title: Development of a Platform to Enable Fully Automated Cross-Titration Experiments.
abstract: In the triage of hits from a high-throughput screening campaign or during the optimization of a lead compound, it is relatively routine to test compounds at multiple concentrations to determine potency and maximal effect. Additional follow-up experiments, such as agonist shift, can be quite valuable in ascertaining compound mechanism of action (MOA). However, these experiments require cross-titration of a test compound with the activating ligand of the receptor requiring 100-200 data points, severely limiting the number tested in MOA assays in a screening triage. We describe a process to enhance the throughput of such cross-titration experiments through the integration of Hewlett Packard's D300 digital dispenser onto one of our robotics platforms to enable on-the-fly cross-titration of compounds in a 1536-well plate format. The process handles all the compound management and data tracking, as well as the biological assay. The process relies heavily on in-house-built software and hardware, and uses our proprietary control software for the platform. Using this system, we were able to automate the cross-titration of compounds for both positive and negative allosteric modulators of two different G protein-coupled receptors (GPCRs) using two distinct assay detection formats, IP1 and Ca2+detection, on nearly 100 compounds for each target.
authors.affiliation: 1 Screening & Protein Science, Merck and Co., North Wales, PA, USA.
authors.forename: Jason
authors.initials: J
authors.lastname: Cassaday
authors.affiliation: 1 Screening & Protein Science, Merck and Co., North Wales, PA, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Finley
authors.affiliation: 1 Screening & Protein Science, Merck and Co., North Wales, PA, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Squadroni
authors.affiliation: 1 Screening & Protein Science, Merck and Co., North Wales, PA, USA.
authors.forename: Sylvie
authors.initials: S
authors.lastname: Jezequel-Sur
authors.affiliation: 1 Screening & Protein Science, Merck and Co., North Wales, PA, USA.
authors.forename: Albert
authors.initials: A
authors.lastname: Rauch
authors.affiliation: 1 Screening & Protein Science, Merck and Co., North Wales, PA, USA.
authors.forename: Bharti
authors.initials: B
authors.lastname: Gajera
authors.affiliation: 1 Screening & Protein Science, Merck and Co., North Wales, PA, USA.
authors.forename: Victor
authors.initials: V
authors.lastname: Uebele
authors.affiliation: 1 Screening & Protein Science, Merck and Co., North Wales, PA, USA.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Hermes
authors.affiliation: 1 Screening & Protein Science, Merck and Co., North Wales, PA, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Zuck
mesh_terms: D057205:Automation, Laboratory / Q000295:instrumentation / Q000379:methods*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation / Q000379:methods*; D057166:High-Throughput Screening Assays; D006801:Humans; D043562:Receptors, G-Protein-Coupled / Q000819:agonists; D017186:Titrimetry / Q000295:instrumentation / Q000379:methods*
chemical_list: D043562:Receptors, G-Protein-Coupled
keywords: G protein–coupled receptor; HTS; agonist shift; allostery; automated biology; engineering; high-throughput screening; robotics and instrumentation