High-Throughput Screening of Metalloproteases Using Small Molecule Microarrays
The promise of rapid and cost-effective drug screening assays on solid support is one that may now be realized with the advent of small molecule microarrays. Many of the initial hurdles in library design and microarray fabrication have been overcome over
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. Introduction One of the earliest and most important steps in any successful drug discovery programme is the identification of small molecule leads that are both potent and selective to the targets of interest (1). Small molecule microarrays represent a new paradigm for performing such high-throughput screening in a rapid and cost-efficient manner (2, 3). The miniaturization, automation and throughput offered on microarrays could significantly accelerate pharmaceutical and commercial screening efforts (4). Since the introduction of small molecule microarrays, a plethora of design Sridar V. Chittur (ed.), Microarray Methods for Drug Discovery, Methods in Molecular Biology, vol. 632, DOI 10.1007/978-1-60761-663-4_13, © Humana Press, a part of Springer Science+Business Media, LLC 2010
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and fabrication issues have been addressed and overcome (5). A wide range of combinatorial synthesis protocols, immobilization chemistries, and application guidelines, together with strong infrastructure and commercial support is now available to drive research and future innovation. The platform has hence matured significantly and is now well poised for translation to more routine applications in industry, especially in the areas of pharmaceutical screening and drug discovery. In an effort to further extend the scope and utility of small molecule microarrays, we have developed a protocol for screening and identifying functional inhibitors immediately without the need for downstream revalidation (6–8). It has traditionally been very difficult to distinguish true activity-dependent binding from nonspecific/false positive/ functionally irrelevant binding on microarrays. Hits from microarray experiments frequently have to be retested to establish whether they actually bind to the intended target site. This can significantly limit throughput, especially when hundreds of molecules appear to be positive and each would require retesting. We have thus shown that by employing (a) a target-oriented library of individually synthesized compounds in which every member was characterized prior to spotting on the glass surface; (b) a two-color reciprocal protein labelling/screening strategy; and (c) simultaneous and quantitative measurements of multiple protein ligand-binding interactions, we were able to immediately and reliably elucidate the activity-dependent binding profiles of proteins using small molecule microarrays (6, 7).
2. Materials 2.1. Chemicals and Biochemicals
1. Rink amide-AM resin (GL Biochem).
2.1.1. Combinatorial Synthesis of Small Molecule Library
3. 20 proteinogenic Fmoc protected amino acids (GL Biochem).
2. Hydrochloric acid (HCl; Merck).
4. Fmoc-Lys(Biotin)-OH (GL Biochem). 5. O-Benzotriazole- N,N,N¢,N¢-tetramethyluronium hexafluorophosphate (HBTU; GL Biochem). 6. O-(7-Azabenzotriazole-1-yl)-N,N,N¢,N¢-tetramethyluro nium hexafluorophosphate (HATU; GL Biochem). 7. N-Hydroxybenzotriazole (HOBT; GL Biochem). 8. 2,4,6-Collidine (Sigma-Aldrich). 9. Piperidine (Acros Organics). 10. Trifluoroacetic
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