Solid-phase biological assays for drug discovery.

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Review in Advance first posted online on June 5, 2014. (Changes may still occur before final publication online and in print.)

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Annual Review of Analytical Chemistry 2014.7. Downloaded from www.annualreviews.org by Otterbein University on 06/30/14. For personal use only.

Solid-Phase Biological Assays for Drug Discovery Erica M. Forsberg, Clémence Sicard, and John D. Brennan Biointerfaces Institute, McMaster University, Hamilton, Ontario L8S 4L8, Canada; email: [email protected]

Annu. Rev. Anal. Chem. 2014. 7:20.1–20.23

Keywords

The Annual Review of Analytical Chemistry is online at anchem.annualreviews.org

bioaffinity chromatography, protein microarrays, membrane proteins, kinases

This article’s doi: 10.1146/annurev-anchem-071213-020241 c 2014 by Annual Reviews. Copyright All rights reserved

Abstract In the past 30 years, there has been a significant growth in the use of solidphase assays in the area of drug discovery, with a range of new assays being used for both soluble and membrane-bound targets. In this review, we provide some basic background to typical drug targets and immobilization protocols used in solid-phase biological assays (SPBAs) for drug discovery, with emphasis on particularly labile biomolecular targets such as kinases and membrane-bound receptors, and highlight some of the more recent approaches for producing protein microarrays, bioaffinity columns, and other devices that are central to small molecule screening by SPBA. We then discuss key applications of such assays to identify drug leads, with an emphasis on the screening of mixtures. We conclude by highlighting specific advantages and potential disadvantages of SPBAs, particularly as they relate to particular assay formats.

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1. INTRODUCTION


Biological assays are ubiquitous in a wide range of areas, from clinical diagnostics to environmental analysis to food and beverage testing, and include such techniques as enzyme assays, immunoassays, and assays using DNA hybridization, often coupled to the polymerase chain reaction (1). Although many such assays have been reported for biosensor and other biodetection applications, far less emphasis has been placed on solid-phase biological assays (SPBAs) in the area of drug discovery, particularly for small molecule screening to identify drug leads (2, 3). This is slowly changing, as there are several advantages to moving small molecule screening assays to the solid phase by immobilizing one or more assay components onto a solid support such as plastic, metal, or glass. For example, solid-phase assays make it possible to utilize numerous new assay formats (microarrays, columns, microfluidic chips, etc.), use novel surface-dependent signaling methods [surface plasmon resonance (SPR), total internal reflection fluorescence, etc.], and provide a facile means of performing flow injection or multi-step reactions that require washing steps or addition of reagents to generate signals (2, 4, 5). In some cases, SPBA can provide unique advantages such as multiplexed screening via microarrays (6) and facile extraction of bioactive compounds from mixtures using chromatographic bioextraction methods (7), as well as a platform to increase selectivity by introducing marker compounds that allow competitive assays for site-specific targeting (8). Such assays can also increase assay throughput given that many assays can be designed to utilize small molecule mixtures as inputs, and can be automated by integrating them with advanced liquid handlers or autosamplers (9). To create an SPBA, one must have a biorecognition element (e.g., a biomolecule that is the drug target), a method to immobilize it on or within a suitable surface, and a mechanism to transduce the binding event between the biomolecule and bioactive compound into a measurable signal (Figure 1). Additional steps, such as purification or preconcentration (10), may also be required, and in many cases a method to amplify the initial signal (11) may be required to obtain suitable detection limits for compounds that may be present at low levels in complex mixtures. Although the immobilization of biomolecules, and particularly soluble proteins, on a solid support has been utilized for decades as a method to aid in the detection of biological interactions, there are still many challenges in the field. Reasons for this include a lack of biomolecule stability once immobilized (3, 9), difficulty with immobilizing labile biomolecules such as membrane receptors (3, 12), poor quantitative responses for certain assay formats, which makes it hard to rank hits in order of potency, and potential issues with false positives and negatives owing to nonspecific binding to the solid support (13). In this review, we first provide a description of the biological targets that have been most widely used to develop SPBAs for drug discovery and the typical immobilization methods used to fabricate various devices for solid-phase assays (microwell plates, arrays, columns). We then provide an overview of the various SPBA formats and signal transduction methods used in drug discovery and highlight some of the more recent state-of-the-art approaches that aim to address key challenges and advance the field. Given the breadth of the field, we do not attempt to be comprehensive. Rather, we provide selected examples of recent SPBA applications in the area of drug discovery, which are among the most important in advancing the field. Throughout the review, we highlight specific advantages and potential disadvantages of SPBA, specifically as they relate to particular assay formats or applications. Finally, we discuss key areas for future development in the area of SPBA.

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Figure 1 An overview of solid-phase biological assays used in small molecule drug discovery in which the biorecognition element is immobilized on a surface then probed with small molecules. Detection modes such as fluorescence, mass spectrometry, or surface plasmon resonance can be coupled to the screening platform to characterize ligand interactions. Abbreviations: GPCR, G protein–coupled receptor; GST, glutathione S-transferase.

2. BIOLOGICAL TARGETS Any SPBA requires a biological target with which the small molecule interacts to generate a signal. For the purposes of this review, we specify that the biological element is immobilized and is responsible for interaction with the compound(s) of interest. Typical biorecognition elements used in drug discovery are those that are part of biological pathways related to disease states, either human pathways related to dysfunction of cellular processes (i.e., those involved in cancer, diabetes, dementia, etc.) or those derived from infectious organisms such as bacteria or viruses. The most common examples are proteins, which are typically enzymes, regulatory proteins, or membrane-bound receptors. Alternatively, one can immobilize live cells that link specific targets to promoters that control expression of reporter genes or act as platforms for live/dead or other assays; however, this topic was recently reviewed (12) and will not be covered here in detail.

2.1. Enzymes Enzymes are biological catalysts that accelerate the rate of biological reactions by converting substrates to products. Typically, small molecules modulate enzyme activity by directly binding to the active site (competitively or noncompetitively), binding at an allosteric site, or blocking binding of essential cofactors. The use of enzymes for solid-phase assays in the drug-discovery arena dates back more than 30 years, where Kawuchi and coworkers (14) used an immobilized enzyme electrode for inhibitor screening against glucose oxidase. Since this time, a wide range www.annualreviews.org • Solid-Phase Biological Assays for Drug Discovery


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of soluble enzymes, including, but not limited to, various oxidoreductases (15), proteases (16), esterases (17), kinases (18), and dehydrogenases (19), has been immobilized or entrapped in various materials and used for screening of inhibitors. Most enzymes are soluble [with the exception of receptor tyrosine kinases (RTKs), as discussed in Section 2.2], and thus the polarity of the solid phase must be designed to be relatively hydrophilic to optimize native conformation. However, some classes of clinically relevant soluble enzymes are quite labile and thus challenging to immobilize, such as kinases. Given that kinases are involved in signaling pathways and regulate processes such as gene transcription, cell cycle, apoptosis, and differentiation through phosphorylation of various substrates, these enzymes are considered a major drug target (20, 21). The immobilization of kinases generally requires very biocompatible conditions to avoid loss of function and the presence of appropriate cofactors, such as Mg2+ , to ensure phosphorylation of the substrate. Other key enzymes for drug discovery in the solid phase are proteases and transcriptases, both of which are important in the development of HIV treatments and in cancer treatments (22, 23). Indeed, the caspases are key enzymes involved in apoptosis and thus in the regulation of cancer (24). These enzymes act on either DNA- or protein-based substrates and regulate processes related to infection and cell cycle. As such, these are important targets for drug discovery.


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2.2. Membrane Proteins The most challenging targets for development of small molecule screening assays, particularly for SPBAs, are membrane-bound proteins. Membrane proteins are incredibly diverse and serve in a plethora of cellular functions. Membrane proteins, and more specifically membrane-bound receptors, serve in transduction and amplification of signals across the cell membrane and allow cells to signal growth or apoptosis (25), or release chemicals in response to a physical or chemical stimulus for extracellular signaling (26, 27), making them desirable as targets for small molecule screening. Membrane receptors can be grouped into several major classes including ion-channel receptors, G protein–coupled receptors (GPCRs) (28), and enzyme-linked receptors (29). Ion-channel receptors may be stimulated in response to binding an endogenous ligand [in the case of ligand-gated ion channels (30)], membrane depolarization [in the case of voltage-gated ion channels (VGIC) (31)], or photostimulation [e.g., bacteriorhodopsin (32)]. Due to their roles in neurophysiology, ligand-gated ion channels are targets for therapies ranging from antidepressants to antipsychotics to nicotine addiction (30). GPCRs are perhaps the most important class of target, as they control numerous cell signaling events and can be stimulated in response to binding various ligands, including peptides, glycoproteins, hormones, and other endogenous molecules. The core GPCR structure consists of seven transmembrane α-helical segments joined by intracellular and extracellular loop regions where ligand-binding events are most likely to occur (28). The difficulty in analyzing interactions with this class of protein is their low natural abundance and difficulty in retaining activity outside a cell membrane. To maintain maximal functionality, transmembrane proteins require both the hydrophobic interactions of the lipid bilayer, as well as the hydrophilic interactions that occur on either side of the cell wall. The most common enzyme-linked receptors are RTKs, which serve as mediators for several growth hormone pathways (33). RTKs have a single transmembrane-spanning domain as well as hydrophilic extracellular and cytosolic domains. Initially monomeric, the RTK dimerizes to form an enzymatic kinase on the intracellular surface upon binding to an endogenous ligand (33). The kinase then functions by autophosphorylation of the tyrosine residues on the intracellular 20.4

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domain, initiating a cellular signaling cascade (34). Abnormalities originating from deregulation within these pathways can result in malignant cancer cell proliferation, making them an attractive target for cancer therapies (34).


3. IMMOBILIZATION METHODS When designing an immobilization protocol, it is important to consider several factors. These factors include the density and activity of the biomolecules, the orientation of the biomolecule on the surface, the long-term stability of the biological agent, the accessibility of the small molecules to the biomolecule, fast response times and the ability to reduce leaching or fouling of the immobilized biomolecules, as well as any changes in the binding constants, rate constants, or electrostatic environment of the biomolecule, which can alter interactions with charged species (35–37). As all of these factors may be interrelated and their dependence varies between biomolecules, there is no single immobilization method that is suitable for all biomolecules.

3.1. Immobilization of Soluble Proteins There are several different methods by which soluble biomolecules can be immobilized. The most common methods include physical adsorption, covalent binding, affinity-based techniques, and encapsulation within porous polymeric or composite organic-inorganic materials. Physical adsorption involves the use of weak interactions such as hydrogen bonding, hydrophobic interactions, or electrostatic interactions between the biomolecule and the surface. Although physical adsorption is relatively easy to perform, there is little control over biomolecule orientation; biomolecules may undergo undesirable conformational changes upon adsorption; and minor alterations in solvent conditions, pH, or temperature can lead to desorption of the biomolecule (38). Such issues can be partially overcome by entrapping the protein in films prepared by the layer-by-layer method, which involves the formation of separate films of opposing charge sandwiching the biorecognition element. This approach gives good control over film thickness, and molecular architecture but must be tuned to preserve protein orientation and accessibility (39). Covalent immobilization methods prevent leaching or desorption of the biomolecule as they involve a crosslinking reagent that conjugates a reactive functional group on the surface of the folded protein, such as cysteines (thiols) or lysines (amines), with a suitably activated surface including aldehyde-, carboxylic acid-, amine-, or hydroxyl-modified surfaces. Common crosslinkers include glutaraldehyde, carbonyldiimidazole, N-hydroxysuccinimidyl ester, maleimides, epoxides, and photoreactive species (37). A limitation of this method is that most biomolecules can contain multiple cysteine or lysine residues, making it difficult to control the orientation of the biomolecule, leading to a loss in active-site accessibility. Affinity-based immobilization techniques make use of natural biomolecular binding interactions to specifically tether the biomolecule to a suitably modified substrate. Examples include biotin-avidin interactions, aptamer capture, antibody capture, hexahistidine tags to bind to Ni2+ -nitrilo triacetic acid–derivatized surfaces, and glutathione-S-transferase tags to bind to glutathione-modified surfaces (38). These affinity methods often allow some control over orientation but require recombinant proteins and typically are not amenable to intrinsic or extrinsic membrane proteins or highly hydrophobic proteins such as lipases, which tend to aggregate upon immobilization. A final method is entrapment into polymeric materials, which can include various organic polymers (carboxymethylated dextran, agarose, chitosan, alginate, polypyrrole), acrylate-based hydrogels, and inorganic or hybrid materials, most often obtained by the sol-gel method using www.annualreviews.org • Solid-Phase Biological Assays for Drug Discovery


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alkoxysilanes and related species (i.e., diglyceryl silane, sodium silicate, silicic acid). Such materials are generally formed by condensation of monomers around a biomolecule and can be cast as films, monoliths, or columns; printed as microarrays or as coatings on surfaces; or formed as biomoleculedoped particles (40). The advantages of such an approach to immobilization is the versatility of surface chemistries, ability to control pore sizes to produce a size exclusion barrier (which can prevent degradation of entrapped species by proteases or nucleases), tunable surface chemistry, and the ability to add dopants to provide biomolecule stability or to allow signal development (i.e., via optical dyes, metal particles). However, optimizing such materials can be a time- and laborintensive process, although recent high-throughput optimization processes have helped accelerate the development of such materials (36).


3.2. Immobilization of Membrane Proteins In contrast to soluble proteins, membrane proteins reside either completely or partially embedded within cellular lipid membranes. Therefore, the distinguishing characteristic of membrane proteins is the presence of both surface-exposed lipophilic and membrane-associated hydrophilic amino acid residues (3). As a result, immobilization techniques for membrane proteins need to accommodate both the hydrophilic and hydrophobic portions of the protein. The major problems limiting the development of devices containing membrane receptors arise due to the inherently low abundance and the low stability of membrane receptors, the necessity to stabilize an amphiphilic biological membrane (41) as well as significant difficulties associated with transducing a receptor–ligand binding event into a measurable signal. There have been numerous strategies demonstrated for the immobilization of bilayer lipid membranes (BLMs) (42), including physical adsorption of a bilayer onto a solid surface (43), covalent attachment of a monolayer or bilayer of phospholipids to a solid surface (44, 45), attachment via avidin-biotin linkages (46), and membrane protein–specific antibodies (47), as shown in Figure 2. The early strategies for membrane immobilization involved adsorption of a phospholipid bilayer on top of a planar surface by immersing a freshly prepared metal surface into a phospholipid-organic solvent solution to promote spontaneous adsorption (48). However, detachment of the membrane from the surface and the absence of an aqueous surface between the lipid bilayer and the solid support are key disadvantages (42). These shortcomings have been addressed through (a) covalent attachment of the lipid membrane to the solid support (e.g., silica, gold) to aid adhesion (49) and through (b) insertion of water-soluble polymers between the solid surface and the lipid bilayer to provide a hydrated pseudointracellular environment. Such methods retained receptor functionality and allowed screening of interactions between species such as the nicotinic acetylcholine receptor and α-bungarotoxin, a peptide-based ligand. An alternative immobilization method reported by Wainer and coworkers (50) involved binding of proteoliposomes and cell fragments onto immobilized artificial membrane (IAM) beads. The premise of the immobilization technique is adsorption of detergent-solubilized receptors onto glass beads that have been modified with covalently bound phospholipids. The immobilized receptor was able to bind prototypical ligands with the correct affinities, and the receptor could be used repeatedly for periods of more than 50 days. Most recently, this approach has been extended to in vitro expression of receptors in block copolymersomes, which has been used to express and probe dopamine receptors (51). This approach presents a facile method of functional membrane-protein expression directly in the proteoliposomes without requiring reconstitution into BLMs. This format has the potential to develop a reliable platform for fabricating membrane receptor-based microarrays.

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Figure 2 Immobilization methods for membrane proteins or fragments include (a) adsorption directly on a surface, (b) self-assembly of lipid or polymer layers with the biomolecule, (c) covalent attachment of the membrane protein to the surface, (d ) attachment with affinity tags, or (e) via an immobilized antibody. Abbreviations: BSA, bovine serum albumin; GST, glutathione S-transferase.

4. SOLID-PHASE ASSAYS FOR SMALL MOLECULE SCREENING The drug-discovery process is multifaceted and can involve target discovery, identification of novel interactions between proteins and other biomolecules, and modulation of such interactions by various ligands, or rational design of inhibitors by in silico screening (52–54). Here we focus specifically on small molecule interactions with an immobilized protein, with an emphasis on enzymes or membrane-bound receptors, as these are the main classes of targets that have been used in such assays. Other solid-phase assay methods include immobilization of peptides or small molecules to identify targets, probe substrate specificity, or identify new inhibitors (55–58). However, the immobilization of a small molecule may result in hindered access to the biomolecule, particularly when the active site is buried deep within the protein structure. We refer the interested reader to recent reviews on these topics (55–58). Below we focus on three main areas in which SPBA is used in drug discovery. These are microwell plate–based solid-phase assays, microarrays, and column-based methods that use immobilized proteins as a stationary phase for liquid chromatography (LC)–based assays. In each case, we provide examples of screening studies performed with these methods and highlight advantages and disadvantages of these assay formats.

4.1. Microwell Plate–Based Solid-Phase Assays Microwell plate–based screening methods are the standard assay format in the pharmaceutical industry and are highly amenable to solution assays that monitor changes in absorbance, fluorescence intensity, and polarization, or chemiluminescence (53). Despite the introduction of many

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competing platforms, the infrastructure investment in microtiter plate–based platforms by most major pharmaceutical companies has made such technologies indispensable (53). Although plates have been widely used as a platform for solid-phase immunoassays, and particularly enzyme-linked immunosorbent assays, these approaches have been far less popular as a platform for enzyme assays or those involving membrane receptors. In part, this is because many plate-based screening assays are homogeneous in nature (i.e., mix and read), and thus there is no need for washing or other steps that require the use of a solid support. In addition, immobilization of some classes of biomolecules, such as membrane receptors, is difficult and can result in low activity (59). In addition, the immobilization of a monolayer of material at the bottom of a microwell can be detrimental in terms of total protein concentration, resulting in direct-binding assays involving membrane receptors or regulatory proteins of low sensitivity. Most solution-phase microwell plate assays utilize a label of some kind, typically a radiolabel or fluorophore for membrane-protein assays, or a fluorogenic or chromogenic reagent for enzyme assays. Numerous solid-phase plate-based assays have been developed using such labels, often in conjunction with sol-gel immobilization methods, which provide large protein loading levels for microwell-based assays. Early studies on enzyme activity and inhibition in such materials demonstrated that although a range of enzymes could be utilized in this format (Factor Xa, DHFR, COX II, γ-GT, Src kinase), the KM and kcat values tended to be altered upon entrapment (60, 61). However, the inhibition constants for a range of inhibitor/enzyme combinations were within error of those obtained in solution, providing evidence for the potential of this assay format for small molecule screening. An example of the potential benefits of sol gel–based microwell assays is the ability to create layered structures so that assays occur sequentially in the vertical direction through the well. This was demonstrated using adenosine deaminase and the adenosine binding structure-switching aptamer, with adenosine deaminase in a top layer and the aptamer in a lower layer. Addition of substrate adenosine to the well resulted in conversion to product and no percolation of adenosine to the aptamer, causing a low fluorescence signal. Inhibition of the enzyme allowed adenosine to penetrate through the upper layer and bind the aptamer, resulting in displacement of a quencher and an increase in fluorescence signaling. Failure to separate the enzyme and aptamer resulted in an unworkable assay owing to the effects of substrate depletion upon binding the aptamer (62). Sol gel–based microwell assays could also be adapted for radioligand assays of entrapped membrane receptors, where the monolithic protein-doped materials were fabricated in filter plates. In this case, the proteoliposomes containing either nAChR or dopamine receptors could be incubated with radioligands and competitive inhibitors, and the displacement of the radioligand could be monitored after passing through the monolith and filter plate, allowing quantitative assessment of competitive binding (63). The advantage of this method was the ability to retain the receptors in a functional state within a matrix that had inherent size exclusion properties, with receptors remaining functional for more than a month in these materials. Scintillation proximity assays make up another format that allow microwell-based radioassays to be performed on membrane receptors. In this case, fluoromicrospheres containing immobilized GPCRs and a scintillant have been assayed for ligand binding (64) using radioactive isotope–labeled substrates that stimulate fluorescence from the bead surface upon binding. The advantage of this method is that the radioisotope does not interfere with the binding kinetics, as can be the case when small molecules are labeled with larger probes such as fluorophores. One of the more interesting areas for solid-phase plate-based assays is the development of new transduction methods that couple surface-sensitive detection methods with plate-based assays, allowing for label-free detection of binding interactions. Plate-based SPR assays have been reported using gold-coated wells that are excited from below, which can probe the changes in


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refractive index and thickness at the surface upon ligand binding. However, such assays are not widely used in drug discovery given that they require high protein density and activity and also suffer from mass transport limitations to the sensor surface (65). An alternative method is to use R R Epic microwell plate–based screening system. This platform consists of a plate the Corning with a grating-based optical biosensor in each well that detects shifts in resonant wavelength upon ligand binding (66). Immobilization of carbonic anhydrase II onto the plates via covalent linkage to an amine functionalized sensor surface allowed screening against a series of 80 compounds, with identification of the known inhibitor acetazolamide (67). Although the assay was robust, concentrations required for screening were high at 10 mM, as full saturation of the protein was required to produce a measurable signal. Vela et al. (68) developed a related plated-based label-free screening method for the identification of the direct binding of small molecules to the ligand-binding domain of the estrogen-related R photonic crystal plates that meareceptor gamma, which was immobilized to SRU Biosystems sure biomass changes at the biosensor surface via alterations in local refractive index. Binding of a peptide segment of protein RIP140 validated the response of the biosensor giving a Kd < 100 nM and showed good correlation to other methods. This assay was then able to screen small molecules for agonist/antagonist activity, and could detect binding at concentrations up to 25 μM. A potential drawback of these label-free methods is that they require discrete molecules, as they cannot distinguish specific molecules in a mixture. These methods can also suffer from nonspecific responses arising from changes in temperature or nonspecific binding. Furthermore, binding of low levels of potent inhibitors will not elicit a sufficient shift in wavelength, and thus these methods require relatively high concentrations of ligand to generate signals.

4.2. Microarrays The immobilization of proteins in microarray format is an attractive alternative to plate-based screening. Microarrays contain hundreds or thousands of highly ordered elements, typically with 100–300-μm diameters, containing the target(s) to be probed along with all the necessary controls immobilized on a glass slide or a polymer-, metal-, nitrocellulose-, or hydrogel-coated surface. The miniaturized format provides a method for increased sample throughput and multiplexed analyte detection, and requires smaller reaction volumes, which allows for increased sample concentrations and reaction kinetics versus plate-based assays (9). In addition, microarrays can be printed into 96 well plates to allow integration with conventional liquid handling systems and can be imaged by a range of techniques, including colorimetry (69), fluorescence (70, 71), SPR (72, 73), and mass spectrometry (MS) (74, 75), providing the ability to design a wide range of assays to study protein-ligand interactions. The earliest report of a protein microarray was in 2000, when MacBeath & Schreiber (76) described the contact printing of three different proteins onto an amine-coated glass slide to produce a protein microarray, which was used to study protein-protein interactions in the target of rapamycin pathway. Shortly afterward, Zhu and coworkers (77) reported the first high density protein microarrays for high-throughput proteomics studies, where 93.5% of the yeast proteome (∼5,800 proteins) was covalently immobilized onto aminosilanized glass slides and used to probe interactions with calmodulin. Since these initial reports, functional protein microarrays have been used in many areas of biomedical research, including proteomics and biomarker analysis. In addition, printing methods have evolved to include both contact and noncontact (piezoelectric) printing methods, and all conventional immobilization methods have been utilized to produce microarrays (78–82). www.annualreviews.org • Solid-Phase Biological Assays for Drug Discovery


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4.2.1. Small molecule screening with protein microarrays. An early example of screening on microarrays utilized the ligand-binding domain of ErbB4, a receptor kinase that protects against some cancers, and probed its interaction with a fluorescently labeled phosphopeptide substrate in the presence of various oncogenic proteins (83). The displacement of the substrate upon binding of the oncogenes provided insight into the protective role of ErbB4 in cancer, which was shown to involve the formation of heterodimers with some oncogenes used in the screen. Microarrays have also been used for determining protein substrates of kinases (84) and ribosyltransferases (85), which is important in understanding posttranslational modifications and cell signaling pathways. The discovery of novel small molecule inhibitors, agonists, or antagonists of clinically relevant drug targets via protein microarray technology is not as well utilized, and to date only a few reports exist, most of which are proof-of-concept studies. In one early example, coimmobilization of the α-catalytic subunit of cAMP protein kinase A with the substrate Kemptide was done using a sol gel–derived microarray. Overprinting of known inhibitors, followed by staining of the microarray with a phosphoprotein binding dye, allowed determination of inhibition based on a reduction in fluorescence (86). This study demonstrated that it was possible to perform full quantitative inhibition assays on the array with determination of accurate IC50 values using less than 1 nL of reagents per assay. Through further optimization of sol gel–derived materials, multiple kinases were entrapped on a single high-density multiplexed array. The four kinases p38α/SAPK2a, epidermal growth factor receptor (EGFR), MAPK2, and GSK-3β were probed with the general kinase inhibitor staurosporine and found to appropriately report inhibition of kinase function (87). Direct site-specific immobilization of kinases to slide surfaces has also been used to probe for small molecule interactions using a calixcrown prolinker layer to immobilize anti-glutathione S-transferase (GST) antibodies and GST-tagged Polo-box domains of polo-like kinase 1 (Plk1), a serine/threonine kinase domain targeted for cancer therapy (18). Using a Cy5-labeled Bora peptide as a substrate, it was possible to detect kinase activity and inhibition based on blocking of the peptide-binding event, without the need to directly probe enzyme activity (Figure 3). Thus far, there is only one study that has used a microarray format for a high-throughput screen (HTS) of small molecule–ligand interactions. In this case, the enzyme acetylcholinesterase was pin-printed within a sol gel–derived microarray after a preliminary screen to identify optimal materials for pin-printing the enzyme. A fluorescence assay utilizing a disulfide-linked dimeric BODIPY-FL dye and acetylthiocholine was developed, wherein production of thiocholine cleaves the self-quenched dimer, resulting in a large increase in fluorescence intensity. Overprinting the dye, substrate, and inhibitor demonstrated that 1,000 bioactive molecules could be screened on the array to identify hits and that the array could then be used to determine quantitative inhibitor binding constants (88). The immobilization of membrane receptors in microarray elements is more difficult to achieve. One successful report of multiplexed GPCR microarrays by Hong et al. (89) showed the immobilization of neurotensin receptor 1, cholinergic receptor muscarinic 2, opioid receptor, and cannabinoid receptor 1 on porous glass slides, which were assayed with GDP and Eu2+ -labeled GTP. Binding of Eu-GTP could be disrupted by the addition of an antagonist in a cocktail of agonists. In another study, the same group developed a multiplexed array containing ten different GPCR fragments used to characterize binding of fluorescently labeled ligands (6). However, small molecule screening was not reported. Microarrays of membrane-associated cytochrome P450s, formed by entrapping the enzymes into hydrophobic sol gel–based materials, have also been used for high-throughput analysis of drug metabolism, which is an important part of lead discovery and optimization (90). More recently, three different CYP450s were stamped over Hep3B cells in alginate DataChipsTM to screen for


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Figure 3 Microarrays for kinase screening are immobilized using a ProteoChip coated with a calixcrown layer that adsorbs anti-GST antibodies. The surface is blocked with BSA, followed by immobilization of GST-tagged kinase Plk1 to the antibodies. The Cy5-labeled Bora peptide is then used to detect inhibition of kinase function. Inhibition was observed with compound E8 during a small screen. Abbreviations: BSA, bovine serum albumin; GST, glutathione S-transferase; Plk1, polo-like kinase 1. Figure adapted from Lee & Kang (18) with permission. Copyright Springer, 2013.

cytotoxicity (91). This assay was capable of performing drug and metabolite toxicity screening in high-throughput and generating dose-response curves for each drug. Overall, the use of microarray technologies for small molecule screening is still an emerging field. Advantages such as multiplexing and low-volume screening have been demonstrated for enzymes and membrane receptors. However, there is only one report of a medium-throughput screen of small molecules, and at present the infrastructure needed for microarray-based assays is not commonly available. Even so, this format currently represents the leading edge in miniaturized assay formats and is expected to further develop as a screening platform of choice.

4.3. Column-Based Assays Chromatographic stationary phases containing immobilized biomolecules are a major platform for solid-phase small molecule screening assays. These are typically used either as enzyme reactors, where inhibitors are infused along with the substrate of the enzyme and screened based on changes in product-to-substrate ratios, or as affinity phases, where potential inhibitors are identified by www.annualreviews.org • Solid-Phase Biological Assays for Drug Discovery


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Time or elution volume Figure 4 Bioaffinity columns can be used in various chromatographic assay formats, including (a) immobilized enzyme reactor, (b) zonal or nonlinear, (c) frontal affinity, and (d ) competitive displacement chromatography.

shifts in retention time or based on their ability to displace marker compounds. Advantages of chromatographic assays include versatility, as many different protein classes can be studied, and the ability to screen mixtures of compounds and extract strong binders from mixtures, as well as interface with detectors such as tandem mass spectrometers to allow screening and deconvolution of mass-encoded libraries. Some typical assay formats for small molecule drug discovery are outlined in Figure 4. Column materials containing an immobilized protein can be made using many different techniques. The three major methods are (a) functionalized particles conjugated with the protein of interest, which are packed into Microbore columns, giving bed volumes on the order of 0.1– 0.5 mL and compatibility with standard LC systems (50); (b) encapsulation in three-dimensional 20.12

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monoliths derived from organic (92), inorganic (93), or hybrid materials (94), providing bed volumes of 1–10 μL suitable for nanoflow LC; or (c) direct immobilization of proteins on the inner surface of an open tubular capillary for nanoLC (46, 95). Detection is most commonly performed by optical methods (absorbance, fluorescence), radioligand-based methods, or MS. Protein-doped columns, usually containing antibodies, have also been used for sample preconcentration and cleanup methods (10) or as bioreactors (96). In addition, there are numerous reports on columns with immobilized ligands that are used to identify protein-based targets on the basis of their retention on the column, an area commonly referred to as affinity proteomics (97, 98). However, this section focuses on screening applications for the discovery and analysis of protein-binding ligands using enzyme reactor or various affinity methods. 4.3.1. Immobilized enzyme reactor assays. One of the key properties of enzymes that can be exploited for small molecule screening is their ability to convert a substrate to a product. Immobilized enzyme reactor (IMER) columns can be used to probe enzyme function by the coinjection of both the substrate and the potential ligand and to monitor the turnover of the substrate. IMER columns can be prepared by any of the methods outlined above and have been coupled to absorbance, fluorescence, and MS-based detectors. Operating modes are based either on injection of sample plugs or continuous flow of substrate through the column. Early work on IMER was done in the 1970s by Ramachandran & Perlmutter (99, 100) and proved that flow-based assays using immobilized enzymes could provide useful information on enzyme properties and serve as a platform for inhibitor screening. Early studies of enzyme inhibition by IMER used optical methods to follow the reaction and generally used small injections of substrate and inhibitors. For example, Wainer and coworkers (101, 102) reported on the combination of an IMER with a reversed-phase LC system. They used absorbance-based detection as a method for examining the activity of immobilized enzymes, including enzymes that were immobilized onto hydrophobic beads. Massolini et al. (103) developed monolithic columns with covalently bound enzymes to create an IMER that was used in conjunction with absorbance detection. Palm & Novotny (104) have used enzyme reactors interfaced with off-line matrix-assisted laser desorption/ionization (MALDI)/MS for evaluation of PNGase F activity. More recent IMER studies have examined monoamine oxidase A and B, prepared using an IAM stationary phase and used to study the kinetics of kynuramine turnover (15). In subsequent studies using a covalent immobilization strategy, glyceraldehyde-3-phosphate dehydrogenase (19) and purine nucleoside (105) were immobilized in open tubular columns and used with the IMER format for on-line inhibitor screening. In all these studies, reversed-phase columns were required to resolve the substrate and product peaks via UV-Vis detection following the IMER study. An example of direct on-line monitoring of an immobilized enzyme reaction by MS was provided by Vanzolini et al. (106), who used MS to monitor product formation upon introduction of a plug of substrate into an immobilized enzyme column. This method provided a label-free method to assess enzyme activity via MS but required multiple injections of various levels of substrate and inhibitor to allow construction of a Lineweaver-Burke plot to extract KI values. Furthermore, the system did not operate under conditions of steady-state equilibrium at the injected substrate concentration. Our group reported on a continuous-flow nanoLC IMER system interfaced to electrospray ionization mass spectrometry for screening inhibitors of adenosine deaminase, which was entrapped in capillary scale monolithic silica columns derived by the sol-gel process (107). The technique involved infusing substrate directly in the mobile phase and as such operated under equilibrium conditions. It was shown that mixtures of inhibitors, also present in substrate solutions, could be infused from an autosampler and produce changes in product-to-substrate ratios depending on www.annualreviews.org • Solid-Phase Biological Assays for Drug Discovery


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inhibitor concentration and potency. A follow-up study using acetylcholinesterase within monolithic columns demonstrated the ability to rapidly screen more than 1,000 compounds, delivered as mixtures of 20 compounds each, in a matter of hours, followed by deconvolution of bioactive mixtures to identify inhibitors (17). Advantages of IMER, and particularly when coupled to MS, include the ability to use low concentrations of protein owing to inherent signal amplification resulting from substrate turnover to obtain accurate IC50 and KI values, even when apparent KM and kcat values are altered (60), and to obtain information about mode of action of inhibitors; moreover, the signal derived is based on changes in protein function, which are not significantly affected by nonspecific binding. However, such methods require enzymes with relatively high turnover numbers, as contact time on a column can be quite short—this can be a problem for some kinases that have turnover numbers of pmol min−1 relative to esterases that can have turnover numbers of μmol min−1 . In addition, when screening mixtures, it is necessary to do manual deconvolution by IMER or another technique to find the active compound. Furthermore, if an inhibitor is noncompetitive or has a very slow off rate, it can cause column fouling.


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4.3.2. Zonal affinity chromatography. Although IMER has been shown to be capable of medium-throughput screening, the method requires that the target biomolecule have catalytic function. When the biomolecule is a membrane-receptor, regulatory, or transport protein, it is necessary to develop solid-phase assays that are based on binding affinity, often with variations in ligand retention time being the signal output. Early work in this area involved zonal affinity or nonlinear chromatography (NLC), wherein ligand binding was assessed by injecting a plug of the test compound, followed by analysis of the peak profile for asymmetrical tailing that is proportional to the ligand concentration. Chaiken (108), Carr (109), and coworkers were of the first to covalently immobilize proteins in columns to study protein-ligand binding kinetics by their zonal elution profiles. This method is also capable of determining on/off rates as well as the equilibrium constant for ligand binding (5, 110, 111). Recent studies of NLC used in conjunction with frontal affinity chromatography (FAC; see below) have characterized binding of sulfonylurea drugs to human serum albumin (112) and screening of botanical extracts with estrogen-related receptor columns (113). Although this method has found some utility in the study of proteinligand interactions, it is difficult to perform high-throughput assays of many mixtures, and the method is prone to false signals related to nonspecific binding of test compounds to the stationary phase. 4.3.3. Frontal affinity chromatography. FAC is a widely used screening format first developed in the 1980s by Kasai, Ishii and coworkers (114) using a RNAse T1 -doped column interfaced to a UV detector. In this method, a mixture of ligands is continuously infused through a column with an immobilized protein using either a syringe pump or nanoLC to produce flow rates of 1–10 μL min−1 . Ligands with affinity for the protein will show a shift in retention volume proportional to their concentration and Kd . The method can be used with a range of detectors but is most widely used in combination with MS, as this allows identification of any ligands that bind through measurement of their mass. The method has often been used in a direct mode, where mixtures of ligands at identical concentrations are infused and rank-ordered on the basis of affinity, with the highest affinity species eluting last. It has also been used with a marker compound, where a higher affinity ligand will cause displacement and a roll-up of a lower affinity ligand that has previously eluted through the column and occupied a fraction of the binding sites (115). This latter mode is well suited to radioactivity or fluorescence detectors, but such detectors will not allow direct identification of binders within a mixture. 20.14

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Studies with FAC or FAC/MS have used various soluble and membrane-associated proteins, as well as ligand-binding domains of membrane receptors. Soluble proteins have included enzymes such as dihydrofolate reductase (116) and N-acetylglucosaminyltransferase V (117), the plasma protein human serum albumin (118), antibodies (119), and lectins for glycan characterization (120). Bioaffinity columns can be fabricated using covalent immobilization (120), affinity-based immobilization such as biotin-streptavidin (117), or entrapment of protein into monolithic columns (116). Such studies were most often used to probe the nature of protein-ligand interactions. For example, Hage and coworkers (118) determined human serum albumin binding site affinities for the drug imipramine with frontal analysis in the absence and presence of warfarin (Sudlow site I) and L-tryptophan (Sudlow site II). Schriemer and coworkers (117) used FAC with the enzyme N-acetylglucosaminyltransferase V to rank-order the affinity of ligands within mixtures. Fractions were collected from the N-acetylglucosaminyltransferase V columns and then separated by hydrophilic interaction chromatography prior to MS analysis (117). Perhaps the most relevant study in terms of HTS was a screen of 1,000 compounds by Ng et al. (13), who used a computational approach to rapidly identify elution times for compounds in a mixture of potential human estrogen receptor β inhibitors, with a rank-ordering of compounds being determined by ESI/MS analysis. An issue with direct infusion of eluents into ESI/MS during FAC is that infusion of several compounds simultaneously can cause ion suppression of analyte signals. Another drawback of ESI/MS is the need for elution buffers containing low concentrations of volatile buffers that may not be compatible with immobilized proteins. This issue can be addressed in two ways. The first is to perform an LC separation following FAC, as noted above. This method allows higher mixture complexity but significantly increases assay time. The second is to mix the output of the FAC column with matrix solution and directly deposit the mixture onto a MALDI plate by nebulization-assisted electrospraying (121). The resulting track is read by a MALDI-QqQ instrument, and it is possible to use volatile buffers at an ionic strength of up to 100 μM for the elution. An important area where FAC has been of significant utility is the study of membrane-associated proteins. One of the first membrane proteins to be studied by FAC was the glucose transporter protein, which was immobilized within proteoliposomes onto agarose beads (122, 123). In these studies FAC was used to provide thermodynamic and kinetic binding data for protein-ligand interactions, rather than as a screening tool (124). FAC has also been used extensively in inhibitor screening, including many GPCRs, ligand-gated ion channels, and transmembrane drug transporters (50). Membrane receptors often require immobilization strategies that suit both the hydrophobicity of the receptor as well as the polarity of the analytes. To retain receptor activity on columns, our group developed nAChR-doped monolithic silica in the presence of poly(ethylene glycol) and 3-aminopropyltriethoxysilane, both of which were shown to improve receptor activity for FAC-MS/MS studies with epibatidine (125). Wainer’s (126) group has studied the α3β4 nicotinic acetylcholine receptor, where the ligands tend to be polar and therefore entrapment in cellular membrane affinity columns is optimal (126), whereas screening for ligands of cannabinoid receptors requires immobilization via biotin-streptavidin linkage to prevent nonspecific binding to the IAM stationary phase (46). Advantages of FAC (and FAC/MS) include the wide range of protein types that can be employed, the fact that continuous infusion provides an equilibrium environment that allows accurate determination of binding constants, the ability to interface to numerous detectors and run in different modes, and the potential to interface FAC with two-dimensional LC assays to allow prefractionation of complex libraries prior to FAC analysis. Disadvantages of FAC are the need for high levels of protein to maximize shifts in retention time (127), as well as the need for several control experiments to address issues regarding nonspecific binding, difficulty in obtaining shifts www.annualreviews.org • Solid-Phase Biological Assays for Drug Discovery


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4.3.4. Competitive displacement chromatography. Displacement assays provide a means by which to address the drawbacks of FAC, including the need for high-protein concentrations and controls to account for retention based on nonspecific binding, as well as submicromolar binding affinities. The basic approach is to include a known ligand with a high to moderate binding constant as part of the assay. In the zonal chromatography approach, the elution time of a marker ligand depends on its affinity and concentration. When a competing ligand is included in the sample, the new ligand will occupy a certain fraction of binding sites, and hence the retention of the marker ligand will be reduced, leading to an earlier elution time. The shift in retention time of the marker can be used to determine the binding constant of the competing ligand as long as the concentration of this species is known. An advantage of the competitive displacement assay is the presence of an internal control compound, which will not get displaced if the test ligand does not bind directly to the protein at the same site. This overcomes some of the issues with nonspecific binding. In addition, the use of a marker ligand allows numerous different detection modes, as the marker can be radiolabelled or otherwise tagged with a probe to allow detection. This approach has been widely used for membrane receptors using radioflow assays that employ a constant flow of known radioligand that can be measured against infusion of a competitive inhibitor, as demonstrated recently for the breast cancer resistance protein using [3 H]-etoposide as a marker (128) (Figure 5). Another example of a displacement technique involves the use of columns prepared with immobilized EGFR to screen

N +

O –O

P O

80

O

70 O

O (CH2)12 O (H2C)12 HN

CMAC or NMAC

CH3 O O

Si

Relative signal


from weak binders, and a relatively low throughput owing to issues with mixture complexity. In addition, all compounds in a test mixture must be present at the same concentration to allow ranking of binding affinity. As such, complex or heterogeneous extracts cannot be analyzed by FAC. When integrated into ESI/MS, the method must also be used with low ionic strength, volatile buffers, which may not be compatible with all types of immobilized proteins, and the test ligands must be compatible with the ESI method in order to be detected. These issues have led to the development of newer methods for screening, as described below.

60 50 40

E D

30

C B

20

A

10

O

Membrane fragment

O

IAM-PC

0 2

4

6

8

10

12

14

16

Minutes Figure 5 Frontal affinity chromatography using immobilized affinity membranes with phosphatidylcholine to bind either cellular or nuclear membrane fragments containing transmembrane proteins. In this case, elution profiles of (A) 1 μM, (B) 2 μM, (C) 5 μM, (D) 10 μM, and (E) 20 μM etoposide were collected using immobilized breast cancer resistance protein columns coupled to mass spectrometric detection to determine the ligand inhibition constant. Figure adapted from Habicht et al. (128) with permission. Copyright Elsevier, 2013. 20.16

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herbal medicinal compounds for antitumor activity. Compounds to be analyzed were injected in a plug against a constant infusion of either competitive ligand or in the presence of the negative control compound. A decrease in retention when the competitive ligand was present in the mobile phase was indicative of binding to EGFR in a dose-dependent manner (129). Although zonal assays using competitive displacement have shown some advantages, they still require a significant amount of protein and rely on a shift in retention time, which can be difficult to detect when analyzing low affinity ligands. An alternative method for competitive displacement chromatography uses a continuous-flow assay, wherein a high affinity competitive ligand is included in the mobile phase and is constantly eluted through the column (8). This ligand reaches equilibrium with the immobilized receptor and elutes at the same concentration as it is infused. The addition of a plug of competing ligand displaces a fraction of the marker, resulting in a spike in the signal. The area under the spike is determined by the concentration and affinity of the competing ligand. Because the total concentration of displaced ligand elutes as a sharp spike, only very small amounts of ligand need to be displaced. As such, the amount of protein needed on the column is much less than that needed for FAC or zonal chromatography assays that rely on shifts in retention time, and ligands with weaker affinity can be detected. This assay was demonstrated using a monolithic column (5-μL bed volume) with entrapped nAChR operated in nanoflow LC mode with tandem MS detection. Only 2 pmol of receptor was needed to provide a signal based on displacement of epibatidine upon interaction with nicotine (Kd ∼ 1 μM) (8). The epibatidine peak area was dependent on the injected nicotine concentration, and the peak heights as a function of nicotine concentration could be used to determine the Kd of the competing ligand.

5. CONCLUSIONS The use of solid-phase assays is emerging as a viable technique for small molecule screening. These assays provide a means to integrate novel detection methods, such as SPR or photonic crystal biosensors, with well-established formats such as microwell plates, allow for miniaturization and multiplexing of assays via microarrays, or provide a method to screen mixtures using a variety of flow-based chromatographic assays. In all cases, the key issue is to immobilize the protein of interest in a manner that retains its biological function; several methods now exist to address this need, and many are amenable to both soluble and membrane-bound proteins. Once accomplished, assays can be developed using turnover of substrate (for enzymes), direct binding of ligands, or displacement of marker compounds to allow screening of libraries. Both microarrays and column-based methods (IMER and FAC) have been shown to be amenable to medium-throughput screening of ∼1,000 compounds, but no method has yet moved to the realm of true HTS (10,000+ compounds per day). As such, SPBAs currently find application in secondary screening, where rank-ordering of affinity or mode of action are determined. Further advances in automation and mixture deconvolution are needed to extend these methods toward HTS. In addition, further developments are needed to expand the number of biomolecular targets that can be utilized in each of the three assay formats, and in particular the use of membrane receptors in multiplexed assays involving microwell plates or microarrays.

SUMMARY POINTS 1. Current developments for HTS center around three key formats, all of which have unique advantages.



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2. SPBAs have mainly utilized enzymes and membrane receptors as targets and can utilize turnover of substrate, direct binding, or displacement assays to identify hits. 3. The choice of SPBA format will depend on the nature of the target, concentration of target available, and the type of library (discrete compounds or mixtures).

FUTURE ISSUES


1. There remains a need to implement many of the methods outlined above for more advanced screening of extracts or much larger libraries (>10,000 molecules per day) 2. Further development is needed to enhance stability, reduce nonspecific binding, and develop new formats for screening more complex mixtures. 3. Extension to membrane proteins is important but is still relatively limited in terms of formats, being based mainly on column-based assays.

DISCLOSURE STATEMENT The authors are not aware of any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this review. LITERATURE CITED 1. Tyagi S, Kramer FR. 1996. Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. 14:303–8 2. Berrade L, Garcia AE, Camarero JA. 2011. Protein microarrays: novel developments and applications. Pharm. Res. 28:1480–99 3. Tiefenauer L, Demarche S. 2012. Challenges in the development of functional assays of membrane proteins. Materials 5:2205–42 4. Kwok KC, Cheung NH. 2010. Measuring binding kinetics of ligands with tethered receptors by fluorescence polarization and total internal reflection fluorescence. Anal. Chem. 82:3819–25 5. Sanghvi M, Moaddel R, Wainer IW. 2011. The development and characterization of protein-based stationary phases for studying drug-protein and protein-protein interactions. J. Chromatogr. A 1218:8791– 98 6. Hong YL, Webb BL, Pai S, Ferrie A, Peng JL, et al. 2006. G-protein-coupled receptor microarrays for multiplexed compound screening. J. Biomol. Screen. 11:435–38 7. Mallik R, Yoo MJ, Briscoe CJ, Hage DS. 2010. Analysis of drug-protein binding by ultrafast affinity chromatography using immobilized human serum albumin. J. Chromatogr. A 1217:2796–803 8. Sharma J, Besanger TR, Brennan JD. 2008. Assaying small-molecule-receptor interactions by continuous flow competitive displacement chromatography/mass spectrometry. Anal. Chem. 80:3213–20 9. Lebert JM, Forsberg EM, Brennan JD. 2008. Solid-phase assays for small molecule screening using sol-gel entrapped proteins. Biochem. Cell Biol. 86:100–10 10. Cichna-Markl M. 2006. Selective sample preparation with bioaffinity columns prepared by the sol-gel method. J. Chromatogr. A 1124:167–80 11. Wark AW, Lee J, Kim S, Faisal SN, Lee HJ. 2010. Bioaffinity detection of pathogens on surfaces. J. Ind. Eng. Chem. 16:169–77 12. Yarmush ML, King KR. 2009. Living-cell microarrays. Annu. Rev. Biomed. Eng. 11:235–57 13. Ng W, Dai JR, Slon-Usakiewicz JJ, Redden PR, Pasternak A, Reid N. 2007. Automated multiple ligand screening by frontal affinity chromatography-mass spectrometry (FAC-MS). J. Biomol. Screen. 12:167–74 20.18

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ARI

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AC07CH20-Brennan

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Forsberg

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Sicard

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Brennan



AC07CH20-Brennan

ARI

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20.23

Epigenetic assays for chemical biology and drug discovery.

The importance of epitope binning for biological drug discovery.

In Vitro Transcription Assays and Their Application in Drug Discovery.

Versatility of homogeneous time-resolved fluorescence resonance energy transfer assays for biologics drug discovery.

Transmembrane drug transporter - taxonomy, assays, and their role in drug discovery.

Drug discovery FAQs: workflows for answering multidomain drug discovery questions.

Atlas for drug discovery.

Biological standards for hepatitis B virus assays.

Hyperpolarized NMR probes for biological assays.

Computational approaches for drug discovery.

Community-Reviewed Biological Network Models for Toxicology and Drug Discovery Applications.

Biological Networks for Cancer Candidate Biomarkers Discovery.

Fluorescence polarization assays in high-throughput screening and drug discovery: a review.

Advantages of cell-based high-volume screening assays to assess nuclear receptor activation during drug discovery.

Application and challenges in using LC-MS assays for absolute quantitative analysis of therapeutic proteins in drug discovery.

Recent developments in cell-based assays and stem cell technologies for botulinum neurotoxin research and drug discovery.

Using reverse-phase protein arrays as pharmacodynamic assays for functional proteomics, biomarker discovery, and drug development in cancer.

Synthetic biology for pharmaceutical drug discovery.

Open drug discovery for the Zika virus.

Current tools for norovirus drug discovery.

Animal models for anti-emphysema drug discovery.

Epigenetic drug discovery for Alzheimer's disease.

Epigenetic approaches for bipolar disorder drug discovery.

Repurposing strategies for tropical disease drug discovery.