Kinetic identification of protein ligands in a 51,200 small-molecule library using microarrays and a label-free ellipsometric scanner

James P. Landry; Andrew P. Proudian; Galina Malovichko; Xiangdong Zhu

doi:10.1117/12.2003374

22 February 2013 Kinetic identification of protein ligands in a 51,200 small-molecule library using microarrays and a label-free ellipsometric scanner

James P. Landry, Andrew P. Proudian, Galina Malovichko, Xiangdong Zhu

Author Affiliations +

Proceedings Volume 8587, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XI; 85871V (2013) https://doi.org/10.1117/12.2003374
Event: SPIE BiOS, 2013, San Francisco, California, United States

Abstract

Drug discovery begins by identifying protein-small molecule binding pairs. Afterwards, binding kinetics and biofunctional assays are performed, to reduce candidates for further development. High-throughput screening, typically employing fluorescence, is widely used to find protein ligands in small-molecule libraries, but is rarely used for binding kinetics measurement because: (1) attaching fluorophores to proteins can alter kinetics and (2) most label-free technologies for kinetics measurement are inherently low-throughput and consume expensive sensing surfaces. We addressed this need with polarization-modulated ellipsometric scanning microscopes, called oblique-incidence reflectivity difference (OI-RD). Label-free ligand screening and kinetics measurement are performed simultaneously on small-molecule microarrays printed on relatively inexpensive isocyanate-functionalized glass slides. As a microarray is reacted, an OI-RD microscope tracks the change in surface-bound macromolecule density in real-time at every spot. We report progress applying OI-RD to screen purified proteins and virus particles against a 51,200-compound library from the National Cancer Institute. Four microarrays, each containing 12,800 library compounds, are installed in four flow cells in an automated OI-RD microscope. The slides are reacted serially, each giving 12,800 binding curves with ~30 sec time resolution. The entire library is kinetically screened against a single probe in ~14 hours and multiple probes can be reacted sequentially under automation. Real-time binding detection identifies both high-affinity and low-affinity (transient binding) interactions; fluorescence endpoint images miss the latter. OI-RD and microarrays together is a powerful high-throughput tool for early stage drug discovery and development. The platform also has great potential for downstream steps such as in vitro inhibition assays.

Citation Download Citation

James P. Landry, Andrew P. Proudian, Galina Malovichko, and Xiangdong Zhu "Kinetic identification of protein ligands in a 51,200 small-molecule library using microarrays and a label-free ellipsometric scanner", Proc. SPIE 8587, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XI, 85871V (22 February 2013); https://doi.org/10.1117/12.2003374

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