Small molecule microarrays: recent advances and applications

Mahesh Uttamchandani1, Daniel P Walsh3, Shao Q Yao1,2 and Young-Tae Chang3

Directed or exploratory drug development programs constantly seek robust screening platforms for the high delity identication and validation of potential targets. Smallmolecule microarrays (SMMs) have risen to this call by elegantly forging the capability of combinatorial chemistry in producing myriad compounds with the powerful throughput afforded by microarrays. This synergism offers scientists a versatile tool for rapid compound analysis and discovery. Microarrays of small molecules have already been successfully applied in important areas ranging from protein proling to the discovery of therapeutic leads. Recent interesting developments towards improved immobilization strategies and library creation methods, together with novel advances herein described, have set the stage for SMMs to take on wider and more routine applications in academia and industry. As a rapidly maturing technology, SMMs pave the way forward in high-throughput exploration, both in the identication of biologically signicant natural and synthetic small molecules and in harnessing their vast potential towards medicinal and diagnostic applications.
Addresses 1 Department of 3 Science Drive 2 Department of 3 Science Drive 3 Department of 10003, USA Biological Sciences, National University of Singapore, 3, Singapore 117543 Chemistry, National University of Singapore, 3, Singapore 117543 Chemistry, New York University, New York, New York,

provide unprecedented capability for the analysis of thousands of biochemical interactions simultaneously and unravel, in a single step, all possible associations [1]. Microarray-based technologies have now been extended to the high-throughput analysis of a host of biomolecules, including DNA/RNA and oligonucleotides [2], proteins [3] including membrane proteins [4], peptides [5], carbohydrates [6] small molecules [7], tissue [8] and live cells [9], with each technology possessing distinctive characteristics while providing unique opportunities. Though small-molecule microarrays (SMMs) have colloquially described small molecules immobilized on a variety of slide substrates, recent developments using small molecules indirectly sequestered on oligonucleotide microarrays have greatly extended the ways in which small molecules are deployed, thereby expanding the scope and applicability of SMMs [10,11,1214]. Small chemical molecules are efcacious in selectively and specically perturbing cellular signaling and the functions of biological targets (e.g. genes or proteins). Like mutational analysis, where the gene product is functionally perturbed, chemical genetics using small molecules gives a handle to modulate proteins and DNA, allowing various cellular mechanisms and biological pathways to be better understood, and thereby controlled [15]. Though both natural and synthetic small molecules form the backbone of contemporary therapeutics, those most successful in chemical genetic screens have been natural products. Notwithstanding, modern synthetic approaches are valuable not only in generating large repertoires of complementary compounds for parallel analysis, but also for the systematic large-scale diversication of extant natural compounds for improved efcacy [16]. Solid-phase synthesis and combinatorial chemistry have taken root as fundamental organic tools, and the hordes of compounds now available draw impetus towards even more robust screening methods, dening a movement towards greater discovery-driven, in place of hypothesis-driven, research [17]. Biological evaluation of small molecules is an important rst step in the drug discovery pipeline, and the application of a robust screening tool, such as SMMs, is thus foundational for the pursuit of drug candidates and biologically relevant compounds. MacBeath et al. rst described SMMs in 1999 [7]. The following two years of pioneering work led to alternative immobilization chemistries and the introduction of diversity-oriented synthesis (DOS) approaches [18]. In 2002, the same group successfully employed DOS in an
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Corresponding authors: Chang, Y-T (yt.chang@nyu.edu) and Yao, SQ (chmyaosq@nus.edu.sg)

Current Opinion in Chemical Biology 2005, 9:413 This review comes from a themed issue on Proteomics and genomics Edited by Benjamin F Cravatt and Thomas Kodadek Available online 8th January 2005 1367-5931/$ see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2004.12.005

Introduction
Biological systems comprise a labyrinthine network of biomolecules in the elaborate orchestration of life. The detailed and individual dissection of this cellular circuitry at the molecular level, both spatially and temporally, has traditionally been difcult. However, in the past decade, high-throughput screening methods using microarray technologies have resulted in a paradigm shift in modern biology, in which miniaturized analytical tools can now
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Small molecule microarrays: recent advances and applications Uttamchandani et al.

important early development by generating a library of 3780 encoded small molecules [19], in which the compounds were made using the so-called one-bead onecompound combinatorial approach originally developed for peptide libraries [20]. Upon being immobilized on the glass slide, these small molecules were screened against a yeast transcriptional inhibitor, Ure2p, and one compound on the microarray was found to positively bind. This hit (Kd = 7.5 mM) was subsequently found to be capable of specically inhibiting Ure2p activity in vivo and derepressing a downstream glucose-sensitive transcriptional pathway. This discovery clearly endorsed SMMs for identifying putative binders against virtually any protein of interest. Another early development in 2001 related to SMMs was that of peptide nucleic acid (PNA)-encoded small molecule libraries, developed by Schultz and coworkers. PNA tags were used to encode small molecules, thus enabling their subsequent identication by hybridization on an oligonucleotide microarray [10]. The strategy was used to screen peptide acrylates that were irreversible inhibitors of several target members of the cathepsin and caspase families [11]. A detection limit of 0.2 pmol of puried caspase-3 was attained, demonstrating the high sensitivity of the assay, which was further applied to monitoring activity of the enzyme under cytotoxic lymphocyte-mediated cell death. The method provides an effective protocol for comparative proling of multiple enzymes in a convenient miniaturized format. This burgeoning eld has since seen a urry of activity, with many developments offering improved strategies for small-molecule immobilization and array creation, synthetic strategies for diverse library generation and novel technological developments inspired by creative ideas. This opinion reviews the recent work that has made signicant progress in each of these themes and highlights areas that will benet from further development.

approach by Germeroth et al. synthesized the libraries directly on the substrate [21]. By adopting SPOTTM synthesis [22], which is typically used to synthesize peptides, the researchers synthesized 1,3,5-triazines directly on membranes (arrays). The triazine libraries were synthesized on amino-functionalized polypropylene and cellulose membranes. The synthesis took advantage of the known reactivity differences upon subsequent substitutions to cyanuric chloride, and the reagents were discreetly pipetted onto the membrane to form the microspots. For compound characterization, the molecules were released from the membrane by cutting out the desired spot and performing an acidic cleavage step. However, a disadvantage of this method is the low purity of the compounds that limits these molecules to a primary screening role. By contrast, the bulk of libraries used in SMMs are synthesized cleanly before attachment to the microarray. As mentioned earlier, DOS-generated libraries were used to great effect by Schreiber et al. in dissecting a glucose signaling pathway by identifying ligands of the protein Ure2p [19]. The library was a collection of 3780 1,3dioxane small molecules possessing a high degree of diversity. The molecules were prepared using a one bead-one stock solution split-pool approach [2325]. As a hallmark of DOS libraries, the overall library design was structurally unbiased. The 1,3-dioxane scaffold was selected for its rigidity, a property desirable in pharmacophores, its high degree of stereocontrol, and the highly pure synthesis possible with many diverse ancillary groups (Figure 1a-1) [24]. The hydroxyl group exposed after cleavage provides the anchoring point needed to immobilize the compounds onto the array. The Schreiber group expanded DOS to include compounds that have dramatic differences in their skeletal structures. 6336 phenol-containing fused bicycles and tetracycles from a six step stereoselective synthesis afforded products with two to four rings and up to six stereocenters through a one bead- one stock method. (Figure 1a-2) [26,27,28]. These compounds were coupled with an array substrate functionality that chemoselectively reacts with acidic proton-bearing heteroatoms, in this case phenol. Schreiber again exploited hydroxyl functionalized molecules for immobilization with chlorinated glass slides [29]. A combination of three libraries was employed for a total of 12 396 immobilized species on the array. One class of compounds stemmed from the previously seen 1,3-dioxane synthetic pathway (Figure 1a-1). The two other libraries included biaryl medium rings (Figure 1a-3) synthesized from a one bead-one stock solution and spatially segregated dihydropyrancarboxamides synthesized from a DOS pathway incorporating asymmetric catalysis (Figure 1a-4) [30,31].
Current Opinion in Chemical Biology 2005, 9:413

Library design and synthesis

It is anticipated that effective patterning of small molecules on an array in a dened manner using virtually any functional group class will soon become possible, with diminished concern over their binding compatibility with the substrate surface. As substrate technologies advance in tandem with the sophisticated immobilization chemistries, this hope may one day materialize as the upcoming sections resoundingly afrm, recent contributions have already made signicant headway toward this goal. Until a truly universal substrate platform can be developed, however, a serious limitation is that library constituents must possess some functional group that can react predictably and chemoselectively with a corresponding functional group on the substrate surface, thus necessitating ligand design consideration. Several strategies exist for developing libraries compatible with SMM immobilization strategies. One early
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6 Proteomics and genomics

Figure 1

(a)

H2N n = 0,1 O X R2 R1 O

To immobilization on the microarray O H H O H Me N N Me O H O H H OH HO HO 2

R3 R2 R1 O N

R3

To immobilization on the microarray

To immobilization on the microarray

(b)
R3 N N N N NH X1

O R5 O N R4 R3 O R2 R1 OH

Cleavage from solid support

R4

H2N Linker

4 To immobilization on the microarray (c) To immobilization on the microarray

R1 Rn
PGN x B HN N O N H N O O N H H N O H2N O FITC-TTGCCCGTAATG NH
Current Opinion in Chemical Biology

NH2 N O O N

Scaffold

O N H O O O

H N

O NH2

Example compound

Illustration of cleavage and immobilization points in various libraries. Diversity elements are illustrated in red. (a) 1, 1,3-dioxane library [24]; 2, representative compound [26,27,28]. This library included complex variation of the skeletal structure making the scaffold itself a diversity element and an illustration of a diversified core cumbersome. Thus, a representative compound was shown. 3, biaryl-containing library; 4, dihydropyrancarboxamide library [2931]. (b) Tagged linker triazine library [33]. (c) PNA-encoded libraries and example compound. [10,11]. Rn, diversity element; B, base of the peptidonucleic acid; x, bases encoding a single diversity element; PG, protective group.

Also mentioned earlier, Schultz et al. have taken another approach in generating libraries for SMMs involving PNA tags [10,11]. This method eliminates the need for direct immobilization of the probes to the slide surface for
Current Opinion in Chemical Biology 2005, 9:413

screening purposes. Thus, the necessity to have a predetermined functional group for immobilization is removed, but this does require that the ligand of choice possess some chemical functionality available for
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Small molecule microarrays: recent advances and applications Uttamchandani et al.

conjugation with the PNA tag; a process of great synthetic exibility (Figure 1c). PNA was chosen to encode the ligand because of its hybridization properties with complementary strands of DNA, synthetic exibility and robustness [32]. The PNA-conjugated ligand libraries are incubated with the target off the array, in contrast to all other methods, with the identity of any positive hits determined by spatially addressable PNA-hybridization to its complementary oligonucleotide sequence on the array itself (Figure 1c). In addition, the synthetic exibility enables uorophores to be incorporated into the PNAligand conjugate [10,11]. These libraries and PNA tags were synthesized using procedures reported previously. Chang et al. chose 1,3,5-triazines as the library scaffold because these molecules enable temperature controlled, versatile modications, their threefold symmetry removes regiochemical worries during subsequent substitutions and they have shown themselves to be valuable for uncovering biological targets [33,34,35]. The library synthesis utilized the tagged linker strategy incorporating a variety of amino-functionalized ligands for immobilization to the array substrate. The tagged linker strategy is desirable because it incorporates the linker as an intrinsic part of the molecule from the beginning. This eliminates concerns of any downstream linker or tether effect that often results as compounds require modications to or removal of the linker. In addition, it enables the same compound to be seamlessly moved from one assay to another, whether in solution or immobilized on the solid phase [3638]. The parallel, solid-phase synthesis of the library takes advantage of the decreased reactivity of the triazine scaffold upon subsequent addition (Figure 1b). The steps in the pathway are completely orthogonal to each other, thus eliminating the problems of impurity carry-over seen in traditional solution-phase triazine synthesis. This enables libraries of very high purity. Simultaneous resin cleavage and Boc deprotection afforded a primary amine functionalized compound ready for immobilization.

The former may be additionally advantageous, as no specic functional groups may need to be incorporated on the small molecules. In the discovery of drug candidates, the crucial factor is the ability to retain the biological activity observed in vitro in an in vivo setting (such as an animal model or clinical trials). We found it useful to include linkers as an intrinsic part of the library design, allowing optimal hit selection for solution-phase screening, thus ensuring a quicker hit turnaround [33].

Covalent immobilization
Developing new surfaces for immobilization of small molecules provides avenues towards the development of diverse applications. One of the simplest ways in improving sensitivity of an array application is in increasing the surface density of active sites available for anchoring the compounds. Accordingly, Puskas and co-workers developed six different surfaces for covalent immobilization of nucleic acids, proteins and small molecules with acrylic and epoxy reactive surfaces [39]. Using dendrimer and triamino linker systems, increased immobilization efciency was demonstrated with reduced backgrounds. This strategy further opens up the possibility of modifying the surface hydrophobicity or hydrophilicity to generate surfaces with alternative specicities, and in generating varied substrates and surfaces for SMM application. Waldmann and co-workers have established a chemoselective method for attachment of compounds on arrays using mild conditions [40]. This is desirable, especially as it utilizes a pair of reactive functional groups that are biologically inert. It comprises a Staudinger ligation that couples azide-functionalized molecules with a phosphane-derivatized glass substrate (Figure 2a). The application of a Kenner-type linker allows the azide group to be introduced in the safety-catch cleavage step in the synthetic route for the small molecules. This strategy is attractive as it allows reaction in the presence of water and oxygen and is compatible with a wide variety of solidphase synthetic strategies. Also, Schreiber et al. recently developed diazobenzilidene-functionalized glass slides for the covalent capture of phenols and other molecular classes with comparable acidity [27].

Immobilization of small molecules on microarrays

Several methods have been described for the construction of small-molecule microarrays, namely covalent immobilization, photoactivatable cross-linking and in situ synthesis. Immobilization is critical in SMM preparation as it determines the way in which the molecules are oriented for interaction with target compounds. It also determines the chemical groups that should be incorporated during synthesis for homogeneous immobilization of small molecules. For certain applications such as binding epitope analysis, it may be desirable to randomly immobilize the small molecules on an array to expose all possible binding sites, yet in other cases a homogenous orientation is preferred, as in enzyme proling for uniform labeling.
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Photoactivation strategies
Mrksich et al. utilized the Diels-Alder reaction with photoactivation of self-assembled alkanethiolate monolayers (SAMs) consisting of nitroveratryloxycarbonyl (NVOC)-protected hydroquinone groups on gold-coated substrates [41]. Upon UV irradiation using photomasks or an optical microscope, selected sites are activated to free the hydroquinone that will then undergo chemical or electrochemical oxidation to benzoquinone. This reaction renders these surfaces reactive to cyclopentadienetagged ligands (Figure 2bii). The technique is able to easily generate gradients and patterns with excellent exibility, and in an increasingly miniaturized format with submicrometer precision using AFM and near eld
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8 Proteomics and genomics

Figure 2

(a) Covalent immobilization N3 P Ph O O Staudinger ligation N O HN Molecule

O Molecule

Ph

HN

(b) Photoactivation strategies Small molecules

UV irradiation (i) Non-selective immobilization OMe O O HO hv (365 nm) O O Reduction OMe O O2N HO Oxidation O OH O O

Ready for reaction

O Diels-alder reaction R O

(ii) Selective immobilization

(c) In situ synthesis R1 N H

O Glycolic acid coupling R1 N O O O O O NO2 O R1 N O O S O

UV irradiation O R1 N OH

Repeat cycle

O R1 N

R2 N

Peptoid coupling H

Activation

Current Opinion in Chemical Biology

Various methods for generating SMM. (a) Covalent tethering [40]. (b) Photoactivated capture [41,42]. (c) Sequential assembly in situ [43].

scanning optical microscopy (NSOM). In a creative approach towards non-selective immobilization, Kanoh et al. used a photoinduced cross-linking reaction to anchor small molecules on array [42]. A diazirin based photoafnity linker was coated on the slide surface, and was activated by UV irradiation after spotting the small moleCurrent Opinion in Chemical Biology 2005, 9:413

cules (Figure 2bi). The reactive species generated would react to proximate small molecules in a functional-groupindependent manner. This method eliminates the need for incorporating specic functional groups amongst library members and is particularly suited for naturally derived repertoires of compounds. The method also
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Small molecule microarrays: recent advances and applications Uttamchandani et al.

allows multiple facets of the immobilized molecules to be exposed for interaction.

In situ synthesis
A strategy developed to avoid the lengthy process of encoding and subsequent immobilization would entail the synthesis of small molecule microarrays in situ, very much in the same fashion that high-density oligonucleotide chips are generated. Demonstrating this concept on peptoids (oligomers of N-substituted glycines), Kodadek and co-workers used MeNPOC-protected glycolic acid and digital photolithography to sequentially activate and couple residues containing EDANS and Dabsyl dyes [43]. Quenching of this FRET pair across patterned regions indicated successful peptoid synthesis (Figure 2c). Stepwise synthesis in this fashion can potentially enable rapid creation of huge diversities of compounds on array within a few synthetic steps [44]. This method is of considerable utility in the eld of SMM as it allows rapid generation of arrays of spatially addressed compounds. It is also amenable to automation, reducing the time taken for array generation and potentially increasing the surface density, and thus correspondingly the number of features that may be assessed simultaneously on a single glass slide.

human IgG [33]. Contrasting from aforementioned DOS strategies where identication of each hit necessitates a further deconvolution effort, the synthetic approach we adopted enabled the identity of each compound to be known before printing, allowing immediate post-screening identication of hits. The motivation behind selecting IgG as the target for analysis was the discovery of safe and cost-effective small-molecule alternatives of Protein A and Protein G for industrial antibody purication and production. The hits obtained on array were conrmed using SPR, identifying the tightest binder with a Kd of 2.02 mM. In a recent development, Schreiber et al. synthesized 18 000 enantioenriched and skeletally distinct 1,3-dioxanes that enabled, for the rst time, stereochemically diverse sets of small molecules to be evaluated concurrently on array [45]. The libraries were encoded by both mass and the assignment of a unique combination of diazoketone tags to each building block for unequivocal identication of the synthesized compounds. Subsets of this library were anchored using diisocyanate capture chemistry and screened against calmodulin as the candidate protein. Interestingly, patterned incorporation of heptamethyleneimine elements in the molecular scaffold was implicated in sequestering calmodulin, identifying ligands with dissociation constants in the range of 10 20 mM. Further phenotypic screens revealed library members that inhibited heart function in an enantiomer-dependent manner. These reports both demonstrate the valuable expediency of SMM as a chemical genetic tool and showcase its importance in propelling discoverydriven research.

Successful application of small-molecule microarrays

In recent years, microarrays have been effectively applied to the discovery of small-molecule ligands for a variety of protein targets. After their original discovery of a small molecule that inhibits the yeast transcription factor Ure2p using SMM [19], Schreiber et al. further extended the strategy in the identication of functional inhibitors of Hap3p, a component of the yeast transcription factor complex Hap2/3/4/5p involved in nutrient-response signaling and aerobic respiration [29]. 12 396 mass-encoded compounds derived from three different DOS pathways were printed on chlorinated slides. The arrays were screened against Hap3p (expressed as a GST fusion protein) and visualized using Cy-5-labeled anti-GST antibody. Of the two hits obtained from the dihydropyrancarboximide libraries, only one was deemed a true binder to Hap3p (with Kd of 5.03 mM), while the other was discovered to bind GST. Introducing this ligand in vivo demonstrated by transcriptional proling experiments that it functionally inhibits Hap3p. Systematic modication of this molecule led to discovery of an even stronger ligand of Hap3p with a Kd of 0.33 mM. The same group successfully developed a DOS-derived SMM of 6336 phenol containing fused bicycles and tetracycles on diazobenzylidine slides [27]. These microarrays were interrogated against Cy-5-tagged calmodulin and produced 16 putative hits, of which 13 were validated using SPR, with the strongest ligand binding with a Kd of 0.12 0.03 mM. In a further extension, we generated arrays with 2688 triazines that were queried for novel ligands against
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Novel developments
As is characteristic in a eld that is rapidly emerging, sophisticated methodologies are progressively being introduced to enhance and extend execution of SMM towards novel applications. In an effort to move beyond the study of non-covalent biochemical interactions, we developed a SMM strategy to ngerprint and characterize proteins on the basis of their enzymatic activities [46]. The method employs small-molecule coumarin derivatives conjugated with an enzyme recognition domain. In the conjugated state, the small molecule is virtually nonuorescent; but upon enzymatic recognition and catalysis, the coumarin moiety is liberated, restoring its uorescence for easy detection. Probes were successfully designed for four different enzyme classes, namely epoxide hydrolases, esterases, proteases and phosphatases, and were immobilized in a microarray format for parallel analysis against their representative enzymes (Figure 3a). The signicant advantage of this approach is the labelfree analysis of proteins, setting the foundation towards high-throughput proling of disease states using SMM as a potential diagnostic tool a modern dipstick equivalent. In an earlier development, Ellman et al. utilized a
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10 Proteomics and genomics

Figure 3

(a)

Enzyme recognition head X

Fluorogenic moiety O O
E

HX

O Fluorescent

Enzyme mediated catalysis

X = O or NH

(b) Libraries printed in glycerol droplets Br Br Br N N O N N

Each spot represents one small molecule

Aerosolized deposition of enzyme and fluorogenic substrate

Small-molecule inhibitor of caspase activity

Current Opinion in Chemical Biology

Recent developments in SMM strategy. (a) Hydrolytic enzyme detection strategy [46]. (b) Solution-phase screening strategy [48].

complementary approach generating uorogenic peptide arrays for proling protease activity [47]. In an interesting development that abrogates the attachment of molecules covalently on array, Diamond and Gosalia used nanoliter droplets of glycerol as microreactors that were applied to the discovery of a novel caspase inhibitor [48]. A 352-member combinatorial library was printed on microarrays in a manner that retained the wetness of the droplets such that the small molecules were suspended in solution phase. An aerosolized application of caspases 2, 4 and 6, followed by their respective uorogenic substrates allowed parallel analysis of the inhibitory effects that each small molecule had on the enzymes activity (Figure 3b). This strategy led to the discovery of a caspase inhibitor against all three caspase isoforms screened. The greatest advantage of this strategy is the ability to perform miniaturized high-throughput solution-phase screening of reactions at extremely low reaction volumes (1.6 nl per spot) without physical immobilization of any of the components. However, the necessary use of low-volatile mediums such as glycerol or DMSO may limit the overall applicability in scenarios where reactions require predominantly aqueous environments. The creation process of arrays involving rst the synthesis of thousands of different molecules and second the addressable display of these compounds on slide for
Current Opinion in Chemical Biology 2005, 9:413

parallel screening has invariably been the greatest bottleneck of any microarray endeavor. One solution rides on the now routinely applied and commercially mature oligonucleotide microarray, making it cost-effective and attractive to augment this platform with large-scale smallmolecule screening, with a DNA hybridization-based deconvolution approach for identifying small-molecule interactions of interest. This alternative is conceptually similar to the PNA approach described earlier [10,11], and affords the advantage that interactions are examined in solution phase, eliminating immobilization requirements to solid support that further decelerate reaction kinetics [1214]. A further development to accelerate fabrication of microarrays has been an extension of in situ synthesis, specically a one-step polymerization of libraries of monomeric subunits. All these are discussed in the following paragraphs.

Small-molecule microarray applications involving DNA microarrays

We recently developed expression display for the highthroughput discovery of enzymes (Figure 4a): by using small-molecule probes to target multiple enzymes expressed with the coding mRNAs in a ribosome display format, we were able to isolate and identify subclasses of proteins with desired activities [13]. Multiple target enzymes of the protein tyrosine phosphatase family were simultaneously isolated and identied on a DNA
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Small molecule microarrays: recent advances and applications Uttamchandani et al. 11

Figure 4

(a) In vitro transcription DNA pool mRNAs In vitro translation

RT-PCR
H yb

mRNA elution

rid

iz

at

io

DNA microarray

Small molecule probe

(b) Target Biopanning and PCR enrichment Complementary region Hybridization onto DNA microarrays

3' 5'

Unique oligonucleotide tag

Current Opinion in Chemical Biology

SMM application involving DNA microarrays. (a) Enzymatic screening using activity-based small-molecule probes [13]. (b) Self-assembling chemical libraries [12].

microarray. The use of ribosome display is advantageous as it allows the in vitro expression of a huge pool of proteins in a cell-free format, while incorporating mRNA tags for each of the protein members for decoding. By incubating the libraries with biotinylated activity-based small-molecule probes, target proteins were captured on streptavidin beads and subsequently identied by RT-PCR of their coding mRNA and hybridization to a DNA microarray. We demonstrated that expression display is highly versatile, able to classify proteins on the basis of their enzymatic activities, which furnishes a proteomic tool with promising applications in genomewide proling of various classes of proteins en masse. Where traditional array methodologies have been limited to exploring a binary interaction between a small molecule and the selected target, a creative extension by Neri et al. has demonstrated the ability to screen multivalent assemblies of small molecule against targets of interest (Figure 4b) [12] The technology uses libraries of organic molecules covalently linked to oligonucleotide tags that
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mediate self-assembly of the library and incorporates a code to uniquely identify each component. Panning the libraries against human serum albumin and bovine carbonic anhydrase, followed by PCR amplication and decoding of the sequestered assemblies, revealed binders with sub-micromolar dissociation constants. Elegantly, constructing libraries in this format exponentially expands the variety of scaffolds that may be generated and screened from a single dened library, greatly enriching its utility and value. The strategy is also particularly adept in the identication of chelated small molecules with highly specic interaction with various targets, for developing inhibitors of multipocketed enzymes such as kinases and proteases. Recently, Liu and co-workers have also creatively adopted a similar strategy for the evaluation of bond-forming combinations amongst small-molecules libraries [14].

In situ polymerization
Rapid generation of molecules on array is desirable to quickly move into the screening process in microarray
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tio

12 Proteomics and genomics

applications. Langer and co-workers generated 576 acrylate polymers by simply printing combinations of 25 different acrylate, diacrylate, dimethylacrylate and triacrylate monomers on pHEMA-coated slides [49]. Long-wave UV irradiation initiated polymerization and the entire process was carried out in an oxygen-free atmosphere (as oxygen was inhibitory to the polymerization process). The concatenated small molecules were screened for their ability to support embryonic stem cell attachment and proliferation. The strategy is promising in rapidly building architectures of monomeric compounds in miniaturized microarray format for high-throughput cell-based screenings in the discovery of molecules that can control cell growth and proliferation, as well as in the programmable patterning of different cell types on slides based on their afnities for different surfaces.

2.

Schena M, Shalon D, Davis RW, Brown PO: Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 1995, 270:467-470. MacBeath G, Schreiber SL: Printing proteins as microarrays for high-throughput function determination. Science 2000, 289:1760-1763. Fang Y, Frutos AG, Lahiri J: Membrane protein microarrays. J Am Chem Soc 2002, 124:2394-2395. Min DH, Mrksich M: Peptide arrays: towards routine implementation. Curr Opin Chem Biol 2004, 8:554-558. Shin I, Cho JW, Boo DW: Carbohydrate arrays for functional studies of carbohydrates. Comb Chem High Throughput Screen 2004, 7:565-574. MacBeath G, Koehler AN, Schreiber SL: Printing small molecules as microarrays and detecting protein-ligand interactions en masse. J Am Chem Soc 1999, 121:7967-7968. Kononen J, Bubendorf L, Kallioniemi A, Barlund M, Schraml P, Leighton S, Torhost J, Mihatsch MJ, Sauter G, Kallioniemi OP: Tissue microarrays for the high-throughput molecular proling of tumour specimens. Nat Med 1998, 4:844-847. Ziauddin J, Sabatini DM: Microarrays of cells expressing dened cDNAs. Nature 2001, 411:107-110.

3.

4. 5. 6.

7.

8.

Conclusions
Within just ve years of its inception, the great strides taken in the eld of SMM have brought it to the forefront of high-throughput technology. Recent work has improved methods for SMM preparation and landmark publications have demonstrated various ways in which the platform may be exploited from the prospective discovery of novel small-molecule ligands, to the study of biological samples and macromolecules with specic activity proles. More importantly, there is concerted afrmation amongst its proponents of its expanding utility in high-throughput proling, and this heralds the even more promising role SMM is to play in future discovery-driven research. As the technology increases in popularity, remarkable insights will be gained into the pathways and biological processes targeted by small molecules. This knowledge may then be channeled to the development of improved targets for medicine. With greater commercial availability of diverse libraries of compounds, it is only a matter of time before industry places SMM on the assembly line in convenient kit-based formats for any researcher interested and willing. Further developments should see SMM taking on applications in bedside diagnosis as well as in dening biologically useful hits more effectively using predictive bioinformatics applications in addition to in vivo studies. Notwithstanding, the implications of smallmolecule microarrays have already been many and important, and are likely to be even more noteworthy in future.

9.

10. Winssinger N, Harris JL, Backes BJ, Schultz PG: From split-pool  libraries to spatially addressable microarrays and its application to functional proling. Angew Chem Int Ed Engl 2001, 40:3152-3155. The rst successful application of a PNA-encoded library of small molecules in the discovery of inhibitors of Cathepsin L. 11. Winssinger N, Ficarro S, Schultz PG, Harris JL: Proling protein function with small molecule microarrays. Proc Natl Acad Sci USA 2002, 99:11139-11144. 12. Melkko S, Scheuermann J, Dumelin CE, Neri D: Encoded  self-assembling chemical libraries. Nat Biotechnol 2004, 22:568-574. An exciting development that utilizes DNA linked small molecules to generate multivalent assemblies of chemical structures for target binding. 13. Hu Y, Chen GYJ, Yao SQ: Activity-based high-throughput  screening of enzymes using DNA microarray. Angew Chem Int Ed Engl 2005, DOI: 10.1002/anie.200461612. This paper describes an exciting method with which to exploit small molecules in the high-throughput discovery of enzymes. An expression display strategy was conceived where huge pools of proteins, each linked to its coding mRNA, could be rapidly dissected to characterize and identify protein subclasses with desired activities. 14. Kanan MW, Rozenman MM, Sakurai K, Snyder TM, Liu DR:  Reaction discovery enabled by DNA-templated synthesis and in vitro selection. Nature 2004, 431:545-549. In a unique extension of SMM application, the authors make use of DNAtemplated synthesis and in vitro selection to discover novel bond-forming substrate combinations within libraries of DNA-conjugated small molecules. 15. Schreiber SL: The small-molecule approach to biology: chemical genetics and diversity-oriented organic synthesis make possible the systematic exploration of biology. Chem Eng News 2003, 81:51-61. 16. Burke MD, Berger EM, Schreiber SL: Generating diverse skeletons of small molecules combinatorially. Science 2003, 302:613-616. 17. Schreiber SL: Target-oriented and diversity-oriented organic synthesis in drug discovery. Science 2000, 287:1964-1969. 18. Hergenrother PJ, Depew KM, Schreiber SL: Small-molecule microarrays: covalent attachment and screening of alcohol containing small molecules on glass slides. J Am Chem Soc 2000, 122:7849-7850. 19. Kuruvilla FG, Alykhan FS, Sternson SM, Hergenrother PJ,  Schreiber SL: Dissecting glucose signaling with diversityoriented synthesis and small molecule microarrays. Nature 2002, 416:653-657. www.sciencedirect.com

Acknowledgements
Funding support was provided by the National University of Singapore (NUS) and the Agency for Science, Technology and Research (A*STAR) of Singapore.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:  of special interest  of outstanding interest 1. Chen GYJ, Uttamchandani M, Lue RYP, Lesaicherre M, Yao SQ: Array-based technologies and their applications in proteomics. Curr Top Med Chem 2003, 3:705-724.

Current Opinion in Chemical Biology 2005, 9:413

Small molecule microarrays: recent advances and applications Uttamchandani et al. 13

This was a pioneering example in which SMM was fruitfully deployed in a biological context, with the discovery of ligands that could modulate the function of the target protein in vivo. 20. Lam KS, Salmon SE, Hersh EM, Hruby VJ, Kazmierski WM, Knapp RJ: One bead, one peptide: a new type of synthetic peptide library for identifying ligand-binding activity. Nature 1991, 354:82-84. 21. Scharn D, Wenschuh H, Reineke U, Schneider-Mergener J, Germeroth L: Spatially addressed synthesis of amino- and amino-oxy-substituted 1,3,5-triazine arrays on polymeric membranes. J Comb Chem 2000, 2:361-369. 22. Frank R: Spot synthesis an easy technique for the positionally addressable, parallel chemical synthesis on a membrane support. Tetrahedron 1992, 48:9217-9232. 23. Blackwell HE, Perez L, Stavenger RA, Tallarico JA, Eatough EC, Foley MA, Schreiber SL: A one-bead, one-stock solution approach to chemical genetics: part 1. Chem Biol 2001, 8:1167-1182. 24. Sternson SM, Louca JB, Wong JC, Schreiber SL: Split-pool synthesis of 1,3-dioxanes leading to arrayed stock solutions of single compounds sufcient for multiple phenotypic and protein-binding assays. J Am Chem Soc 2001, 123:1740-1747. 25. Clemons PA, Koehler AN, Wagner BK, Sprigings TG, Spring DR, King RW, Schreiber SL, Foley MA: A one-bead, one-stock solution approach to chemical genetics: part 2. Chem Biol 2001, 8:1183-1195. 26. Kwon O, Park SB, Schreiber SL: Skeletal diversity via a branched pathway: Efcient synthesis of 29 400 discrete, polycyclic compounds and their arraying into stock solutions. J Am Chem Soc 2002, 124:13402-13404. 27. Barnes-Seeman D, Park SB, Koehler AN, Schreiber SL:  Expanding the functional group compatibility of smallmolecule microarrays: discovery of novel calmodulin ligands. Angew Chem Int Ed Engl 2003, 42:2376-2379. The paper describes the successful execution of the SMM approach with a unique immobilization strategy applied to the discovery of novel calmodulin ligands. 28. Tallarico JA, Depew KM, Pelish HE, Westwood NJ, Lindsley CW, Shair MD, Schreiber SL, Foley MA: An alkylsilyl-tethered, high-capacity solid support amenable to diversity-oriented synthesis for one-bead, one-stock solution chemical genetics. J Comb Chem 2001, 3:312-318. 29. Koehler AN, Shamji AF, Schreiber SL: Discovery of an inhibitor of a transcription factor using small molecule microarrays and diversity-oriented synthesis. J Am Chem Soc 2003, 125:8420-8421. 30. Spring DR, Krishnan S, Blackwell HE, Schreiber SL: Diversityoriented synthesis of biaryl-containing medium rings using a one bead/one stock solution platform. J Am Chem Soc 2002, 124:1354-1363. 31. Stavenger RA, Schreiber SL: Asymmetric catalysis in diversityoriented organic synthesis: Enantioselective synthesis of 4320 encoded and spatially segregated dihydropyrancarboxamides. Angew Chem Int Ed Engl 2001, 40:3417-3421. 32. Nielsen PE, Egholm M, Berg RH, Buchardt O: Sequenceselective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science 1991, 254:1497-1500. 33. Uttamchandani M, Walsh DP, Khersonsky SM, Huang X, Yao SQ,  Chang YT: Microarrays of tagged combinatorial triazine libraries in the discovery of small-molecule ligands of human IgG. J Comb Chem 2004, 6:862-868. The authors introduce a powerful approach in constructing SMM, where the identity of each compound is known before printing, allowing immediate identication of hits. 34. Park HE, Lee SY, Ahn HY, Shin JC, Chang YT, Joe YA: Effects of tubulyzines, novel microtubule-binding triazine molecules on

endothelial progenitor cell differentiation. J Appl Pharmacol 2003, 11:85-90. 35. Lowik DWPM, Lowe CR: Synthesis of macrocyclic, triazine-based receptor molecules. Eur J Org Chem 2001: 2825-2839. 36. Moon HS, Jacobson EM, Khersonsky SM, Luzung MR, Walsh DP, Xiong WN, Lee JW, Parikh PB, Lam JC, Kang TW et al.: A novel microtubule destabilizing entity from orthogonal synthesis of triazine library and zebrash embryo screening. J Am Chem Soc 2002, 124:11608-11609. 37. Bork JT, Lee JW, Chang YT: Palladium-catalyzed crosscoupling reaction of resin-bound chlorotriazines. Tetrahedron Lett 2003, 44:6141-6144. 38. Bork JT, Lee JW, Khersonsky SM, Moon HS, Chang YT: Novel orthogonal strategy toward solid-phase synthesis of 1,3,5-substituted triazines. Org Lett 2003, 5:117-120. 39. Hackler LJ, Dorman G, Kele Z, Urge L, Durvas F, Puskas LG: Development of chemically modied glass surfaces for nucleic acid, protein and small molecule. Mol Divers 2003, 7:25-36. 40. Kohn M, Wacker R, Peters C, Schroder H, Soulere L, Breinbauer R, Niemeyer CM, Waldmann H: Staudinger ligation: a new immobilization strategy for the preparation of small-molecule arrays. Angew Chem Int Ed Engl 2003, 42:5830-5834. 41. Dillmore WS, Yousaf MN, Mrksich M: A photochemical method for patterning the immobilization of ligands and cells to selfassembled monolayers. Langmuir 2004, 20:7223-7231. 42. Kanoh N, Kumashiro S, Simizu S, Kondoh Y, Hatakeyama S,  Tashiro H, Osada H: Immobilization of natural products on glass slides by using a photoafnity reaction and the detection of protein small molecule interactions. Angew Chem Int Ed Engl 2003, 42:5584-5587. The paper presents a non-selective immobilization approach by photoinitiated cross-linking, allowing a randomized presentation of molecules on array with multiple facets exposed for interaction. 43. Li S, Bowerman D, Marthandan N, Klyza S, Luebke KJ, Garner HR, Kodadek T: Photolithographic synthesis of peptoids. J Am Chem Soc 2004, 126:4088-4089. 44. Fodor SP, Read JL, Pirrung MC, Stryer L, Lu AT, Solas D: Light-directed, spatially addressable parallel chemical synthesis. Science 1991, 251:767-773. 45. Wong JC, Sternson SM, Louca JB, Hong R, Schreiber SL:  Modular synthesis and preliminary biological evaluation of stereochemically diverse 1,3-dioxanes. Chem Biol 2004, 11:1279-1291. The rst report of using a sterochemically diverse set of compounds in an SMM format for the discovery of ligands in an enantiomer-dependent manner. 46. Zhu Q, Uttamchandani M, Li D, Lesaicherre ML, Yao SQ:  Enzymatic proling system on a small-molecule microarray. Org Lett 2003, 5:1257-1260. Fluorogenic substrates against various enzymatic classes were designed and arrayed to ngerprint enzymes on the basis of their activity proles. 47. Salisbury SM, Maly D, Ellman J: Peptide microarrays for the determination of protease substrate specicity. J Am Chem Soc 2002, 124:14868-14870. 48. Gosalia DN, Diamond SL: Printing chemical libraries on  microarrays for uid phase nanoliter reactions. Proc Natl Acad Sci USA 2003, 100:8721-8726. The paper presents a creative solution-based approach for microarray screening by creating arrays of nanoliter droplets that eliminate the need for covalent attachment of any of the reaction components. 49. Anderson DG, Levenberg S, Langer R: Nanoliter scale synthesis  of arrayed biomaterials and application to human embryonic stem cells. Nat Biotechnol 2004, 22:863-866. This paper successfully demonstrates the feasibility of employing a onestep polymerization to generate large arrays displaying multiple smallmolecule monomers. It is clearly a promising strategy for rapid array generation using UV initiated in situ polymerization.

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Current Opinion in Chemical Biology 2005, 9:413