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PERSPECTIVES IN PHARMACOLOGY

Ligand Discovery Using Small Molecule Microarrays

Department of Pharmacology, Weill Medical College, Cornell University, New York, New York

Received for publication August 31, 2004
Accepted November 9, 2004.

	Abstract

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Small molecule microarrays have recently been used to identify ligands for several proteins, and several themes regarding screening strategies and limitations have emerged. In this review, some of the technical issues related to the manufacture and screening of small molecule microarrays, as well as prospects for small molecule microarrays in several areas of drug discovery and chemistry, are discussed.

	Historical Foundation of Small Molecule Microarrays

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On-Bead Screening
Small molecule microarrays merge the relatively mature technology of on-bead library screening with microarray technology, the latter of which has become an especially powerful tool used primarily by molecular biologists in the context of DNA microarrays. Screening immobilized libraries of small molecules on beads was described in one of the original reports that described the technique of split-pool library synthesis (Lam et al., 1991). The split-pool approach (also known as "portioning-mixing" and related names) involves dividing polymeric supports (i.e., the "beads") into portions for coupling to modular units (i.e., "diversity elements") and then recombining the beads (Furka et al., 1991; Houghten et al., 1991; Lam et al., 1991). Once combined, the beads are washed, deprotected, etc., and the process of splitting the beads and pooling the beads is repeated for several cycles to make a large collection of molecules. Thus, each bead will contain molecules whose structure is a consequence of the history of the diversity element coupling steps to which the bead was subjected. This method is also known as the one bead-one compound method (Lam et al., 1991) since each bead contains one type of molecule, although each bead will contain >10¹³ copies of each molecule.

To screen the molecules on the bead, a labeled protein is incubated with the beads. Beads that contain a molecule that interacts with the labeled protein are then detected via the label on the protein. For example, the protein may be labeled with biotin, and protein-bound beads may be detected by incubating the beads with streptavidin-alkaline phosphatase (AP). Incubation of the AP-bead complex with a chromogenic AP substrate results in the generation of a purple dye that partitions into the hydrophobic interior of the bead, resulting in the beads turning purple (Lam et al., 1991). The beads can then be detected visually and isolated to determine the identity of the molecule on the bead.

This and related approaches have been used successfully to find peptide ligands for several proteins such as streptavidin (Lam et al., 1991) and the src SH3 domain (Morken et al., 1998), ligands for RNAs (Hwang et al., 2003), and peptoid ligands for mdm2 and glutathione S-transferase (Alluri et al., 2003). Up to 1 to 2 million compounds per day can be queried on beads. Once a bead has been identified as possessing a molecule that binds to a specific protein probe, the structure of the molecule on the bead has to be determined. For peptide and peptoid libraries, the molecule on the bead can be eluted and determined by Edman degradation. However, for other libraries, determining the identity of the molecule on the bead can be very difficult—typically not enough is available for NMR and mass spectometry can be ambiguous. A popular approach is to use an encoding scheme during the synthesis of the libraries. Thus, during the steps where diversity elements are coupled, a chemical tag that reflects the identity of the diversity element is covalently coupled to the bead. At the end of the synthesis, the history of the coupling steps can be determined by liberating the tags and detecting them chromatographically (Ohlmeyer et al., 1993). If encoding can be readily performed, on-bead screening followed by deconvolution of the encoding tag can be an exceptionally high-throughput method for ligand discovery.

Microarray-Based Screens
A second emerging technology that helped to bring about small molecule microarrays was the DNA microarray technology that was popularized in the 1990s. Microarray technologies involving several types of macromolecules have existed since the mid 1980s (Ekins et al., 1996; Ekins and Chu, 1999) but have recently become highly popularized and accessible with the advent of DNA microarrays. Microarrays consist of small spots ("microspots") of a few micrometers in diameter, each containing specific molecules, such as DNA, proteins, or small molecules. These spots are arrayed on a surface, most commonly silica or glass, due to the ease with which it can be functionalized and its chemical stability and low intrinsic fluorescence. Although glass has been the principle support for small molecule microarrays, molecules have been immobilized on a wide variety of other materials, such as gold, ceramics, polyacrylamide, and nitrocellulose, and these can also be used for the generation of microarrays.

Microarrays have specific benefits that have made them useful for analytical studies. These include the ability to mass-produce large numbers of arrays, the ability to perform massively parallel binding assays, the rapid kinetics of binding that occurs upon miniaturization, and the high sensitivity of detection of binding to microarrays that results from concentrating analyte-binding molecules to a microspot.

Experiments with DNA microarrays exemplify the beneficial features of microarray technology. DNA microarrays permit the quantification of up to tens of thousands polynucleotides in a sample in a single experiment. Thus, the DNA microarray contains thousands of microspots, each containing a specific DNA sequence that can detect a complementary analyte. These microspots are arranged in a grid such that each DNA is spatially addressable. The binding of fluorescently labeled polynucleotides, prepared enzymatically from cellular lysates, allows quantification of the abundance of specific polynucleotides by quantifying the fluorescence at each microspot. This quantification can be assessed by microarray-scanning instruments that rapidly quantitate the fluorescence at each microspot.

Although microarray screening is massively parallel, a key property of this type of screen is that it permits a higher degree of sensitivity than can be obtained by other binding assays. The high degree of sensitivity derives from the high density of receptor on the glass surface in the microspot. Thus, the labeled analyte is detected in a small area in which the background nonspecific signal is minimized. Furthermore, the degree of binding is predictably related to the concentration of labeled probe. As the size of the spot shrinks, the fractional occupancy of the receptors in the microspot, F, is related to the K_D of the analyte, such that F is equal to [A]/(K_D + [A]), where [A] is the analyte concentration (Ekins, 1998).

	Manufacture of Small Molecule Microarrays

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Small Molecule Microarrays from Combinatorial Libraries
Small molecule microarrays can be prepared by either first synthesizing the molecules and then immobilizing them on the surface or by synthesizing them directly on the surface. In the first approach, stock solutions of library members are arrayed using a commercial microarraying robot such as those used for preparing DNA microarrays. High-capacity synthetic beads can be used to generate microliter volume stock solutions with concentrations >1 mM (Tallarico et al., 2001). Subnanoliter volumes are dispensed to generate spots with diameters typically between 50 and 500 µm. These molecules can be covalently coupled to the glass surface if the small molecule contains chemical moieties that react with functional groups that have been incorporated onto the glass surface. For example, Schreiber and colleagues (Hergenrother et al., 2000) describe the generation of alcohol-reactive glass slides that were activated with silyl chloride. Alcohol-containing small molecules were generated on 500-µm polystyrene macrobeads, eluted and resuspended at a concentration of 5 mM in 5 µl of dimethyl fluoride, and then immobilized on the glass slide using a microarraying robot that dispensed 1 nl per spot (Hergenrother et al., 2000). Thus, each stock solution is sufficient to produce a spot on several thousand microarrays. In another study, thiol-containing molecules were immobilized on maleimide-functionalized glass surfaces (MacBeath et al., 1999). If the library member has more than one hydroxyl in the first case or thiol in the second case, the library member may be displayed heterogeneously throughout the spot, limiting the number of hydroxyls that can be present in library members.

The strategy taken by Lam and colleagues (Falsey et al., 2001) seems to reduce these limitations on library construction by employing chemoselective ligation to covalently couple the small molecule to the glass surface. In this case, the authors used the selective reaction of an N-terminal cysteine with immobilized glyoxal to link peptides to glass via a thiazolidine. Small molecule libraries that contain cysteine-like moieties would also be expected to couple to these surfaces. A variety of other chemoselective ligation reactions exist that could be used for coupling small molecules to solid supports (Kohn et al., 2003).

Once the microarray is prepared, the microspots can be screened using a labeled probe. In most cases thus far, this has been a fluorescently tagged protein. Microspots that become fluorescent after incubation with the probe indicate a small molecule-probe interaction. When the microarray is prepared using a split-pool library, the identity of the small molecule spotted at any position in the microarray can be determined by recovering the specific bead that generated the molecule at that position and then by eluting the encoding tags that were used during the library synthesis (Kuruvilla et al., 2002; Barnes-Seeman et al., 2003; Koehler et al., 2003).

Screening these microarrays can be problematic due to steric hindrance from the surface. One factor that could affect the binding is the length of the linker used to separate the glass surface from the small molecule (Barnes-Seeman et al., 2003). Alternatively, the chemical properties of the linker may affect binding. In one study of linkers for on-bead screening, it was found that cationic linkers had a substantially beneficial effect on ligand accessibility (Thorpe and Walle, 2000).

	Ligand Identification Using Small Molecule Microarrays

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Screening Microarrays for Hits Using Binding Assays
The product of a small molecule microarray screen is a molecule that interacts with the protein that is used as the probe. However, these molecules will not be useful if they do not bind to functional sites on the protein. Indeed, proteins have large surface areas, and the relevant active site is a small fraction of this area. Thus, the likelihood that a ligand would bind this particular area on the surface of a protein is presumably very small. Despite this, data from several varied approaches suggest that ligands identified by binding may preferentially target functionally important sites.

One example is selective enrichment of ligands by exponential enrichment. This is a highly effective approach for generating RNA ligands for target proteins using binding to select for ligands (Famulok et al., 2000). In this procedure, 10¹⁵ individual RNAs are generated, and RNAs that bind a protein target are recovered and amplified by reverse transcriptase-polymerase chain reaction. Frequently, these ligands bind with nanomolar or subnanomolar affinities (Gold et al., 1995). When RNA ligands are generated against an enzyme, they frequently bind to the active site (Faria and Ulrich, 2002). For example, several RNA ligands were generated that bind to HIV reverse transcriptase, and despite the fact that they lacked sequence similarity to each other, each was found to be a potent inhibitor of the enzyme, several with IC₅₀ values in the mid-nanomolar range (Tuerk et al., 1992; Allen et al., 1996). The crystal structure of an aptamer-HIV reverse transcriptase complex confirmed that the RNA binds to the active site (Jaeger et al., 1998).

Another ligand-generating approach that uses binding assays to select for ligands is phage display (Cwirla et al., 1990; Devlin et al., 1990; Scott and Smith, 1990). In this procedure, peptides are presented on the surface of filamentous bacteriophage. To identify peptide ligands that bind a specific protein, a phage-binding assay is used to recover phage that interact with an immobilized protein target. One study systematically explored the ability of phage display-derived peptides to target active sites in enzymes (Hyde-DeRuyscher et al., 2000). In this study, several diverse enzymes were queried: alcohol dehydrogenase, tyrosyl- and prolyl-tRNA synthetases, carboxypeptidase, -glucosidase, hexokinase, and glycogen phosphorylase. In all cases, except for carboxypeptidase, phage display peptides were recovered that inhibited enzyme activity, each in the high-nanomolar to mid-micromolar range (Hyde-DeRuyscher et al., 2000).

In addition to these approaches, ligands generated from screening small molecule microarrays directly demonstrate that ligands discovered by screening with protein probes bind to functionally important sites on proteins. In a study to find ligands for Ure2p, a yeast protein that binds certain transcription factors, a screen of 3780 compounds based on a functionalized 1,3-dioxane skeleton resulted in the identification of eight ligands for Ure2p, one of which inhibited Ure2p's repressive activity in yeast cells (Kuruvilla et al., 2002). This ligand, uretupamine A, bound to Ure2p with a K_D of 18.1 µM. The other seven ligands failed to inhibit Ure2p, but this may have been due to poor cell permeability. Alternatively, Ure2p may have multiple functionally important sites, only one of which was assayed in the derepression assay, and the other ligands bind to one of these sites. In another study, a microarray of 12,396 compounds was used to find ligands for Hap3p, a yeast transcription factor (Koehler et al., 2003). In this study, a single ligand was identified as binding the Hap3p fusion protein, and this compound inhibited Hap3p-dependent transcription in cellular assays. In both these cases, the ligand presumably blocked a protein-protein interaction, which is considered exceptionally difficult to target (Spencer, 1998). These data further suggest that binding assays used by small molecule microarrays are capable of generating ligands that target functionally important sites, although each ligand needs to be individually tested to determine whether it targets the desired site on the protein.

These studies raise the intriguing question: Why do screens that use binding to recover ligands result in ligands that target functional sites? Binding sites typically are depressions on the surface of proteins, have a higher degree of exposure of hydrophobic groups on the surface than average, have residues that are highly mobile compared with residues elsewhere on the surface of the protein, and contain water molecules that are not tightly bound to the protein (Ringe, 1995; Ringe and Mattos, 1999). Ringe and associates have proposed that these displaceable water molecules provide the gain in entropy that propels the ligand-binding reaction (Ringe and Mattos, 1999). Thus, the unique properties of binding sites may account for the tendency of binding experiments to yield ligands that interact with protein binding sites.

This concept is supported by structural and thermodynamic studies of ligand-protein interactions (Arkin and Wells, 2004). In these studies, the binding energy for a ligand-protein is not distributed evenly across the interface but is localized to small regions of the protein. These regions are characterized by charged and hydrophobic residues, often less than five amino acids, that represent energetic focal points of the interaction (Clackson and Wells, 1995; Clackson et al., 1998; DeLano et al., 2000). Importantly, phage display-derived ligands bind to hot spots for protein-protein interactions (Fairbrother et al., 1998; DeLano et al., 2000). Furthermore, small molecules that bind interleukin-2, bind to the hot spot that mediates its interaction with the interleukin-2 receptor (Thanos et al., 2003). Thus, small molecules and peptides target hot spots. In addition to having hot spots that have a propensity to interact with ligands, proteins may have undergone evolutionary selection to reduce the "stickiness" of nonbinding portions of protein surfaces to reduce irrelevant interactions that may occur due to the high concentration of intracellular molecules in the cytosol.

To "force" a small molecule microarray screen to yield ligands that target a particular site, one could imagine simple adjustments to the screening protocol. For example, to confirm that ligands are binding to an active site, the screen can be performed with two proteins, one containing a mutation or deletion at the active site. Each of the proteins could be labeled with a different fluorescent tag, e.g., Cy3 and Cy5. Thus, microspots that show binding of both proteins would represent ligands that do not bind to the region of interest.

Affinity Limits in Microarray Screening
A fundamental difference between microarray binding assays and activity assays is that low-affinity binding cannot be detected in a microarray format. In most library screens using activity assays, K_D values of hits in the range of 0.1 to 1.0 µM would be considered successful. However, in microarray screens, hits with K_D values in this range may be so weak and transient that the labeled protein will be lost in the washes. This phenomenon is due to the high rate of dissociation for interactions with K_D values in this range. The K_D is the ratio of two kinetic parameters, i.e., k_off/k_on. Although k_on varies for different ligands, k_on values generally tend to be within an order of magnitude of 10⁶ M^–1s^–1. Thus, for a protein-ligand interaction with a K_D of 100 nM and a k_on = 1 x 10⁶ M^–1s^–1, the value of k_off is 1 x 10^–1 s^–1. The dissociation half-life is 0.693/k_off, meaning that the dissociation half-life is 6.93 s. Thus, if a microarray is washed for as little as 3 min, over 25 half-lives of dissociation will occur. This will completely deplete all the bound protein bound to a microspot. Thus, certain classes of interactions are selected when using a microarray approach: nanomolar-affinity interactions, interactions that have large k_on, or interactions that are associated with additional conformational changes that stabilize the initial receptor-ligand complex. Because of the relationship between k_off and K_D, high-affinity interactions seen on microarrays can be distinguished from lower affinity interactions in part by their stability to washing.

Since a library may be devoid of any ligand that binds a protein probe with the requisite affinity to survive washings, dimeric probes can be used to detect ligands that bind with lower affinity. As opposed to monomeric probes, dimeric probes exhibit a higher effective affinity when binding to a surface. This avidity effect is due to the simultaneous binding of immobilized ligand by the dimeric probe. For example, in the screen to identify Hap3p ligands, Hap3p was fused to the dimeric glutathione S-transferase moiety for screening (Koehler et al., 2003). Screens using monomeric protein, e.g., His-tagged fusion proteins, can lead to the identification of ligands that have higher affinity.

Factors Affecting the Success of the Small Molecule Microarray Screens
Library Design. Although small molecule microarrays may permit screening tens of thousands of compounds, it is still far from guaranteed that a library screen will identify a small molecule that binds to a target with high affinity. The problem is compounded when little is known about likely structural features of the protein's physiologic interacting partners, thus necessitating the use of an unbiased library. How can the probability of success be improved? Probably the factor that may most affect the success of a screen is the size of the library. This idea makes sense—if you screen enough molecules, sooner or later you will come upon a hit. However, the relationship between the size of a library and the number of hits remains to be established. For example, when a library screen that uses 10⁵ members fails, is it because the specific skeletal structure used by the library is wholly incapable of generating a hit, or would the use of 10⁶,10⁷, or even 10⁸ members have resulted in a hit? Another approach is the design of libraries that have features of natural products (Nicolaou et al., 2000; Breinbauer et al., 2002; Kissau et al., 2003). The rationale for these approaches is based in the concept of the "privileged structure" or "privileged substructure" (Evans et al., 1988), which postulates that certain structures (such as benzodiazepines and benzazepines) have an intrinsic ability to bind multiple, unrelated classes of proteins. Microarrays that use libraries of privileged structures will help to address whether these molecules are more likely to generate ligands that other types of libraries.

Choice of Target. The success of the screen will also depend on the target. In a review of 150 (nonmicroarray) screens at Pfizer, Inc. to determine hit rates, it was shown that the hit rate was 1 per 120,000 compounds for enzymes and significantly less for small molecules that targeted protein-protein interactions (Spencer, 1998). The difficulty in obtaining ligands that affect protein-protein interactions may arise from the nature of protein interfaces, which often are flat and large (from 800 Ų to 4660 Ų) (Clackson and Wells, 1995; Lo Conte et al., 1999). Despite these considerations, small molecule microarrays have been successful in generating ligands that seem likely to disrupt these types of interactions (Kuruvilla et al., 2002; Barnes-Seeman et al., 2003; Koehler et al., 2003).

Sensitivity and Specificity in Solid-Phase Screening. Another factor affecting the success of the screen is the sensitivity and specificity of the signal. The sensitivity of the method is determined by the nonspecific binding of the protein used as the probe to the microarray. Nonspecific binding could be to the solid support (e.g., the functionalized glass) or the specific chemical entity that is present on the microspot. In the former case, specific blocking agents could be tested and identified that reduce nonspecific binding. In the latter case, each microspot can be considered as a chemically different surface with its own nonspecific-binding properties. Resolving this nonspecific binding may be a much more difficult task. Nonspecific interactions occur when a protein makes weak contacts with multiple immobilized individual small molecules. These multiple weak nonspecific interactions produce an avidity effect leading to tenacious protein binding to the microspot. Methods to reduce nonspecific binding need to be chemically complex to deal with the varied and idiosyncratic nonspecific binding that will be encountered at each microspot. In some cases, bovine serum albumin was used, whereas in others, highly complex proteinaceous blocking agents such as milk and clarified Escherichia coli lysate (Alluri et al., 2003) were required to reduce nonspecific binding, as well as incubation buffers and salt washes as high as 1 M (Alluri et al., 2003). These approaches will presumably also interfere with specific binding, thus reducing the likelihood of success with this approach. Nevertheless, if the libraries are constructed with the idea of reducing nonspecific binding and appropriate blocking techniques are implemented, blocking techniques can allow sensitive detection of binding.

In cases where background binding can be resolved, sensitivity can be improved by novel detection methods. Fluorescence tags typically cannot be used for screening on-bead libraries due to the intrinsic fluorescence of most polystyrene-based synthetic beads. However, newer quantum dot-based labels seem to overcome this problem (Olivos et al., 2003) and have the potential to increase the sensitivity of screening on small molecule microarrays as well. Microarray screens that use fluorescence microscopy techniques such as those based on evanescent wave microscopy have the potential to substantially increase the sensitivity of these screens (Neuschafer et al., 2003).

Although issues regarding library size and features affect the success of any screen—on a microarray, on beads, or in a conventional solution-phase screening assay—small molecule microarray screening has a distinct advantage over the other approaches in terms of the ease of screening. Thus, multiple copies of a microarray can be fabricated, and each can be screened against a different protein. Thus, even if the library on the microarray is too small to be assured of finding a ligand for a given protein, the likelihood of coming up with a ligand for some protein is improved by the ease of screening multiple proteins in parallel. In the case of on-bead screening, beads could conceivably be washed after being screened by individual proteins, but the screening must be accomplished serially, and detecting hits is more tedious.

	Future Prospects

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Manufacturing Small Molecule Microarrays by Photolithography
An alternative approach for the preparation of small molecule microarrays is in situ synthesis of the small molecules on the glass surface. This has been performed using photolithographic synthesis. In the most common implementation of this method, chemical synthesis is achieved by initiating the removal of photolabile protecting groups by directing ultraviolet light to specific sites using a chrome/glass mask. After deprotection at specific sites, the entire array is exposed to a reactant, but only the deprotected sites react. Next, other sites that were not deprotected are deprotected in a spatially specific manner using the mask. This procedure is limited by the need to generate specific masks to match the pattern of photodeprotection, which can be a costly and time-consuming process. Nevertheless, small molecule microarrays consisting of peptides (Fodor et al., 1991), oligocarbamates (Cho et al., 1993), nucleotides (Pease et al., 1994), and peptoids (Li et al., 2004) have been described.

The technique of maskless photolithography has been described previously (Singh-Gasson et al., 1999). In this technique, a digital micromirror array, such as those used in computer display projection systems, is used to project ultraviolet light in a specific pattern on the microarray to initiate highly spatially specific photodeprotection. This method has been used to synthesize oligonucleotide (Nuwaysir et al., 2002) and peptoid (Li et al., 2004) microarrays. In another approach using a digital micromirror array, acid-labile protecting groups and photogenerated acids were used to achieve photolithography of oligonucleotides (LeProust et al., 2000).

Although photolithography is clearly becoming more rapid and inexpensive, its long-term viability for small molecule microarrays remains unclear, especially for chemistries less robust than peptide and oligonucleotide synthesis. In the case of nucleotide microarrays, the fidelity of the synthesis can be readily determined in situ by the ability of the microarray to recognize specific oligonucleotides; however, for the synthesis of other types of small molecules, confirmation of the synthesis, yield, and byproducts are especially difficult considering the small amounts of material on the microarray. Conceivably subjecting the microarray to matrix-assisted laser desorption ionization-based analysis, assuming matrix-assisted laser desorption ionization-labile linkers are used in the synthesis, may assist in validating the synthesis of other types of small molecules. A simpler approach is the spotting of presynthesized molecules obtained through the more routine split-pool approach. Spotting of these molecules would presumably be performed once the split-pool chemistry has been optimized. Furthermore, the in situ synthesis protocol seems exceptionally low-throughput considering that each microarray needs to be individually synthesized. Synthesizing the library by a split-pool procedure once and then making many microarray replicas is likely to be more efficient and robust.

Screening Chemical Libraries on DNA Microarrays
An innovative and fundamentally different approach for screening libraries using microarrays was described by Winssinger et al. (2001). Molecules are incubated with their target proteins in solution, and bound ligands are separated from unbound ligands using gel filtration chromatography. To couple the small molecule to an array, this group used a prefabricated spatially addressable DNA microarray. The key to this approach was the bifunctional nature of their library: one component of the molecule was the protein-interacting moiety, and the other component was a fluorescently tagged protein nucleic acid (PNA) sequence. PNAs are constructed using standard amide bond-forming chemistries yet interact in a sequence-specific manner with specific DNA sequences. The bifunctional molecule was constructed from a lysine residue such that the moiety off the amine is the protein-binding component and the moiety off the amine is the PNA. The PNA component serves to both target the small molecule to specific sites on the microarray and encode the identity of the protein-binding moiety. After recovery of the protein-bound molecules, the molecules were hybridized to the array. The fluorescence at a specific localization on the array indicates the identity of a specific molecule in the protein-bound fraction (Winssinger et al., 2001). This approach reduces the nonspecific surface binding compared to a surface comprised of a specific small molecule and reduces the steric hindrance issues associated with protein binding to immobilized small molecules. Although exceptionally clever, this approach is limited by the solubility of these fairly massive bifunctional molecules, possible binding of the target protein to the PNA component, and the compatibility of PNA synthesis with the synthetic steps required for nonpeptidic small molecule libraries.

Living Microarrays
Most small molecule microarrays have been screened with protein probes. An alternative way to screen is with living cells that can produce a detectable response to certain immobilized small molecules. Lam and colleagues described microarrays of immobilized peptides that were screened with WEHI231 lymphoma cells (Falsey et al., 2001). Adhesion of these cells to the microarray was monitored and resulted in the identification and confirmation of peptide molecules that adhere to these cells. This technology could be used to generate materials that promote cell adhesion or cell repulsion. Many devices and materials that are implanted in the body undergo a fibrotic reaction or endothelialization that impairs their function. For example, stents that are used to open narrowed coronary arteries are limited by their propensity to undergo restenosis as cells adhere and occlude the stent. Alternatively, physiologic assays that use green fluorescent protein, such as subcellular localization assays or transcription assays, could be designed, and microarrays could be screened by fluorescence microscopy. An alternative type of living microarray involves microarrays of noncovalently bound small molecules embedded in a high-density matrix. These microarrays would permit diffusion of small molecules into cells and could be used to study the effects on cells that grow above each microspot.

Identification of DNA, RNA, and Carbohydrate Ligands
Although the published work with small molecule microarrays has involved screening with labeled proteins, other molecules such as DNAs, RNAs, carbohydrates, and peptides could clearly be used in screens. Indeed, on-bead screening with RNA probes have resulted in the identification of ligands for the TAR RNA sequence (Hwang et al., 2003). Similarly, small molecule microarrays might be useful to identify molecules that interact with DNA in a sequence-specific manner. The relative prevalence of literature with protein probes compared with nonprotein probes may be related to the ease with which proteins can be labeled with affinity tags, such as biotin. Newer RNA-labeling techniques (Babendure et al., 2003) may facilitate screens using biosynthetically prepared polynucleotides.

Identification of Catalysts and Sensors Using Small Molecule Microarrays
Although small molecule microarrays are typically screened against labeled proteins, small molecule microarrays are likely to have utility in the identification of novel small molecule sensors and catalysts. Rather than probing small molecule arrays with proteins, microarrays can be screened with small molecules as the solution phase-screening agent. Molecules on the microarray that interact with the analyte could be identified via tags on the analyte, e.g., fluorescent tags; alternatively, environmentally sensitive fluorophores within the microarrayed molecules could be used with unlabeled solution-phase analyte probes. Screening for microspots with analyte-dependent changes in fluorescence excitation/emission properties could identify sensors that could serve as novel diagnostics. Suslick and colleagues describe the use of arrays of metalloporphyrins that change color upon exposure to various odorants (Rakow and Suslick, 2000). These odorants form complexes with the metal ion in the macrocycle. These microarrays comprise molecules that exhibit analyte-dependent patterns of reactivity. In an alternative approach, small molecule microarrays have been used to profile enzymatic activities in tissues. Gao and colleagues have prepared arrays of various chromogenic dye derivatives (Zhu et al., 2003) that become fluorescent when hydrolyzed upon incubation with solutions containing specific enzymes. Conceivably, these arrays could be used to obtain an "enzymatic profile" of different biological samples.

Similarly, novel catalysts could be detected using a microarray-based approach. Morken and colleagues (Taylor and Morken, 1998) have described approaches for thermographic selection of catalysts from an on-bead library. They exploited the fact that most chemical reactions have a measurable heat of reaction (H), and thus the progress of a reaction can be measured by monitoring beads that have undergone a temperature change. Using this principle, the authors searched for bead-bound molecules that catalyzed the hydrolysis of acetic anhydride with an infrared camera. Conceivably, reactions catalyzed by small molecules on microarrays may induce highly localized and concentrated temperature changes, permitting screening with thermal imaging.

	Footnotes

 
This work was supported by a grant from the New York Academy of Medicine, Speaker's Fund for Biomedical Research.

doi:10.1124/jpet.104.076943.

ABBREVIATIONS: AP, alkaline phosphatase; HIV, human immunodeficiency virus; PNA, PNA, protein nucleic acid.

Address correspondence to: Dr. Samie R. Jaffrey, Department of Pharmacology, Weill Medical College, Cornell University, 1300 York Avenue, Box 70, New York, NY 10021. E-mail: srj2003{at}med.cornell.edu

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