Abstract
We describe herein a new method for the high-throughput identification of affinity-based probes (AfBPs) using a small molecule microarray (SMM) approach. A hydroxylethylene-based small molecule library was first generated by solid-phase combinatorial synthesis. The library was tagged with biotin to facilitate immobilization on avidin-coated slides. Preliminary screening with γ-secretase (both the recombinantly purified protein as well as cellular lysates overexpressing the enzyme) was carried out, in order to identify potential small molecule binders, which were subsequently redesigned into AfBPs. Several specific and potent probes for γ-secretase were thus identified through the binding profiles observed on the SMMs. The SMM platform was able to sensitively and conveniently report activity-based binding interactions between aspartic proteases and their small molecule inhibitors. This new approach thus provides a potentially more rapid and efficient method for developing AfBPs using SMMs.
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Acknowledgments
We gratefully acknowledge financial support by MOE (R143-000-394-112), BMRC (R143-000-391-305), CRP (R143-000-218-281), and DSO National Laboratories. We thank R. Yada (University of Guelph, Canada) and M. Wolfe (Harvard) for the kind gifts of HAP (and mutants) and γ-30 cell line, respectively.
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Shi, H., Uttamchandani, M., Yao, S.Q. (2010). A Method for Small Molecule Microarray-Based Screening for the Rapid Discovery of Affinity-Based Probes. In: Uttamchandani, M., Yao, S. (eds) Small Molecule Microarrays. Methods in Molecular Biology, vol 669. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-845-4_5
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DOI: https://doi.org/10.1007/978-1-60761-845-4_5
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