- Email: [email protected]
A pipeline for ligand discovery using small-molecule microarrays
A pipeline for ligand discovery using small-molecule microarrays Jay L Duffner, Paul A Clemons and Angela N Koehler Uncovering the functions of thousands of gene products, in various states of post-translational modification, is a key challenge in the post-genome era. To identify small-molecule probes for each protein function, high-throughput methods for ligand discovery are needed. In recent years, small-molecule microarrays (SMMs) have emerged as high-throughput and miniaturized screening tools for discovering protein–smallmolecule interactions. Microarrays of small molecules from a variety of sources, including FDA-approved drugs, natural products and products of combinatorial chemistry and diversity-oriented synthesis, have been prepared and screened by several laboratories, leading to several newly discovered protein–ligand pairs. Addresses Broad Institute of Harvard and MIT, 7 Cambridge Center, Cambridge, MA 02142, USA Corresponding author: Koehler, Angela N ([email protected])
Current Opinion in Chemical Biology 2007, 11:74–82 This review comes from a themed issue on Proteomics and genomics Edited by Matthew Bogyo and Benjamin F Cravatt Available online 13th December 2006 1367-5931/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2006.11.031
Introduction The successful completion of the Human Genome Project provides a new grand challenge for the broader research community in the years to come. Deciphering the information encoded in the functional genome, including thousands of predicted gene products, will require a variety of new scientific tools and methods. Increasingly, small molecules are used as tools to study functions of proteins and cellular processes [1–4]. Cellpermeable small molecules that bind and perturb the functions of proteins can be particularly useful tools in studies that require temporal or spatial control over the protein target. Additionally, specific small-molecule probes might uncover novel therapeutic targets for human disease as well as serve as templates for therapeutic design. To identify small-molecule probes for each protein function, high-throughput methods for ligand discovery or ligand design are needed. In the absence of structural information about most proteins in the proteome, it is currently impossible to design small Current Opinion in Chemical Biology 2007, 11:74–82
molecules that specifically target and perturb the various functions of each protein. High-throughput screening (HTS) methods that require little or no prior information of protein structure or protein function should prove useful in building a small-molecule toolkit to study the proteome. With this goal in mind, Schreiber and coworkers [5] reported a high-throughput and miniaturized proteinbinding assay involving microarrays of small molecules. Taking a cue from the world of complementary DNA microarrays and whole-genome expression profiling [6], collections of small molecules are immobilized, typically covalently, onto glass microscope slides. The smallmolecule microarrays (SMMs) are probed with a protein of interest and binding events are detected using a fluorescence-based readout with a standard microarray scanner (Figure 1). Since the initial report, several laboratories have contributed new twists on the microarray format, including several novel attachment chemistries for the preparation of SMMs, as well as novel screening and profiling applications, reviewed elsewhere [7]. Protein–ligand interactions of varying affinities have been discovered using SMMs [8–12,13,14]. Representative interactions are shown in Figure 2. Small molecules that bind and alter the function of transcriptional regulators have been reported [9,10]. More recently, selfassembled peptide nucleic acid (PNA)-based microarrays were used to identify selective inhibitors that distinguish two closely related cysteine proteases [14]. HsiehWilson and co-workers [13] identified chondroitin sulfate-E (CS-E) tetrasaccharide as a ligand to the cytokine tumor necrosis factor a (TNF-a) and demonstrated that CS-E inhibited a cytokine–cell-surface receptor interaction. Most reported interactions have dissociation constants (KD) in the low micromolar range [8–11]. Although screeners would like to find interactions with high affinities, ligands of modest affinity have been successfully used in chemical genetic studies [9,10]. Kodadek and coworkers [15] have also developed promising methods to transform lead compounds of low affinity into specific and efficient capture reagents for target proteins in the presence of complex mixtures. Synthetic chemistry can also be used to boost the affinity or potency of lead compounds [10]. Recent developments in the preparation of SMMs include the use of on-array, light-directed synthetic methods [16], immobilization of hydrazide-tagged compounds on epoxide-coated surfaces [17], and the www.sciencedirect.com
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Figure 1
Preparation and screening of SMMs. (a) Stock solutions of small molecules are arrayed onto functionalized microscope slides. Microarrays are typically screened by (b) incubation with a protein of interest, which is either purified or from a complex mixture such as a cell lysate, followed by (c) incubation with a fluorescently labeled antibody, either against the protein or an epitope tag. (d) Fluorescent array features indicate putative protein–small-molecule interactions.
use of diazoketone-modified slides to capture phenols or carboxylic acids [18]. Two groups reported a nonselective isocyanate-mediated capture strategy for the immobilization of compounds with a variety of nucleophilic functional groups (Figure 3a) [18,19,20]. Kanoh et al. [21] applied a nonselective approach to immobilization that uses photoactivation on gold-coated chips used in surface plasmon resonance (SPR) imaging (Figure 3b). Finally, Pohl and co-workers [22] described a novel method for the noncovalent capture of fluorous-tagged carbohydrates on slides coated with a fluoroalkylsilane reagent (Figure 3c). This method, which takes advantage of the highly specific fluorous affinity interaction, could www.sciencedirect.com
also be applied to collections of small molecules prepared using the fluorous approach to organic synthesis [23]. The fluorous strategy can be particularly useful for the synthesis and display of small molecules biased toward specific classes of proteins, such as histone deacetylases, where a specific displayed orientation of the small molecule is desired [24]. These recent advances should prove useful to research groups interested in preparing and screening SMMs. Here, we describe the challenges and successes of developing a platform for ligand discovery involving SMMs at the Broad Institute of Harvard and MIT. Current Opinion in Chemical Biology 2007, 11:74–82
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Figure 2
Representative protein–small-molecule interactions of varying affinities discovered using SMMs. Compounds (a)–(e) are products of diversity-oriented synthesis, and compound (f) is a natural product tetrasaccharide. Proposed sites of attachment to the microarray surface are colored in red. (a) FKL-01 binds to human FKBP12 with a KD of 37.2 mM [8]. (b) Uretupamine A binds to yeast Ure2p with a KD of 18.1 mM. The ligand specifically activates a glucose-sensitive transcriptional pathway downstream of Ure2p [9]. (c) Haptamide A binds to the yeast transcription factor Hap3p with a KD of 5.0 mM and inhibits Hap2/3/4/5p-mediated transcription [10]. (d) AMD03 binds to human IgG with a KD of 2.0 mM [11]. (e) Calmoduphilin binds to human calmodulin with a KD of 121 nM [12]. (f) Chondroitin sulfate-E tetrasaccharide binds to TNF-a and antagonizes the interaction of the cytokine with the cell surface receptor [13]. (g) KKLF is a selective inhibitor of cathepsin K [14]. (h) KLLL is a selective inhibitor of cathepsin F [14].
Pipeline for ligand discovery using SMMs at the Broad Institute: a case study The chemical biology program at the Broad Institute of Harvard and MIT aims to develop systematic ways to explore biology using small molecules. Scientists from a range of disciplines, including chemistry, biology, Current Opinion in Chemical Biology 2007, 11:74–82
computational science and engineering, all work cooperatively toward this goal. The use of synthetic chemistry and HTS, coupled with computational analysis, enables biologists to discover new research tools for biology, and enables chemists to understand how specific chemical properties influence particular biological processes. SMM www.sciencedirect.com
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Figure 3
Recent immobilization strategies for the preparation of SMMs. (a) Isocyanate surfaces can react with a variety of nucleophilic functional groups. Compounds containing multiple nucleophilic functional groups have the potential to be displayed in varying orientations in a given printed spot [18,19,20]. (b) Small molecules can be covalently immobilized, using photoactivated capture, onto aryl-diazirine-coated gold surfaces for label-free detection of proteins using SPR [21]. (c) Selective and noncovalent capture of fluorous-tagged compounds onto fluorous-coated glass surfaces takes advantage of the hydrophobic effect [22]. Additional immobilization strategies were reviewed by Uttamchandani et al. [7].
technology is one of the platforms used at the Broad Institute to aid the discovery of new protein-specific probes. We collaborate with groups interested in screening SMMs with collections of proteins relevant to various areas of disease biology, including psychiatric and infectious www.sciencedirect.com
diseases, and functional protein classes, such as transcription factors. Additionally, as part of an interaction with the National Cancer Institute’s Initiative for Chemical Genetics (ICG), SMMs are screened against several proteins that have a role in cancer. A brief description of the SMM pipeline is provided for those who have an interest in screening using this method. Current Opinion in Chemical Biology 2007, 11:74–82
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Compound curation
SMMs prepared at the Broad Institute contain compounds originating from a variety of sources. Traditionally, SMMs have contained compounds prepared using diversity-oriented synthesis (DOS) [25,26]. These compounds are equipped with a specific reactive appendage, such as an alcohol, that mediates attachment to the slide [7,8–10,12]. Recent advances in nonselective capture chemistry have led to an expanded printable collection that includes synthetic compounds, natural products, known bioactives and FDA-approved drugs [19,20]. The active screening collection includes over 100 000 compounds coming from commercial sources [4,27], inhouse synthetic chemistry efforts [28–30] and donations from synthetic chemistry laboratories around North America [4,27,31–36]. The SMMs also contain compounds prepared through the National Institute of General Medical Sciences Centers of Excellence in Chemical Methodologies and Library Development (CMLDs) at Boston University, Harvard University and the University of Pittsburgh (for an overview see: http://www.nigms.nih.gov/Initiatives/CMLD/). SMM preparation
In an effort to expand the functional group compatibility of SMMs, we recently adopted the isocyanate-mediated capture shown in Figure 3a [19]. Isocyanates react with several nucleophilic functional groups, thereby increasing the diversity of small molecules, from natural or synthetic sources, that can be immobilized on a single SMM. To date, we have printed roughly 3000 natural products, 6000 commercial compounds and 17 000 compounds provided by synthetic organic chemists from academia using this approach. A recent analysis of the functional groups present in the compounds from the screening collection at the Broad Institute suggests that roughly 70% of the compounds can be covalently immobilized on SMMs using the isocyanate-functionalized slides. A detailed protocol for making SMMs, which used isocyanate chemistry with the suggested equipment setup, reagent setup and known protein–small-molecule interaction quality controls, was recently published [20]. Executing a screen
Most protein-binding screens involve the use of purified and epitope-tagged proteins and binding is detected using a fluorescently labeled antibody against the tag, as depicted in Figure 1. Typically, proteins are screened at a concentration of 1 mg/ml in a buffer that is most appropriate to preserve stability or activity of the target. Triplicate data are collected for each protein against a given set of compounds. Overall, each screen consumes roughly 1–10 mg of protein sample, depending on the method used to incubate the protein solution with the SMMs [20]. More recently, screens involving complex protein mixtures and mammalian cell lysates have been performed [19,37]. This type of screen is becoming Current Opinion in Chemical Biology 2007, 11:74–82
routine as it removes the need for protein purification. More importantly, proteins obtained from cell lysates are more likely to retain proper conformations as well as possess post-translational modifications or interact with protein partners associated with a specific activity or tertiary structure. Unfortunately, to date a general method for screening integral membrane proteins has not been developed. Assay development projects aimed at integral membrane proteins are underway. Immediately after a new set of compounds is printed, a panel of readily available pure proteins is screened to identify promiscuous binders (Figure 4a,b). Data from the ‘specificity’ set are useful in helping a screener to focus on compounds that are relevant to their protein of interest. The goal is to eventually make composite Z-scores for all SMMs available to all screeners at the Broad Institute through ChemBank (http://www.chembank.broad.harvard.edu/) [4]. Chembank is a publicly available webbased database and application that stores information about the small molecules in the screening collection and data from assays [38,39]. To satisfy the conflicting needs of enabling scientific research and ensuring that experiments developed by the original scientist are free of immediate competition from the entire research community, a time delay of 12 months has been instituted between the time of data collection and public data dissemination. This delay allows the data to be confirmed in secondary assays and typically provides sufficient time for the original screener to publish their findings. All chemists and biologists who participate in the Broad Institute screening program are free to access each other’s data once they sign a data sharing agreement (DSA). This agreement establishes the ground rules for collaboration and proper use of another screener’s data [4]. A second version of ChemBank (DSA ChemBank), which contains information about approximately 1200 assays, is available only to those who sign the agreement. After the one year delay, the DSA ChemBank datasets are migrated to the public ChemBank site. Allowing screeners to access SMM data helps to prioritize candidates for further study based on whether a compound is specific for the target, a promiscuous binder, or a positive in a related assay involving a protein of similar structure or function. Additionally, screeners might find that compounds that score in the SMM assay also score in phenotypic assays. It is our hope that the intersection of SMM data and phenotypic data in ChemBank will lead to target ID hypotheses. Protein-binding data are a relatively new addition to ChemBank and we hope to make more data publicly available in the year to come. Secondary binding assays
The fluorescence intensity of a SMM positive is not a direct indicator of the binding affinity between a given small molecule and the protein target. Although a correlation between fluorescence intensity and affinity is often www.sciencedirect.com
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Figure 4
Characterization of microarray positives in secondary protein-binding assays. (a) A heat map of composite Z-scores sorted by compound ID was constructed for the following ten protein-binding screens involving SMMs: (1) bovine carbonic anhydrase II, (2) streptavidin, (3) fibrinogen, (4) wheat germ agglutinin, (5) concanavalin A, (6) calmodulin with calcium, (7) calmodulin with EGTA, (8) bungarotoxin, (9) bovine serum albumin, and (10) anti-5xHis monoclonal antibody. (b) Selected compounds with high composite Z-scores (>3) for either calmodulin condition (6 and 7) are shown in another heat map. (c) Secondary binding assays involving fluorescence-based thermal shifts were performed for selected positives and the melting curves are shown [44]. (d) SPR binding assays were conducted for a candidate ligand to calmodulin under both screening conditions (6 and 7) [40]. (e) Chemical structure and common name for SMP2_000044 (see also http://chembank.broad.harvard.edu/chemistry/ viewMolecule.htm?cbid=907437).
observed, fluorescence intensity can vary as a function of compound concentration, reactivity toward the slide surface and heterogeneity within printed features caused by multiple displayed orientations of the printed small molecule. Secondary binding assays are routinely used to confirm candidate interactions and measure binding affinities, as well as to rank ligands for further characterization in functional assays. Two types of secondary binding assays used at the Broad Institute to achieve these goals are described hereafter. The first assay involves SPR using a Biacore1 S51 Biosensor. SPR-based interaction analysis is an established method for characterizing the affinities between proteins and small molecules [40,41,42]. Secondary assays using SPR are performed with the same protein and smallmolecule stock reagents used in the primary SMM screen. In contrast to SMM assays, the protein is directly immobilized or captured with an antibody on the biosensor surface and the small molecule is injected onto the surface in solution phase. This assay orientation helps to annotate small molecules of weak affinity that score as positives in the primary assay, possibly because of avidity effects as a consequence of display on the microarray. www.sciencedirect.com
Compound solubility is also evaluated before injection onto the biosensor surface. Compounds that are insoluble at 10 mM in buffer with 5% (v/v) DMSO are rejected before the SPR assay so as to avoid invalidating data from subsequent injections. Compounds with poor solubility under these conditions are unlikely to be soluble under standard conditions for enzymatic or cell-based assays [43]. SPR is used to evaluate SMM positives in two types of assays. In the first assay, called a ‘yes/no’ assay, 1–2 ml of stock solution is cherry-picked from a plate and injected with a limited dilution series so as to increase the number of compounds tested per day. A negative control, such as streptavidin or GST, is tested simultaneously with the target to assess specificity. Positives in the yes/no assay are subjected to a second ‘compound characterization’ SPR assay. The second assay measures the binding with an extensive dilution series and additional replicates to gain information about affinity and binding kinetics (Figure 4d). Benefits of the SPR assay include minimal consumption of reagents, semi-automated sample delivery, diverse Current Opinion in Chemical Biology 2007, 11:74–82
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capture strategies for proteins, and user-friendly control and data analysis software. The high cost of instrumentation and consumables, and sequential or iterative assay development are disadvantages of the assay. Recently, high demand for SPR created a need for a complimentary yes/no assay, preferably with a higher throughput, to confirm putative interactions. The thermal-shift assay, also known as ThermoFluor1, was recently adopted to confirm and rank SMM positives [44]. This technique has been reviewed as a method for detecting biomolecular interactions as part of a drug discovery pipeline [45]. The thermal-shift assay detects protein–ligand interactions by monitoring ligand-dependent thermal stabilization of a target protein (Figure 4c). Protein melting is monitored using an environmentally sensitive dye, such as SYPRO1 Orange, which binds to hydrophobic residues that are normally found in the interior of a folded protein. The thermal-shift assay has been used to identify small-molecule additives to aid in protein crystallization [46] and to confirm and characterize enzyme inhibitors [47,48]. The assay can be carried out in 96-well plates using a quantitative PCR instrument to control temperature and measure fluorescence [46]. Advantages of the thermal-shift assay, relative to the SPR, include higher sample throughput and lower cost of instrumentation and consumables. A typical assay is finished in 60 minutes and enables more samples to be tested, relative to SPR, in the same time frame. It has been reported that compounds that exhibit nonspecific inhibition for different classes of enzymes result in negative melting temperature shifts, thus enabling identification of these compounds early in the discovery process [43,45]. Several disadvantages should be noted. Proteins with solvent-exposed hydrophobic patches, such as bovine serum albumin, result in high initial fluorescence that obscure the melting curve. Proteins composed of several non-energetically connected domains, such as fusion proteins, might unfold at different temperatures. If the melting temperatures are not widely separated, reliable calculation of the melting temperature becomes difficult. Although a mathematical framework has been developed to link the change in melting temperature to a measurement of affinity, the thermal-shift assay is typically applied qualitatively to rank the affinity of compounds [44]. Specific compound classes, such as chelators, can destabilize protein targets and complicate the analysis. Despite the disadvantages, thermal-shift yes/no assays are typically performed when a large number of positives are identified from primary SMM screen. Compounds that perturb the melting curve are subsequently tested in SPR compound characterization assays or functional assays. SPR-based yes/no assays are typically performed for SMM datasets that contain less than 30 positives. HistoriCurrent Opinion in Chemical Biology 2007, 11:74–82
cally, over 80% of SMM positives also score as binders in SPR or thermal-shift assays, and dissociation constants typically fall in the range of 0.1–20 mM. Figure 4 illustrates the pipeline for SMM, thermal-shift and SPR assays, leading to a modest ligand for calmodulin, NPC-15437, which is a known inhibitor of protein kinase C [49]. Additional studies are underway to characterize the nature of the interaction for this representative positive.
Conclusions Small-molecule microarrays (SMMs) have proven to be a general tool in the discovery of new protein–smallmolecule interactions. SMMs are a key component of the ligand discovery pipeline at the Broad Institute and can be used to complement datasets from phenotypic assays involving the same compounds. The assay format is constantly evolving. Many laboratories have developed new methods of preparing the arrays or have reported new screening applications that go beyond simple searches for binding interactions. Several laboratories have reported SMM-based enzyme assays and inhibitor fingerprinting studies [14,50,51]. SMMs show promise for diagnostic applications in the agricultural industry and in the clinic [52,53]. SMMs should be explored further as a platform for generating signatures of cell type or cell state by monitoring changes in protein–small-molecule interactions from cell lysates [19,54]. Once signature ligands (i.e. small molecules that distinguish between two states) are identified, they might be used as affinity reagents to identify protein biomarkers and can be studied further in a broad array of phenotypic assays. Finally, SMMs compatible with use in whole cells are being developed for studying interactions at cell surfaces and as a platform for performing highly miniaturized cell-based screens [55,56]. Hopefully, all of these developments will add to the small-molecule toolkit for deciphering the functional proteome.
Acknowledgements Worked described herein has been funded in whole or in part with Federal funds from the National Cancer Institute’s Initiative for Chemical Genetics, National Institutes of Health(Contract No. N01-CO-12400). The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Service, nor does mention of trade names, commercial products or organizations imply endorsement by the US Government. The authors also acknowledge Nicole Bodycombe, Anna Borodovsky, Scott Eliasof, John McGrath, Olivia McPherson, Martin Serrano and Nicola Tolliday for their contributions to small-molecule microarray screening platform at the Broad Institute.
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proteins for structural studies. Anal Biochem 2006, 357:289-298. This paper describes the use of thermal-shift assays as a high-throughput method for identifying optimal buffer conditions to crystallize various proteins. 47. Lo MC, Aulabaugh A, Jin G, Cowling R, Bard J, Malamas M, Ellestad G: Evaluation of fluorescence-based thermal shift assays for hit identification in drug discovery. Anal Biochem 2004, 332:153-159. 48. Matulis D, Kranz JK, Salemme FR, Todd MJ: Thermodynamic stability of carbonic anhydrase: measurements of binding affinity and stoichiometry using ThermoFluor. Biochemistry 2005, 44:5258-5266. 49. Sullivan JP, Connor JR, Shearer BG, Burch RM: 2,6-Diamino-N([1-(1-oxotridecyl)-2-piperidinyl] methyl)hexanamide (NPC 15437): a novel inhibitor of protein kinase C interacting at the regulatory domain. Mol Pharmacol 1992, 41:38-44. 50. Sun H, Chattopadhaya S, Wang J, Yao SQ: Recent developments in microarray-based enzyme assays: from functional annotation to substrate/inhibitor fingerprinting. Anal Bioanal Chem 2006, 386:416-426. 51. Wang J, Uttamchandani M, Sun LP, Yao SQ: Activity-based high throughput profiling of metalloprotease inhibitors using small molecule microarrays. Chem Comm 2006:717-719. The authors describe a nanodroplet SMM strategy for high-throughput profiling of inhibitors of enzymes. The technology is demonstrated by generating inhibitor fingerprints for two metalloproteases across a 400member small-molecule library. 52. Peng Z, Bang-Ce Y: Small molecule microarrays for drug residue detection in foodstuffs. J Agric Food Chem 2006, 54:6978-6983. 53. Smith AH, Vrtis JM, Kodadek T: The potential of proteindetecting microarrays for clinical diagnostics. Adv Clin Chem 2004, 37:217-238. 54. Borrebaeck CA: Antibody microarray-based oncoproteomics. Expert Opin Biol Ther 2006, 6:833-838. 55. Barrett OJ, Childs JL, Disney MD: Chemical microarrays to identify ligands that bind to pathogenic cells. ChemBioChem 2006, 7:1882-1885. This paper describes the use of SMMs to identify new ligands for whole cells. A library of heterocyclic compounds was interrogated for binding to a panel of pre-stained pathogenic cells including Gram-negative bacteria and fungi. 56. Bailey SN, Sabatini DM, Stockwell BR: Microarrays of small molecules embedded in biodegradable polymers for use in mammalian cell-based screens. Proc Natl Acad Sci USA 2004, 101:16144-16149.
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Current Opinion in Chemical Biology 2007, 11:74–82 This review comes from a themed issue on Proteomics and genomics Edited by Matthew Bogyo and Benjamin F Cravatt Available online 13th December 2006 1367-5931/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2006.11.031
Introduction The successful completion of the Human Genome Project provides a new grand challenge for the broader research community in the years to come. Deciphering the information encoded in the functional genome, including thousands of predicted gene products, will require a variety of new scientific tools and methods. Increasingly, small molecules are used as tools to study functions of proteins and cellular processes [1–4]. Cellpermeable small molecules that bind and perturb the functions of proteins can be particularly useful tools in studies that require temporal or spatial control over the protein target. Additionally, specific small-molecule probes might uncover novel therapeutic targets for human disease as well as serve as templates for therapeutic design. To identify small-molecule probes for each protein function, high-throughput methods for ligand discovery or ligand design are needed. In the absence of structural information about most proteins in the proteome, it is currently impossible to design small Current Opinion in Chemical Biology 2007, 11:74–82
molecules that specifically target and perturb the various functions of each protein. High-throughput screening (HTS) methods that require little or no prior information of protein structure or protein function should prove useful in building a small-molecule toolkit to study the proteome. With this goal in mind, Schreiber and coworkers [5] reported a high-throughput and miniaturized proteinbinding assay involving microarrays of small molecules. Taking a cue from the world of complementary DNA microarrays and whole-genome expression profiling [6], collections of small molecules are immobilized, typically covalently, onto glass microscope slides. The smallmolecule microarrays (SMMs) are probed with a protein of interest and binding events are detected using a fluorescence-based readout with a standard microarray scanner (Figure 1). Since the initial report, several laboratories have contributed new twists on the microarray format, including several novel attachment chemistries for the preparation of SMMs, as well as novel screening and profiling applications, reviewed elsewhere [7]. Protein–ligand interactions of varying affinities have been discovered using SMMs [8–12,13,14]. Representative interactions are shown in Figure 2. Small molecules that bind and alter the function of transcriptional regulators have been reported [9,10]. More recently, selfassembled peptide nucleic acid (PNA)-based microarrays were used to identify selective inhibitors that distinguish two closely related cysteine proteases [14]. HsiehWilson and co-workers [13] identified chondroitin sulfate-E (CS-E) tetrasaccharide as a ligand to the cytokine tumor necrosis factor a (TNF-a) and demonstrated that CS-E inhibited a cytokine–cell-surface receptor interaction. Most reported interactions have dissociation constants (KD) in the low micromolar range [8–11]. Although screeners would like to find interactions with high affinities, ligands of modest affinity have been successfully used in chemical genetic studies [9,10]. Kodadek and coworkers [15] have also developed promising methods to transform lead compounds of low affinity into specific and efficient capture reagents for target proteins in the presence of complex mixtures. Synthetic chemistry can also be used to boost the affinity or potency of lead compounds [10]. Recent developments in the preparation of SMMs include the use of on-array, light-directed synthetic methods [16], immobilization of hydrazide-tagged compounds on epoxide-coated surfaces [17], and the www.sciencedirect.com
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Figure 1
Preparation and screening of SMMs. (a) Stock solutions of small molecules are arrayed onto functionalized microscope slides. Microarrays are typically screened by (b) incubation with a protein of interest, which is either purified or from a complex mixture such as a cell lysate, followed by (c) incubation with a fluorescently labeled antibody, either against the protein or an epitope tag. (d) Fluorescent array features indicate putative protein–small-molecule interactions.
use of diazoketone-modified slides to capture phenols or carboxylic acids [18]. Two groups reported a nonselective isocyanate-mediated capture strategy for the immobilization of compounds with a variety of nucleophilic functional groups (Figure 3a) [18,19,20]. Kanoh et al. [21] applied a nonselective approach to immobilization that uses photoactivation on gold-coated chips used in surface plasmon resonance (SPR) imaging (Figure 3b). Finally, Pohl and co-workers [22] described a novel method for the noncovalent capture of fluorous-tagged carbohydrates on slides coated with a fluoroalkylsilane reagent (Figure 3c). This method, which takes advantage of the highly specific fluorous affinity interaction, could www.sciencedirect.com
also be applied to collections of small molecules prepared using the fluorous approach to organic synthesis [23]. The fluorous strategy can be particularly useful for the synthesis and display of small molecules biased toward specific classes of proteins, such as histone deacetylases, where a specific displayed orientation of the small molecule is desired [24]. These recent advances should prove useful to research groups interested in preparing and screening SMMs. Here, we describe the challenges and successes of developing a platform for ligand discovery involving SMMs at the Broad Institute of Harvard and MIT. Current Opinion in Chemical Biology 2007, 11:74–82
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Figure 2
Representative protein–small-molecule interactions of varying affinities discovered using SMMs. Compounds (a)–(e) are products of diversity-oriented synthesis, and compound (f) is a natural product tetrasaccharide. Proposed sites of attachment to the microarray surface are colored in red. (a) FKL-01 binds to human FKBP12 with a KD of 37.2 mM [8]. (b) Uretupamine A binds to yeast Ure2p with a KD of 18.1 mM. The ligand specifically activates a glucose-sensitive transcriptional pathway downstream of Ure2p [9]. (c) Haptamide A binds to the yeast transcription factor Hap3p with a KD of 5.0 mM and inhibits Hap2/3/4/5p-mediated transcription [10]. (d) AMD03 binds to human IgG with a KD of 2.0 mM [11]. (e) Calmoduphilin binds to human calmodulin with a KD of 121 nM [12]. (f) Chondroitin sulfate-E tetrasaccharide binds to TNF-a and antagonizes the interaction of the cytokine with the cell surface receptor [13]. (g) KKLF is a selective inhibitor of cathepsin K [14]. (h) KLLL is a selective inhibitor of cathepsin F [14].
Pipeline for ligand discovery using SMMs at the Broad Institute: a case study The chemical biology program at the Broad Institute of Harvard and MIT aims to develop systematic ways to explore biology using small molecules. Scientists from a range of disciplines, including chemistry, biology, Current Opinion in Chemical Biology 2007, 11:74–82
computational science and engineering, all work cooperatively toward this goal. The use of synthetic chemistry and HTS, coupled with computational analysis, enables biologists to discover new research tools for biology, and enables chemists to understand how specific chemical properties influence particular biological processes. SMM www.sciencedirect.com
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Figure 3
Recent immobilization strategies for the preparation of SMMs. (a) Isocyanate surfaces can react with a variety of nucleophilic functional groups. Compounds containing multiple nucleophilic functional groups have the potential to be displayed in varying orientations in a given printed spot [18,19,20]. (b) Small molecules can be covalently immobilized, using photoactivated capture, onto aryl-diazirine-coated gold surfaces for label-free detection of proteins using SPR [21]. (c) Selective and noncovalent capture of fluorous-tagged compounds onto fluorous-coated glass surfaces takes advantage of the hydrophobic effect [22]. Additional immobilization strategies were reviewed by Uttamchandani et al. [7].
technology is one of the platforms used at the Broad Institute to aid the discovery of new protein-specific probes. We collaborate with groups interested in screening SMMs with collections of proteins relevant to various areas of disease biology, including psychiatric and infectious www.sciencedirect.com
diseases, and functional protein classes, such as transcription factors. Additionally, as part of an interaction with the National Cancer Institute’s Initiative for Chemical Genetics (ICG), SMMs are screened against several proteins that have a role in cancer. A brief description of the SMM pipeline is provided for those who have an interest in screening using this method. Current Opinion in Chemical Biology 2007, 11:74–82
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Compound curation
SMMs prepared at the Broad Institute contain compounds originating from a variety of sources. Traditionally, SMMs have contained compounds prepared using diversity-oriented synthesis (DOS) [25,26]. These compounds are equipped with a specific reactive appendage, such as an alcohol, that mediates attachment to the slide [7,8–10,12]. Recent advances in nonselective capture chemistry have led to an expanded printable collection that includes synthetic compounds, natural products, known bioactives and FDA-approved drugs [19,20]. The active screening collection includes over 100 000 compounds coming from commercial sources [4,27], inhouse synthetic chemistry efforts [28–30] and donations from synthetic chemistry laboratories around North America [4,27,31–36]. The SMMs also contain compounds prepared through the National Institute of General Medical Sciences Centers of Excellence in Chemical Methodologies and Library Development (CMLDs) at Boston University, Harvard University and the University of Pittsburgh (for an overview see: http://www.nigms.nih.gov/Initiatives/CMLD/). SMM preparation
In an effort to expand the functional group compatibility of SMMs, we recently adopted the isocyanate-mediated capture shown in Figure 3a [19]. Isocyanates react with several nucleophilic functional groups, thereby increasing the diversity of small molecules, from natural or synthetic sources, that can be immobilized on a single SMM. To date, we have printed roughly 3000 natural products, 6000 commercial compounds and 17 000 compounds provided by synthetic organic chemists from academia using this approach. A recent analysis of the functional groups present in the compounds from the screening collection at the Broad Institute suggests that roughly 70% of the compounds can be covalently immobilized on SMMs using the isocyanate-functionalized slides. A detailed protocol for making SMMs, which used isocyanate chemistry with the suggested equipment setup, reagent setup and known protein–small-molecule interaction quality controls, was recently published [20]. Executing a screen
Most protein-binding screens involve the use of purified and epitope-tagged proteins and binding is detected using a fluorescently labeled antibody against the tag, as depicted in Figure 1. Typically, proteins are screened at a concentration of 1 mg/ml in a buffer that is most appropriate to preserve stability or activity of the target. Triplicate data are collected for each protein against a given set of compounds. Overall, each screen consumes roughly 1–10 mg of protein sample, depending on the method used to incubate the protein solution with the SMMs [20]. More recently, screens involving complex protein mixtures and mammalian cell lysates have been performed [19,37]. This type of screen is becoming Current Opinion in Chemical Biology 2007, 11:74–82
routine as it removes the need for protein purification. More importantly, proteins obtained from cell lysates are more likely to retain proper conformations as well as possess post-translational modifications or interact with protein partners associated with a specific activity or tertiary structure. Unfortunately, to date a general method for screening integral membrane proteins has not been developed. Assay development projects aimed at integral membrane proteins are underway. Immediately after a new set of compounds is printed, a panel of readily available pure proteins is screened to identify promiscuous binders (Figure 4a,b). Data from the ‘specificity’ set are useful in helping a screener to focus on compounds that are relevant to their protein of interest. The goal is to eventually make composite Z-scores for all SMMs available to all screeners at the Broad Institute through ChemBank (http://www.chembank.broad.harvard.edu/) [4]. Chembank is a publicly available webbased database and application that stores information about the small molecules in the screening collection and data from assays [38,39]. To satisfy the conflicting needs of enabling scientific research and ensuring that experiments developed by the original scientist are free of immediate competition from the entire research community, a time delay of 12 months has been instituted between the time of data collection and public data dissemination. This delay allows the data to be confirmed in secondary assays and typically provides sufficient time for the original screener to publish their findings. All chemists and biologists who participate in the Broad Institute screening program are free to access each other’s data once they sign a data sharing agreement (DSA). This agreement establishes the ground rules for collaboration and proper use of another screener’s data [4]. A second version of ChemBank (DSA ChemBank), which contains information about approximately 1200 assays, is available only to those who sign the agreement. After the one year delay, the DSA ChemBank datasets are migrated to the public ChemBank site. Allowing screeners to access SMM data helps to prioritize candidates for further study based on whether a compound is specific for the target, a promiscuous binder, or a positive in a related assay involving a protein of similar structure or function. Additionally, screeners might find that compounds that score in the SMM assay also score in phenotypic assays. It is our hope that the intersection of SMM data and phenotypic data in ChemBank will lead to target ID hypotheses. Protein-binding data are a relatively new addition to ChemBank and we hope to make more data publicly available in the year to come. Secondary binding assays
The fluorescence intensity of a SMM positive is not a direct indicator of the binding affinity between a given small molecule and the protein target. Although a correlation between fluorescence intensity and affinity is often www.sciencedirect.com
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Figure 4
Characterization of microarray positives in secondary protein-binding assays. (a) A heat map of composite Z-scores sorted by compound ID was constructed for the following ten protein-binding screens involving SMMs: (1) bovine carbonic anhydrase II, (2) streptavidin, (3) fibrinogen, (4) wheat germ agglutinin, (5) concanavalin A, (6) calmodulin with calcium, (7) calmodulin with EGTA, (8) bungarotoxin, (9) bovine serum albumin, and (10) anti-5xHis monoclonal antibody. (b) Selected compounds with high composite Z-scores (>3) for either calmodulin condition (6 and 7) are shown in another heat map. (c) Secondary binding assays involving fluorescence-based thermal shifts were performed for selected positives and the melting curves are shown [44]. (d) SPR binding assays were conducted for a candidate ligand to calmodulin under both screening conditions (6 and 7) [40]. (e) Chemical structure and common name for SMP2_000044 (see also http://chembank.broad.harvard.edu/chemistry/ viewMolecule.htm?cbid=907437).
observed, fluorescence intensity can vary as a function of compound concentration, reactivity toward the slide surface and heterogeneity within printed features caused by multiple displayed orientations of the printed small molecule. Secondary binding assays are routinely used to confirm candidate interactions and measure binding affinities, as well as to rank ligands for further characterization in functional assays. Two types of secondary binding assays used at the Broad Institute to achieve these goals are described hereafter. The first assay involves SPR using a Biacore1 S51 Biosensor. SPR-based interaction analysis is an established method for characterizing the affinities between proteins and small molecules [40,41,42]. Secondary assays using SPR are performed with the same protein and smallmolecule stock reagents used in the primary SMM screen. In contrast to SMM assays, the protein is directly immobilized or captured with an antibody on the biosensor surface and the small molecule is injected onto the surface in solution phase. This assay orientation helps to annotate small molecules of weak affinity that score as positives in the primary assay, possibly because of avidity effects as a consequence of display on the microarray. www.sciencedirect.com
Compound solubility is also evaluated before injection onto the biosensor surface. Compounds that are insoluble at 10 mM in buffer with 5% (v/v) DMSO are rejected before the SPR assay so as to avoid invalidating data from subsequent injections. Compounds with poor solubility under these conditions are unlikely to be soluble under standard conditions for enzymatic or cell-based assays [43]. SPR is used to evaluate SMM positives in two types of assays. In the first assay, called a ‘yes/no’ assay, 1–2 ml of stock solution is cherry-picked from a plate and injected with a limited dilution series so as to increase the number of compounds tested per day. A negative control, such as streptavidin or GST, is tested simultaneously with the target to assess specificity. Positives in the yes/no assay are subjected to a second ‘compound characterization’ SPR assay. The second assay measures the binding with an extensive dilution series and additional replicates to gain information about affinity and binding kinetics (Figure 4d). Benefits of the SPR assay include minimal consumption of reagents, semi-automated sample delivery, diverse Current Opinion in Chemical Biology 2007, 11:74–82
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capture strategies for proteins, and user-friendly control and data analysis software. The high cost of instrumentation and consumables, and sequential or iterative assay development are disadvantages of the assay. Recently, high demand for SPR created a need for a complimentary yes/no assay, preferably with a higher throughput, to confirm putative interactions. The thermal-shift assay, also known as ThermoFluor1, was recently adopted to confirm and rank SMM positives [44]. This technique has been reviewed as a method for detecting biomolecular interactions as part of a drug discovery pipeline [45]. The thermal-shift assay detects protein–ligand interactions by monitoring ligand-dependent thermal stabilization of a target protein (Figure 4c). Protein melting is monitored using an environmentally sensitive dye, such as SYPRO1 Orange, which binds to hydrophobic residues that are normally found in the interior of a folded protein. The thermal-shift assay has been used to identify small-molecule additives to aid in protein crystallization [46] and to confirm and characterize enzyme inhibitors [47,48]. The assay can be carried out in 96-well plates using a quantitative PCR instrument to control temperature and measure fluorescence [46]. Advantages of the thermal-shift assay, relative to the SPR, include higher sample throughput and lower cost of instrumentation and consumables. A typical assay is finished in 60 minutes and enables more samples to be tested, relative to SPR, in the same time frame. It has been reported that compounds that exhibit nonspecific inhibition for different classes of enzymes result in negative melting temperature shifts, thus enabling identification of these compounds early in the discovery process [43,45]. Several disadvantages should be noted. Proteins with solvent-exposed hydrophobic patches, such as bovine serum albumin, result in high initial fluorescence that obscure the melting curve. Proteins composed of several non-energetically connected domains, such as fusion proteins, might unfold at different temperatures. If the melting temperatures are not widely separated, reliable calculation of the melting temperature becomes difficult. Although a mathematical framework has been developed to link the change in melting temperature to a measurement of affinity, the thermal-shift assay is typically applied qualitatively to rank the affinity of compounds [44]. Specific compound classes, such as chelators, can destabilize protein targets and complicate the analysis. Despite the disadvantages, thermal-shift yes/no assays are typically performed when a large number of positives are identified from primary SMM screen. Compounds that perturb the melting curve are subsequently tested in SPR compound characterization assays or functional assays. SPR-based yes/no assays are typically performed for SMM datasets that contain less than 30 positives. HistoriCurrent Opinion in Chemical Biology 2007, 11:74–82
cally, over 80% of SMM positives also score as binders in SPR or thermal-shift assays, and dissociation constants typically fall in the range of 0.1–20 mM. Figure 4 illustrates the pipeline for SMM, thermal-shift and SPR assays, leading to a modest ligand for calmodulin, NPC-15437, which is a known inhibitor of protein kinase C [49]. Additional studies are underway to characterize the nature of the interaction for this representative positive.
Conclusions Small-molecule microarrays (SMMs) have proven to be a general tool in the discovery of new protein–smallmolecule interactions. SMMs are a key component of the ligand discovery pipeline at the Broad Institute and can be used to complement datasets from phenotypic assays involving the same compounds. The assay format is constantly evolving. Many laboratories have developed new methods of preparing the arrays or have reported new screening applications that go beyond simple searches for binding interactions. Several laboratories have reported SMM-based enzyme assays and inhibitor fingerprinting studies [14,50,51]. SMMs show promise for diagnostic applications in the agricultural industry and in the clinic [52,53]. SMMs should be explored further as a platform for generating signatures of cell type or cell state by monitoring changes in protein–small-molecule interactions from cell lysates [19,54]. Once signature ligands (i.e. small molecules that distinguish between two states) are identified, they might be used as affinity reagents to identify protein biomarkers and can be studied further in a broad array of phenotypic assays. Finally, SMMs compatible with use in whole cells are being developed for studying interactions at cell surfaces and as a platform for performing highly miniaturized cell-based screens [55,56]. Hopefully, all of these developments will add to the small-molecule toolkit for deciphering the functional proteome.
Acknowledgements Worked described herein has been funded in whole or in part with Federal funds from the National Cancer Institute’s Initiative for Chemical Genetics, National Institutes of Health(Contract No. N01-CO-12400). The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Service, nor does mention of trade names, commercial products or organizations imply endorsement by the US Government. The authors also acknowledge Nicole Bodycombe, Anna Borodovsky, Scott Eliasof, John McGrath, Olivia McPherson, Martin Serrano and Nicola Tolliday for their contributions to small-molecule microarray screening platform at the Broad Institute.
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