Assessment of the Integrity of Compounds Stored in Assay-Ready Plates Using a Kinase Sentinel Assay

Abstract

Sentinel assays are a convenient adjunct to LC-MS purity assessment to monitor the integrity of compounds in pharmaceutical screening collections over time. To assess the stability of compounds stored both at room temperature and at -20°C in assay-ready plates that were either vacuum pack-sealed using a convenient industrial vacuum sealing method or individually sealed using conventional foil seals, a diverse collection of ~ 5,000 compounds was assayed using a robust biochemical kinase assay at intervals over a one year period. Assay results at each time point were compared to those of initial assay using a series of correlations of compound Percent of Control (POC) values as well as IC50 values of a subset of compounds in 200 nL or 500 nL plates. The fraction of hits in common between initial assays and assays at later time points ranged from 82% to 95% over one year and remained relatively constant over time with all storage temperatures or sealing methods tested. A majority of the hits that exhibited a consistent gradual trend to lower potency over one year storage were shifted to lower potency upon the rapid removal of DMSO solvent. Compound precipitation rather than compound decomposition is the likely reason for trends to lower potency for most such compounds over the storage period. Plates stored at room temperature featured a significantly higher fraction of hits that exhibited a trend to lower apparent potency than those stored at -20°C suggesting that this lower temperature is preferable for longer-term storage.

Oxidative Reactivities of 2-Furylquinolines: Ubiquitous Scaffolds in Common High-Throughput Screening Libraries

“…Assays were performed in triplicate and deaminase activity was quantified as previously reported. 10 Means ± standard deviations are indicated.…”

“…Annotations of chemical libraries to identify scaffolds prone to hit in unrelated assays have been performed in recent years. These offending compounds, termed Pan Assay Interference Scaffolds or PAINS, result as a consequence of the physical and chemical properties of the small molecule and/or its interactions with certain components of the HTS assay. − False positives can arise through various mechanisms including colloidal aggregation, compromised chemical integrity, or inherent reactivity of the small molecule, in addition to organic and/or inorganic impurities in the compound stock solution. − Maintaining the chemical integrity of small molecule libraries as dimethyl sulfoxide (DMSO) stock solutions upon long-term storage and freeze/thaw cycles remains a fundamental concern. − It is known that a percentage of HTS library members decompose when stored in DMSO, which is the most common solvent employed . A number of decomposition mechanisms are possible, such as hydrolysis, oxidation, isomerization, and rearrangement reactions.…”

Compound Transfer by Acoustic Droplet Ejection Promotes Quality and Efficiency in Ultra-High-Throughput Screening Campaigns

“…Turner et al 12 documented the preferential evaporation of DMSO aliquots in edge wells of 1536-well ARPs as detected by a DMSO-sensitive cytochrome P450 assay. In addition, Chen et al 13 found that more than onethird of a set of 230 compounds in ARPs lost activity, as detected in a sentinel kinase assay upon rapid evaporation of Well contents subject to drying Increased incidence of false-negatives Increased susceptibility to nonspecific inhibition the DMSO solvent. Moreover, their analysis of these compounds by liquid chromatography-mass spectrometry (LC-MS) confirmed that compound purity remained high under these conditions, indicating lack of decomposition and implicating insolubility as the cause.…”