title: Development of the predictor HERG fluorescence polarization assay using a membrane protein enrichment approach.
abstract: The life-threatening consequences of acquired, or drug-induced, long QT syndrome due to block of the human ether-a-go-go-related gene (hERG) channel are well appreciated and have been the cause of several drugs being removed from the market in recent years because of patient death. In the last decade, the propensity for block of the hERG channel by a diverse and expanding set of compounds has led to the requirement that all new drugs be tested for hERG channel block in a functional patch-clamp assay. Because of the need to identify potential hERG blockers early in the discovery process, radiometric hERG binding assays are preferred over patch-clamp assays for compound triage, because of relative advantages in speed and cost. Even so, these radiometric binding assays are laborious and require dedicated instrumentation and infrastructure to cope with the regulatory and safety issues associated with the use of radiation. To overcome these limitations, we developed a homogeneous, fluorescence polarization-based assay to identify and characterize the affinity of small molecules for the hERG channel and have demonstrated tight correlation with data obtained from either radioligand binding or patch-clamp assays. Key to the development of this assay was a cell line that expressed highly elevated levels of hERG protein, which was generated by coupling expression of the hERG channel to that of a selectable cell surface marker. A high-expressing clone was isolated by flow cytometry and used to generate membrane preparations that contained >50-fold the typical density of hERG channels measured by [(3)H]astemizole binding. This strategy enabled the Predictor (Invitrogen, Carlsbad, CA) hERG fluorescence polarization assay and should be useful in the development of other fluorescence polarization-based assays that use membrane proteins.
authors.affiliation: Invitrogen Discovery Sciences, Madison, WI 53719, USA. david.piper@invitrogen.com
authors.forename: David R
authors.initials: DR
authors.lastname: Piper
authors.forename: Steve R
authors.initials: SR
authors.lastname: Duff
authors.forename: Hildegard C
authors.initials: HC
authors.lastname: Eliason
authors.forename: W Jack
authors.initials: WJ
authors.lastname: Frazee
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Frey
authors.forename: Maya
authors.initials: M
authors.lastname: Fuerstenau-Sharp
authors.forename: Christine
authors.initials: C
authors.lastname: Jachec
authors.forename: Bryan D
authors.initials: BD
authors.lastname: Marks
authors.forename: Brian A
authors.initials: BA
authors.lastname: Pollok
authors.forename: Mohammed Saleh
authors.initials: MS
authors.lastname: Shekhani
authors.forename: David V
authors.initials: DV
authors.lastname: Thompson
authors.forename: Pam
authors.initials: P
authors.lastname: Whitney
authors.forename: Kurt W
authors.initials: KW
authors.lastname: Vogel
authors.forename: Stephen D
authors.initials: SD
authors.lastname: Hess
mesh_terms: D016827:CD8 Antigens / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003627:Data Interpretation, Statistical; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004594:Electrophysiology; D051638:Ether-A-Go-Go Potassium Channels / Q000187:drug effects / Q000378:metabolism*; D005434:Flow Cytometry; D005454:Fluorescence Polarization / Q000379:methods*; D005456:Fluorescent Dyes; D005818:Genetic Engineering; D006801:Humans; D007150:Immunohistochemistry; D008564:Membrane Potentials / Q000502:physiology; D008565:Membrane Proteins / Q000502:physiology; D018408:Patch-Clamp Techniques; D011869:Radioligand Assay
chemical_list: D016827:CD8 Antigens; D051638:Ether-A-Go-Go Potassium Channels; D005456:Fluorescent Dyes; D008565:Membrane Proteins