Expert Opinion on Drug Discovery | 2021
Evolution of assay interference concepts in drug discovery
Abstract
Biological assays for small molecules are vulnerable to experimental limitations and artifacts. False-positive activity readouts represent a recurrent and well-appreciated problem for high-throughput screening. Accordingly, hits from a primary high-throughput screen are usually subjected to secondary assays to reevaluate activity signals and establish dose– response behavior of active compounds. Typically, such confirmatory assays eliminate many but not all false-positive hits. While assay artifacts may frequently be attributable to technical issues, they are also caused by compounds that are not specifically active against a target of interest, but fake positive assay signals by a variety of undesired mechanisms. Such chemical entities are often termed assay interference compounds. For drug discovery, major problems arise when such interference compounds are difficult to identify and camouflaged false-positives enter hit-to-lead and lead optimization stages of the discovery pipeline. Significant time and resources might be wasted until their artificial nature is ultimately uncovered, especially if derivatives of pseudo-hits approach the realm of potential candidate compounds. Furthermore, undetected false-positives plague science and hinder progress. For example, in academic settings, new compounds – including those originating from computational screening – are often only investigated in a single assay system. If subtle artifacts occur under such conditions, they are notoriously difficult to detect. Once published, pseudo-hits tend to propagate through the literature and frequently give rise to follow-up studies that are doomed to fail and misleading at best. Over the past decade, it has increasingly been recognized that assay interference effects often are of unexpectedly large magnitude, continue to compromise drug discovery efforts, and pollute the scientific literature [1]. Most of these problems are caused by assay interference compounds, ‘chemical con artists’ [2], which are often rather difficult to uncover [1,2]. Different concepts have been introduced for recognizing and combating assay interference, which are of critical importance for the practice of biological screening and medicinal chemistry. However, these concepts are also controversially viewed for reasons discussed below. To assess their value, different scientific approaches and viewpoints should be taken into consideration.