title: Design of an automated enhanced-throughput platform for functional characterization of positive allosteric modulator-induced leftward shifts in apparent agonist potency in vitro.
abstract: Prosecution of positive allosteric modulator (PAM) targets demands a specialized assay toolset. Many GPCR or ion channel targets are adaptable to functional assays whereby PAM efficacy can be inferred from left or rightward shifts in the concentration-response curves of orthosteric agonist. The inherent emphasis on throughput and occasional paucity of radioligands for a diverse array of allosteric modulator targets yields a need for an enhanced throughput agonist potency shift assay. Here, we describe a process by which such an assay was automated with robust, reproducible in vitro pharmacology. In direct comparison with a manual CRC shift assay, the enhanced throughput automated platform described here delivered near identical rank orders (r(2) = 0.75) at ~4-fold throughput/assay iteration. Correspondingly, average cycle time/plate decreased from 104 to 72 minutes. We also observed reductions in assay interference associated with compounds exhibiting ago-allosterism, which we attribute to preread compound incubation periods which are more precisely time-constrained under automation control. By leveraging automated laboratory technology, we have achieved meaningful throughput with no sacrifice of precision. Rather than to be target-class specific, the present process was specifically designed to serve as a platform template for a variety of cell-based functional allosteric modulation assays.
authors.affiliation: Bristol-Myers Squibb, Wallingford, CT 06494, USA. adam.hendricson@bms.com
authors.forename: Adam
authors.initials: A
authors.lastname: Hendricson
authors.forename: Michele
authors.initials: M
authors.lastname: Matchett
authors.forename: Meredith
authors.initials: M
authors.lastname: Ferrante
authors.forename: Chris
authors.initials: C
authors.lastname: Ferrante
authors.forename: Ed
authors.initials: E
authors.lastname: Hunnicutt
authors.forename: Ryan
authors.initials: R
authors.lastname: Westphal
authors.forename: Walt
authors.initials: W
authors.lastname: Kostich
authors.forename: Yanling
authors.initials: Y
authors.lastname: Huang
authors.forename: Nelly
authors.initials: N
authors.lastname: Masias
authors.forename: Debra
authors.initials: D
authors.lastname: Hong
authors.forename: Rob
authors.initials: R
authors.lastname: Bertekap
authors.forename: Neil
authors.initials: N
authors.lastname: Burford
authors.forename: John
authors.initials: J
authors.lastname: Watson
authors.forename: Andrew
authors.initials: A
authors.lastname: Alt
authors.forename: Jim
authors.initials: J
authors.lastname: Myslik
authors.forename: Litao
authors.initials: L
authors.lastname: Zhang
authors.forename: Ron
authors.initials: R
authors.lastname: Knox
mesh_terms: D057205:Automation, Laboratory / Q000379:methods*; D003584:Cytological Techniques / Q000379:methods*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D007473:Ion Channels / Q000819:agonists*; D013678:Technology, Pharmaceutical / Q000379:methods*
chemical_list: D007473:Ion Channels