title: Evaluation and optimization of compound solubilization and delivery methods in a two-tiered ion channel lead optimization triage.
abstract: Low-volume dispensing of neat dimethyl sulfoxide (DMSO) into plate-based assays conserves compound, assay reagents, and intermediate dilution plate cost and, as we demonstrate here, significantly improves structure-activity relationship resolution. Acoustic dispensing of DMSO solutions into standard volume 384W plates yielded inconsistent results in studies with 2 cell lines because of apparent effects on the integrity of the cell monolayer (increased intracellular Ca⁺⁺ levels as indicated by elevated basal dye fluorescence after acoustic transfer). PocketTip-mediated transfer was successful at increasing apparent potency on a more consistent basis. Notably, the correlation coefficient among fluorescence imaging plate reader (FLIPR):electrophysiology (EP) across a representative ~125 compound collection was increased ~5× via conversion to a PocketTip direct dispensation, indicating a triage assay more predictive of activity in the decisional patch-clamp assay. Very importantly, the EP-benchmarked false-negative rate as measured by compounds with FLIPR EC₅₀ more than the highest concentration tested fell from >11% to 5% assay-wide, and the relative FLIPR:EP rank-order fidelity increased from 55% to 78%. Elimination of the aqueous intermediate step provided additional benefits, including reduced assay cost, decreased cycle time, and reduced wet compound consumption rate. Direct DMSO dispensing has broad applicability to cell-based functional assays of multiple varieties, especially in cases where limit solubility in assay buffer is a recognized impediment to maximizing interassay connectivity.
authors.affiliation: Department of Molecular Sciences and Candidate Optimization/Lead Evaluation, Bristol Myers Squibb, Wallingford, Connecticut 06494, USA. adam.hendricson@bms.co
authors.forename: Adam W
authors.initials: AW
authors.lastname: Hendricson
authors.forename: Liz
authors.initials: L
authors.lastname: Gallagher
authors.forename: Michele
authors.initials: M
authors.lastname: Matchett
authors.forename: Meredith
authors.initials: M
authors.lastname: Ferrante
authors.forename: Steve
authors.initials: S
authors.lastname: Spence
authors.forename: Tony
authors.initials: T
authors.lastname: Paiva
authors.forename: Wilson
authors.initials: W
authors.lastname: Shou
authors.forename: Svetlana
authors.initials: S
authors.lastname: Tertyshnikova
authors.forename: Mike
authors.initials: M
authors.lastname: Krambis
authors.forename: Deborah
authors.initials: D
authors.lastname: Post-Munson
authors.forename: Litao
authors.initials: L
authors.lastname: Zhang
authors.forename: Ron
authors.initials: R
authors.lastname: Knox
mesh_terms: D000162:Acoustics; D002021:Buffers; D002118:Calcium / Q000032:analysis / Q000737:chemistry; D002498:Centrifugation; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D005188:False Negative Reactions; D005470:Fluorometry; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays / Q000295:instrumentation* / Q000379:methods*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007201:Indicator Dilution Techniques; D007473:Ion Channels / Q000187:drug effects*; D018408:Patch-Clamp Techniques; D012995:Solubility; D012996:Solutions; D014867:Water
chemical_list: D002021:Buffers; D007473:Ion Channels; D012996:Solutions; D014867:Water; D002118:Calcium; D004121:Dimethyl Sulfoxide