X-ray Structures of Target–Ligand Complexes Containing Compounds with Assay Interference Potential

Abstract

Pan assay interference compounds (PAINS) have become a paradigm for compound classes that might cause artifacts in biological assays. PAINS-defining substructures are typically contained in larger compounds. We have systematically examined X-ray structures of protein-ligand complexes for compounds containing PAINS motifs. In 2874 X-ray structures, 1107 unique ligands with PAINS substructures belonging to 70 different classes were identified. PAINS most frequently detected in crystallographic ligands included a number of prominent candidates such as quinones, catechols, or Mannich bases. However, on the basis of X-ray data, the presence of specific ligand-target interactions and reactivity under assay conditions were not mutually exclusive. In some instances, reactivity of ligands was likely responsible for complex formation. Different categories of PAINS-containing ligands were distinguished, which aided in the interpretation of specific interactions versus potential assay artifacts. Careful consideration of structural data adds another dimension to the analysis of interference compounds.

5-[(Z)-5-Chloro-2-oxoindolin-3-ylidene]-3-{(E)-[(4-hydroxyphenyl)imino]methyl}-2-thioxothiazolidin-4-one

“…unravel [13]. Therefore, such protein-ligand complex must be more rigorously evaluated using two or more experimental methods to avoid false results.…”

“…The product was obtained pure in 70% of yield by simple washing and filtration from ethanol. The structure of the resulting compound was defined using 1 H, 13 C-NMR spectroscopy; HRMS, and X-ray single crystal analysis (see Supplementary Materials for original spectra).…”

“…Figure 1 shows that the double bond between C(10) and C(11) is of a Z-configuration, while that between N(1) and C(7) is of an E-configuration. The structure of the resulting compound was defined using 1 H, 13 C-NMR spectroscopy; HRMS, and X-ray single crystal analysis (see Supplementary Materials for original spectra).…”

“…Arylidenerhodanines, due to the Michael acceptor functionality, are often claimed as pan assay interference compounds (PAINS) mainly in high throughput screening (HTS) campaigns [10,11]. Despite this, medicinal chemists have not lost interest in them as evidenced by numerous publications in these last few years [12], as the relationships between the inactivity, specific activity, and artificial activity of ligands with potential liabilities are often highly complex and difficult to unravel [13]. Therefore, such protein-ligand complex must be more rigorously evaluated using two or more experimental methods to avoid false results.…”

Targeting the Small GTPase Superfamily through Their Regulatory Proteins

“…[131] We have also identified PA INs motifs in many of the compounds targeting the GTPase regulators.P an assay interference compounds show activity across ar ange of biochemical, biophysical, and cellular assays and proteins, resulting in false positive identification or interference in in vitro or in vivo assays.I ti si mportant to note that by identifying inhibitors containing PA INs,weare not automatically claiming these compounds have been incorrectly identified as inhibitors of the GTPases or their regulators.I ti s important not to "black box" compounds that contain PA INs and immediately ignore them for future studies;t here are FDAi mproved drugs that contain PA INs motifs [45c, 132] and examples of PA INs compounds have been shown crystallographically to bind their putative targets. [133] Indeed, some of the PA INs compounds listed in this review have persuasive in vitro and in vivo data that correlates with the inhibition of stated targets.H owever,i ti si mportant to be aware of the possibility of the compound being an artefact, so that necessary steps are taken to confirm target selectivity and potencysuch as robust biophysical measures of binding or co- crystal structures.C ommon toxicophores have also been highlighted;w hilst these can be acceptable in chemical probes,c are should be taken when using compounds for in vivo evaluation or in therapeutic development. [22] In this review,f ew publications accommodated for PA INs or toxic moieties in their screens,w hich when combined with the aforementioned issues in compound design and use is worrying.…”