for primary and secondary screening of acetylcholine binding protein ligands

Surface plasmon resonance (SPR) biosensors recently gained an important place in drug discovery. Here we present a primary and secondary SPR biosensor screening methodology. The primary screening method is based on a direct binding assay with covalent immobilized drug target proteins. For the secondary screening method, a sequential competition assay has been developed where the captured protein is first exposed to an unknown test compound, followed directly by an exposure to a high-molecular-weight reporter ligand. Using the high-molecular-weight reporter ligand to probe the remaining free binding site on the sensor, a significant signal enhancement is obtained. Furthermore, this assay format allows the validation of the primary direct binding assay format, efficiently revealing false positive data. As a model system, acetylcholine binding protein (AChBP), which is a soluble model protein for neuronal nicotinic acetylcholine receptors, has been used. The secondary assay is lower in throughput than the primary assay; however, the signal-to-noise ratio is two times higher compared with the direct assay, and it has a z′ factor of 0.96. Using both assays, we identified the compound tacrine as a ligand for AChBP.     

Small Molecule Screening in Human Induced Pluripotent Stem Cell-derived Terminal Cell Types

A need for better clinical outcomes has heightened interest in the use of physiologically relevant human cells in the drug discovery process. Patient-specific human induced pluripotent stem cells may offer a relevant, robust, scalable, and cost-effective model of human disease physiology. Small molecule high throughput screening in human induced pluripotent stem cell-derived cells with the intent of identifying novel therapeutic compounds is starting to influence the drug discovery process; however, the use of these cells presents many high throughput screening development challenges. This technology has the potential to transform the way drug discovery is performed.     

Predicting Allosteric Effects from Orthosteric Binding in Hsp90-Ligand Interactions: Implications for Fragment-Based Drug Design

A key question in mapping dynamics of protein-ligand interactions is to distinguish changes at binding sites from those associated with long range conformational changes upon binding at distal sites. This assumes a greater challenge when considering the interactions of low affinity ligands (dissociation constants, K_D, in the μM range or lower). Amide hydrogen deuterium Exchange mass spectrometry (HDXMS) is a robust method that can provide both structural insights and dynamics information on both high affinity and transient protein-ligand interactions. In this study, an application of HDXMS for probing the dynamics of low affinity ligands to proteins is described using the N-terminal ATPase domain of Hsp90. Comparison of Hsp90 dynamics between high affinity natural inhibitors (K_D ~ nM) and fragment compounds reveal that HDXMS is highly sensitive in mapping the interactions of both high and low affinity ligands. HDXMS reports on changes that reflect both orthosteric effects and allosteric changes accompanying binding. Orthosteric sites can be identified by overlaying HDXMS onto structural information of protein-ligand complexes. Regions distal to orthosteric sites indicate long range conformational changes with implications for allostery. HDXMS, thus finds powerful utility as a high throughput method for compound library screening to identify binding sites and describe allostery with important implications for fragment-based ligand discovery (FBLD).