Phantom PAINS: Problems with the Utility of Alerts for Pan-Assay INterference CompoundS

Abstract

The use of substructural alerts to identify Pan-Assay INterference compoundS (PAINS) has become a common component of the triage process in biological screening campaigns. These alerts, however, were originally derived from a proprietary library tested in just six assays measuring protein–protein interaction (PPI) inhibition using the AlphaScreen detection technology only; moreover, 68% (328 out of the 480 alerts) were derived from four or fewer compounds. In an effort to assess the reliability of these alerts as indicators of pan-assay interference, we performed a large-scale analysis of the impact of PAINS alerts on compound promiscuity in bioassays using publicly available data in PubChem. We found that the majority (97%) of all compounds containing PAINS alerts were actually infrequent hitters in AlphaScreen assays measuring PPI inhibition. We also found that the presence of PAINS alerts, contrary to expectations, did not reflect any heightened assay activity trends across all assays in PubChem including AlphaScreen, luciferase, beta-lactamase, or fluorescence-based assays. In addition, 109 PAINS alerts were present in 3570 extensively assayed, but consistently inactive compounds called Dark Chemical Matter. Finally, we observed that 87 small molecule FDA-approved drugs contained PAINS alerts and profiled their bioassay activity. Based on this detailed analysis of PAINS alerts in nonproprietary compound libraries, we caution against the blind use of PAINS filters to detect and triage compounds with possible PAINS liabilities and recommend that such conclusions should be drawn only by conducting orthogonal experiments.

Identification of Compounds That Interfere with High‐Throughput Screening Assay Technologies

“…[27] However,e ven for AlphaScreen, the accuracy is quite low when the filters are applied to our data. As imilar observation was made in as tudy by Capuzzi et al [11] One hypothesis may be that the relativelyl ow accuracy is due to differences between our dataset and the one on which the filters were built, in terms of compound structures as well as screening conditions, such as compound concentration. These hypotheses could not be verified as the PAINS dataset was never publicly disclosed.I ti sa lso possible that specific PAINS substructures were classified as undesirable internally and were thus not part of the screening deck.…”

“…Over the years, the applicability of PAINS filtersh as been discussed and criticized. [5,10,11] It has been reported that the presence of PAINS substructure does not necessarily imply an undesirable mechanism and the applicability domain of the PAINS is limited by the chemical space and the unique assay technologyu sed in buildingt he filters.…”

Targeting the Small GTPase Superfamily through Their Regulatory Proteins

“…It is important to note that by identifying inhibitors containing PAINs, we are not automatically claiming these compounds have been incorrectly identified as inhibitors of the GTPases or their regulators. It is important not to “black box” compounds that contain PAINs and immediately ignore them for future studies; there are FDA improved drugs that contain PAINs motifs and examples of PAINs compounds have been shown crystallographically to bind their putative targets . Indeed, some of the PAINs compounds listed in this review have persuasive in vitro and in vivo data that correlates with the inhibition of stated targets.…”

Targeting der kleinen GTPasen über ihre regulatorischen Proteine

“…Es ist wichtig zu beachten, dass wir bei der Identifizierung von PAINs enthaltenden Inhibitoren nicht automatisch behaupten, dass diese Verbindungen inkorrekterweise als Inhibitoren der GTPasen oder ihrer Regulatoren identifiziert wurden. Es ist wichtig, PAINs enthaltende Verbindungen nicht sofort für zukünftige Studien zu ignorieren; es gibt von der FDA zugelassene Wirkstoffe, die PAINs‐Motive enthalten und Beispiele von PAINs‐Verbindungen, für die kristallographisch gezeigt wurde, dass sie an ihre vermeintlichen Zielstrukturen binden . In der Tat zeigen einige der in diesem Aufsatz beschriebenen Verbindungen überzeugende In‐vitro‐ und In‐vivo‐Daten, die mit der Inhibition der angegebenen Zielstrukturen korrelieren.…”