title: Application of Cassette Ultracentrifugation Using Non-labeled Compounds and Liquid Chromatography-Tandem Mass Spectrometry Analysis for High-Throughput Protein Binding Determination.
abstract: Membrane-based devices typically used for serum protein binding determination are not fully applicable to highly lipophilic compounds because of nonspecific binding to the device membrane. Ultracentrifugation, however, completely eliminates the issue by using a membrane-free approach, although its wide application has been limited. This lack of utilization is mainly attributed to 2 factors: the high cost in acquiring and handling of radiolabeled compounds and low assay throughput owing to the difficulties in process automation. To overcome these challenges, we report a high-throughput workflow by cassette ultracentrifugation of nonradiolabeled compounds followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Twenty compounds with diverse physicochemical and protein binding properties were selected for the evaluation of the workflow. To streamline the working process, approaches of matrix balancing for all the samples for LC-MS/MS analysis and determining free fraction without analytical calibration curves were adopted. Both the discrete ultracentrifugation of individual compounds and cassette ultracentrifugation of all the test compounds followed by simultaneous LC-MS/MS analysis exhibited a linear correlation with literature values, demonstrating respectively the validity of the ultracentrifugation process and the cassette approach. The cassette ultracentrifugation using nonradiolabeled compounds followed by LC-MS/MS analysis has greatly facilitated its application for high-throughput protein binding screening in drug discovery.
authors.affiliation: Discovery Chemistry Platforms, Bristol-Myers Squibb Company, Wallingford, Connecticut 06492.
authors.forename: Kasia
authors.initials: K
authors.lastname: Kieltyka
authors.affiliation: Discovery Infectious Diseases, Bristol-Myers Squibb Company, Wallingford, Connecticut 06492.
authors.forename: Brian
authors.initials: B
authors.lastname: McAuliffe
authors.affiliation: Discovery Infectious Diseases, Bristol-Myers Squibb Company, Wallingford, Connecticut 06492.
authors.forename: Christopher
authors.initials: C
authors.lastname: Cianci
authors.affiliation: Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Company, Wallingford, Connecticut 06492.
authors.forename: Dieter M
authors.initials: DM
authors.lastname: Drexler
authors.affiliation: Discovery Chemistry Platforms, Bristol-Myers Squibb Company, Wallingford, Connecticut 06492.
authors.forename: Wilson
authors.initials: W
authors.lastname: Shou
authors.affiliation: Discovery Chemistry Platforms, Bristol-Myers Squibb Company, Wallingford, Connecticut 06492. Electronic address: jun.zhang1@bms.com.
authors.forename: Jun
authors.initials: J
authors.lastname: Zhang
mesh_terms: D001798:Blood Proteins / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D055808:Drug Discovery / Q000379:methods; D007553:Isotope Labeling / Q000379:methods; D004364:Pharmaceutical Preparations / Q000737:chemistry*; D011485:Protein Binding; D044967:Serum / Q000737:chemistry; D013194:Staining and Labeling / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014461:Ultracentrifugation / Q000379:methods*; D057188:Workflow
chemical_list: D001798:Blood Proteins; D004364:Pharmaceutical Preparations
keywords: LC-MS/MS; cassette; high-throughput; protein binding; ultracentrifugation