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A fluorescence-based high throughput assay for the determination of small molecule−human serum albumin protein binding

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Abstract

Herein, we describe the development of a fluorescence-based high throughput assay to determine the small molecule binding towards human serum albumin (HSA). This innovative competition assay is based on the use of a novel fluorescent small molecule Red Mega 500 with unique spectroscopic and binding properties. The commercially available probe displays a large fluorescence intensity difference between the protein-bound and protein-unbound state. The competition of small molecules for HSA binding in the presence of probe resulted in low fluorescence intensities. The assay was evaluated with the library of pharmacological active compounds (LOPAC) small molecule library of 1,280 compounds identifying known high protein binders. The small molecule competition of HSA−Red Mega 500 binding was saturable at higher compound concentrations and exhibited IC50 values between 3 and 24 μM. The compound affinity toward HSA was confirmed by isothermal titration calorimetry indicating that the new protein binding assay is a valid high throughput assay to determine plasma protein binding.

A high throughput fluorescence-based serum protein binding assay

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Acknowledgments

The work was supported by the University of Wisconsin–Milwaukee, the University of Wisconsin-Milwaukee Research Growth Initiative (RGI), National Institute of Drug Abuse DA031090, the University of Wisconsin-Milwaukee Research Foundation, the Lynde and Harry Bradley Foundation, and the Richard and Ethel Herzfeld Foundation. Further support was enabled by the Molecular Libraries Initiative of the National Institutes of Health Roadmap for Medical Research and the Intramural Research Program of the NHGRI, NIH. We also thank Dr. Frick to enable the use of the ITC instrument.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Wisconsin–Milwaukee, 3210 N. Cramer St, Milwaukee, WI, 53211, USA

    Megan M. McCallum, Alan J. Pawlak, William R. Shadrick & Leggy A. Arnold

  2. NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, 20892-3370, USA

    Anton Simeonov, Ajit Jadhav, Adam Yasgar & David J. Maloney

Authors
  1. Megan M. McCallum
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  2. Alan J. Pawlak
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  3. William R. Shadrick
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  4. Anton Simeonov
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  5. Ajit Jadhav
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  6. Adam Yasgar
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  7. David J. Maloney
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  8. Leggy A. Arnold
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Correspondence to Leggy A. Arnold.

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McCallum, M.M., Pawlak, A.J., Shadrick, W.R. et al. A fluorescence-based high throughput assay for the determination of small molecule−human serum albumin protein binding. Anal Bioanal Chem 406, 1867–1875 (2014). https://doi.org/10.1007/s00216-013-7560-3

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  • DOI: https://doi.org/10.1007/s00216-013-7560-3

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