Design of an automated enhanced-throughput platform for functional characterization of positive allosteric modulator-induced leftward shifts in apparent agonist potency in vitro.

Journal of laboratory automation

PubMedID: 22357567

Hendricson A, Matchett M, Ferrante M, Ferrante C, Hunnicutt E, Westphal R, Kostich W, Huang Y, Masias N, Hong D, Bertekap R, Burford N, Watson J, Alt A, Myslik J, Zhang L, Knox R. Design of an automated enhanced-throughput platform for functional characterization of positive allosteric modulator-induced leftward shifts in apparent agonist potency in vitro. J Lab Autom. 2012;17(2):104-15.
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.