title: A fluorescence-based high throughput assay for the determination of small molecule-human serum albumin protein binding.
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.
authors.affiliation: Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, 3210 N. Cramer St, Milwaukee, WI, 53211, USA.
authors.forename: Megan M
authors.initials: MM
authors.lastname: McCallum
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Pawlak
authors.forename: William R
authors.initials: WR
authors.lastname: Shadrick
authors.forename: Anton
authors.initials: A
authors.lastname: Simeonov
authors.forename: Ajit
authors.initials: A
authors.lastname: Jadhav
authors.forename: Adam
authors.initials: A
authors.lastname: Yasgar
authors.forename: David J
authors.initials: DJ
authors.lastname: Maloney
authors.forename: Leggy A
authors.initials: LA
authors.lastname: Arnold
mesh_terms: D001667:Binding, Competitive; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D057166:High-Throughput Screening Assays; D006801:Humans; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D011485:Protein Binding; D012709:Serum Albumin / Q000378:metabolism*; D054852:Small Molecule Libraries / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D005456:Fluorescent Dyes; D004364:Pharmaceutical Preparations; D012709:Serum Albumin; D054852:Small Molecule Libraries