Ligand Discovery Using Small-Molecule Microarrays

Abstract

Genome-wide association studies and genetic linkage studies have created a growing list of proteins related to disease. Small molecules can serve as useful probes of function for these proteins in a cellular setting or may serve as leads for therapeutic development. High-throughput and general binding assays may provide a path for discovering small molecules that target proteins for which little is known about structure or function or for which conventional functional assays have failed. One such binding assay involves small-molecule microarrays (SMMs) containing compounds that have been arrayed and immobilized onto a solid support. The SMMs can be incubated with a protein target of interest and protein-small molecule interactions may be detected using a variety of fluorescent readouts. Several suitable methods for manufacturing SMMs exist and different immobilization methods may be more or less preferable for any given application. Here, we describe protocols for covalent capture of small molecules using an isocyanate-coated glass surface and detection of binding using purified protein.

Microarray as High Throughput Tool for Tyrosinase Gene Expression Analysis

“…It is being used to detect ligands for new drug discovery. Small molecules including organic compounds, natural products, peptides have been immobilized on microarray slides and a variety of biomolecules can be incubated with these slides [28,29], (Figure 2). …”

Probing Small‐Molecule Microarrays with Tagged Proteins in Cell Lysates

“…The screener should consider whether the surface chemistry used for SMM manufacture results in a linkage that is stable to cellular esterases prior to executing the screen. Please refer to the previously published protocols on SMM manufacture for lysate-compatible arrays (Casalena et al, 2012) or reviews that thoroughly discuss the multitude of options for array manufacture, including surface capture strategies and array layouts (Uttamchandani et al, 2010). Typical SMMs vary in the amount of printed features with a range of 1,000–16,000 printed features.…”

“…Printed small-molecule microarray (SMM) slides, four replicate arrays (Casalena et al, 2012, Uttamchandani et al, 2010). …”

“…The process for manufacturing microarrays also has been reviewed previously (Shi et al, 2010; Uttamchandani et al, 2004; Uttamchandani and Yao, 2010; Vegas and Koehler, 2010; Walsh and Chang, 2004) and is not the focus of this protocol. The reader is referred to previous step-by-step protocols focused on the manufacture of SMMs (Bradner et al, 2006; Casalena et al, 2012). The SMM assay is based on the ability of compounds corresponding to various types of chemical structures to directly bind to soluble proteins, with specificity that depends on the properties of the small molecule as well as the three-dimensional structure of the protein surface.…”

Recent discoveries and applications involving small-molecule microarrays

“…In addition, a variety of biomolecules can be incubated with SMMs, including purified proteins, cell lysates, antibodies, RNAs and even living cells (Figure 1). We refer the reader to several excellent and thorough reviews and volumes published previously that cover approaches to SMM manufacture in great detail [7-11]. This review will focus on selected SMM-facilitated probe discovery stories and functional proteomics applications from the last five years.…”