A small molecule that binds Hedgehog and blocks its signaling in human cells

Small-molecule microarrays as tools in ligand discovery

Small molecules that bind and modulate specific protein targets are increasingly used as tools to decipher protein function in a cellular context. Identifying specific small-molecule probes for each protein in the proteome will require miniaturized assays that permit screening of large collections of compounds against large numbers of proteins in a highly parallel fashion. Simple and general binding assays involving small-molecule microarrays can be used to identify probes for nearly any protein in the proteome. The assay may be used to identify ligands for proteins in the absence of knowledge about structure or function. In this tutorial review, we introduce small-molecule microarrays (SMMs) as tools for ligand discovery; discuss methods for manufacturing SMMs, including both non-covalent and covalent attachment strategies; and provide examples of ligand discovery involving SMMs. Chemical Society Reviews, 37, 1385 – 1394 (2008).

Fluorous-based small-molecule microarrays for the discovery of histone deacetylase inhibitors

Angewandte Chemie International Edition, 46, 7960-7964 (2007).

Detecting binding interactions using microarrays of natural product extracts

Small-molecule microarrays have been used to discover biologically active small molecules in collections of synthetic compounds. Here we utilize a versatile isocyanate chemistry to immobilize extracts from microorganisms in a microarray format. Specific bioactive small molecules are detected in crude extracts of Streptomyces hygroscopicus, and the amount of specific to nonspecific binding of a protein to an immobilized compound on a microarray can be estimated. These natural product-extract microarrays (NPEMs) provide new tools for characterizing the metabolic products of organisms and for discovering biologically active small molecules from nature. Journal of the American Chemical Society, 129, 11346–11347 (2007).

A method for the covalent capture and screening of diverse small molecules in a microarray format

This protocol describes a robust method for the covalent capture of small molecules with diverse reactive functional groups in microarray format, and outlines a procedure for probing small-molecule microarrays (SMMs) with proteins of interest. A vapor-catalyzed, isocyanate-mediated surface immobilization scheme is used to attach bioactive small molecules, natural products and small molecules derived from diversity-oriented synthesis pathways. Additionally, an optimized methodology for screening SMMs with purified proteins and cellular lysates is described. Finally, a suggested model for data analysis that is compatible with commercially available software is provided. These procedures enable a platform capability for discovering novel interactions with potential applications to immunoglobulin profiling, comparative analysis of cellular states and ligand discovery. With the appropriate materials and experimental setup, the printing of SMMs can be completed in 14 hours over 3 days. Screening and data analysis requires 2 days. A detailed timeline is provided. Nature Protocols, 1, 2344 - 2352 (2006).

A robust small-molecule microarray platform for screening cell lysates

Herein we report the expanded functional group compatibility of small-molecule microarrays to include immobilization of primary alcohols, secondary alcohols, phenols, carboxylic acids, hydroxamic acids, thiols, and amines on a single slide surface. Small-molecule diversity microarrays containing nearly 10,000 known bioactive small molecules, natural products, and small molecules originating from several diversity-oriented syntheses were produced by using an isocyanate-mediated covalent capture strategy. Selected printed bioactive compounds were detected with antibodies against compounds of interest. The new surface of the diversity microarrays is highly compatible with approaches involving cellular lysates. This feature has enabled a robust, optimized screening methodology using cellular lysates, allowing the detection of specific interactions with a broad range of binding affinity by using epitope-tagged or chimeric fluorescent proteins without prior purification. We believe that this expanded research capability has considerable promise in biology and medicine. Chemistry and Biology, 13, 493-504 (2006).

Discovery of an inhibitor of a transcription factor using small molecule microarrays and diversity-oriented synthesis

Small molecule microarrays were screened to identify a small molecule ligand for Hap3p, a subunit of the yeast Hap2/3/4/5p transcription factor complex. The compound, named haptamide A, was determined to have a KD of 5.03 μM for binding to Hap3p using surface plasmon resonance analysis. Haptamide A also inhibited activation of a GDH1-lacZ reporter gene in a dose-dependent fashion. To explore structure−activity relationships, 11 derivatives of haptamide A were prepared using the same synthetic route that was developed for the original library synthesis. Analysis of dissociation constants and IC50 values for the reporter gene assay revealed a more potent inhibitor, haptamide B, with a KD of 330 nM. Whole-genome transcriptional profiling was used to compare effects of haptamide B with a hap3Δ yeast strain. Treatment with haptamide B, like the deletion mutant, reduced lactate-induced transcription of several genes from wild-type levels. Profiling the genetic “knockout” and the chemical genetic “knockdown” led to the identification of several genes that are regulated by Hap3p under nonfermentative conditions. These results demonstrate that a small molecule discovered using the small molecule microarray binding assay can permeate yeast cells and reach its target transcription factor protein in cells. Journal of the American Chemical Society, 125, 8420-8421 (2003).

Dissecting glucose signalling with diversity-oriented synthesis and small-molecule microarrays

Small molecules that alter protein function provide a means to modulate biological networks with temporal resolution. Here we demonstrate a potentially general and scalable method of identifying such molecules by application to a particular protein, Ure2p, which represses the transcription factors Gln3p and Nil1p. By probing a high-density microarray of small molecules generated by diversity-oriented synthesis with fluorescently labelled Ure2p, we performed 3,780 protein-binding assays in parallel and identified several compounds that bind Ure2p. One compound, which we call uretupamine, specifically activates a glucose-sensitive transcriptional pathway downstream of Ure2p. Whole-genome transcription profiling and chemical epistasis demonstrate the remarkable Ure2p specificity of uretupamine and its ability to modulate the glucose-sensitive subset of genes downstream of Ure2p. These results demonstrate that diversity-oriented synthesis and small-molecule microarrays can be used to identify small molecules that bind to a protein of interest, and that these small molecules can regulate specific functions of the protein. Nature,  416(6881):653-7 (2002).

Printing small molecules onto microarrays and detecting protein-ligand interactions en masse

Journal of the American Chemical Society, 121, 7697-7698 (1999).