Small Molecule Arrays: a New Forefront in Drug Discovery

Summary Scientists from the laboratory of Professor Stuart Schreiber at Harvard University have developed a novel method of printing small molecule libraries on glass microscope slides. This method represents a next-generation platform for drug discovery.

Small Molecule Printing is simple and efficient: Bioactive small molecules, natural products, and small molecules originating from several diversity-oriented syntheses (DOS) beads are dissolved in a small volume of suitable solvent. A high precision robot picks up only 1nl of solution and repetitively delivers each compound to a defined location on a chemically derivatived glass microscope slide (~150 slides per print run). This results in the formation of microscopic spots 200-250 um in diameter. Importantly, each compound shares a common functional group that mediates covalent attachment to the slide surface. In this way, compounds are arrayed and subsequently immobilized on glass slides at extremely high spatial densities (>1000 spots per cm2). Each slide is probed with a different tagged protein (protein solution only 20ug/ml) and binding events are detected by a fluorescence-linked assay. The presence of a linker connecting the small molecule to the slide enables the selection of compounds with known sites of attachment, which enables purification of target proteins by affinity chromatography and the measurement of binding kinetics by surface plasmon resonance.

Isocyanate Coated Slides Allow Functional Group Diversity: Isocyanates react with a number of nucleophilic functional groups without leaving an acidic byproduct; thus, they increase the diversity of natural or synthetic small molecules that can be immobilized onto a single SMM. The functional group compatibility of isocyanate small-molecule microarrays includes immobilization of primary alcohols, secondary alcohols, phenols, carboxylic acids, hydroxamic acids, thiols and amines on a single slide surface. Small molecule libraries with single functional groups can also be attached to an appropriately activated glass slide. For example, chlorinated slides have been used to print primary- alcohol-containing DOS derived compounds while diasobenzylidene-derivatized glass slides have been used to print compounds containing acidic protons, such as phenols, carboxylic acids, and sulfonamides.

Applications The laboratory has previously reported the use of SMMs to discover ligands for calmodulin (calmoduphilins), the yeast transcriptional corepressor Ure2p (uretupamines), and the Hap3p subunit of the yeast HAP transcription factor complex (haptamides). Each of these screens involved SMMs in which only one DOS library was contained on a given slide. They sought to prepare an SMM that contains sublibraries from various DOS synthetic routes in one array (Figure 1 above). A robotic microarrayer was used to print a series of synthetic FKBP12 ligands that were derivatized so as to present a primary alcohol, secondary alcohol, tertiary alcohol, phenol, methyl ether, carboxylic acid, hydroxamic acid, methyl, thiol, primary amine secondary amine, indole, or aryl amine, onto the isocyanate-derivatized slides. Slides were probed with FKBP12-GST followed by a Cy5â„¢-labeled anti-GST antibody, and scanned for fluorescence. The intensity of fluorescent signals corresponding to FKBP12-GST varied according to both the functional group presented for attachment and concentration of ligand. The primary amines, aryl amine, and thiol appear to have the highest immobilization levels while fluorescence intensities for primary alcohols, phenol, hydroxamic acid, secondary amine, and indole were also consistent with significant immobilization. The secondary alcohol, carboxylic acid, and tertiary alcohols were immobilized in lower amounts.

In separate experiments, more than 300 commercially available bioactive compounds were printed onto isocyanate-functionalized slides and successfully screened with corticosterone, digitoxin, and 17 -estradiol antibodies followed by a fluor-labeled goat anti-rabbit secondary. Similarly, this method was expanded to include the detection of interactions between small molecules and target proteins expressed in mammalian cells without prior purification. SMMs were incubated with cellular lysates bearing over-expressed epitope-tagged proteins of interest. The arrays were serially incubated with a primary anti-epitope antibody, and a Cy5â„¢-conjugated secondary antibody

Publications and Patents

James E. Bradner, Olivia M. McPherson, Ralph Mazitschek, David Barnes-Seeman, John P. Shen, Jasmeet Dhaliwal, Kristen E. Stevenson, Jay L. Duffner, Seung Bum Park, Donna S. Neuberg, Paul Nghiem, Stuart L. Schreiber, Angela N. Koehler. A Robust Small-Molecule Microarray Platform for Screening Cell Lysates. In Review

Gavin MacBeath, Angela N. Koehler, Stuart L. Schreiber. Printing Small Molecules as Microarrays and Detecting Protein-Ligand Interactions en Masse'', J. Am. Chem. Soc. 1999,121, 7967-7968.

Paul J. Hergenrother, Kristopher M. Depew, Stuart L. Schreiber. Small Molecule Microarrays: Covalent Attachment and Screening of Alcohol-Containing Small Molecules on Glass Slides, J. Am. Chem. Soc. 2000, 122, 7849-7850

Finny G. Kuruvilla, Alykhan F. Shamji, Scott M. Sternson, Paul J. Hergenrother, Stuart L. Schreiber. Dissecting glucose signalling with diversity-oriented synthesis and small-molecule microarrays'', Nature 2002, 416, 653-657

David Barnes-Seeman, Seung Bum Park, Angela N. Koehler, and Stuart L. Schreiber. Expanding the Functional Group Compatibility of Small-Molecule Microarrays: Discovery of Novel Calmodulin Ligands'', Angewandte Chemie, 2003, 42, 2376-2379

Angela N. Koehler, Alykhan F. Shamji, Stuart L. Schreiber. Discovery of an inhibitor of a transcription factor using small molecule microarrays and diversity-oriented synthesis. J. Am. Chem. Soc., 2003, 125, 8420-8421

US Patent # 6,824,987 For Further Information Please Contact the Director of Business Development Laura Brass Email: [email protected] Telephone: (617) 495-3067

Inventor(s): Schreiber, Stuart L.

Type of Offer: Licensing



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