High-throughput screening (HTS) hits include compounds with undesirable properties. Many filters have been described to identify such hits. Notably, pan-assay interference compounds (PAINS) has been adopted by the community as the standard term to refer to such filters, and very useful guidelines have been adopted by the American Chemical Society (ACS) and subsequently triggered a healthy scientific debate about the pitfalls of draconian use of filters. Using an inhibitory frequency index, we have analyzed in detail the promiscuity profile of the whole GlaxoSmithKline (GSK) HTS collection comprising more than 2 million unique compounds that have been tested in hundreds of screening assays. We provide a comprehensive analysis of many previously published filters and newly described classes of nuisance structures that may serve as a useful source of empirical information to guide the design or growth of HTS collections and hit triaging strategies.

As part of an overall systems approach to generating highly accurate screening data across large numbers of compounds and biological targets, we have developed and implemented streamlined methods for purifying and quantitating compounds at various stages of the screening process, coupled with automated "traditional" storage methods (DMSO, -20 degrees C). Specifically, all of the compounds in our druglike library are purified by LC/MS/UV and are then controlled for identity and concentration in their respective DMSO stock solutions by chemiluminescent nitrogen detection (CLND)/evaporative light scattering detection (ELSD) and MS/UV. In addition, the compound-buffer solutions used in the various biological assays are quantitated by LC/UV/CLND to determine the concentration of compound actually present during screening. Our results show that LC/UV/CLND/ELSD/MS is a widely applicable method that can be used to purify, quantitate, and identify most small organic molecules from compound libraries. The LC/UV/CLND technique is a simple and sensitive method that can be easily and cost-effectively employed to rapidly determine the concentrations of even small amounts of any N-containing compound in aqueous solution. We present data to establish error limits for concentration determination that are well within the overall variability of the screening process. This study demonstrates that there is a significant difference between the predicted amount of soluble compound from stock DMSO solutions following dilution into assay buffer and the actual amount present in assay buffer solutions, even at the low concentrations employed for the assays. We also demonstrate that knowledge of the concentrations of compounds to which the biological target is exposed is critical for accurate potency determinations. Accurate potency values are in turn particularly important for drug discovery, for understanding structure-activity relationships, and for building useful empirical models of protein-ligand interactions. Our new understanding of relative solubility demonstrates that most, if not all, decisions that are made in early discovery are based upon missing or inaccurate information. Finally, we demonstrate that careful control of compound handling and concentration, coupled with accurate assay methods, allows the use of both positive and negative data in analyzing screening data sets for structure-activity relationships that determine potency and selectivity.

The process of drug discovery has evolved considerably since the advent of high-throughput screening (HTS) in the 1980s. Experts and opinion leaders today are agreeing that the current trend in the field is a focus on increasing overall quality (target, screening, and compounds), use of multiple screening approaches for lead discovery, and more flexibility in the process. The associated need for increased flexibility and quality control to support existing HTS paradigms as well as lower throughput approaches such as fragment screening, computational chemistry, focused library building, and centralized lead optimization support has required an evolution in compound management (CM, aka sample management or library management). Although there is much less published peer-reviewed data in CM, due to its historical links to HTS, it has followed very similar trends. In recent years, the focus in CM has been increasingly in compound quality and increased flexibility of the process, as opposed to number of compounds dispensed and speed of dispensing, which were standard metrics and indicators used not so long ago. Ideally, to screen the highest quality sample for every assay, one would start with a correct identity and pure solid, make a correct concentration solution in water or water-soluble/assay-compatible solvent that would allow 100% solubilization, and screen it immediately in a biological assay. Neither CM nor screening has advanced sufficiently to deliver this ideal scenario, but many significant advancements have been made in recent years both in terms of quality of compounds in stores and flexibility of the process, which will be reviewed herein.

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