Dark chemical matter – could “inactive” compounds be good starting points for lead discovery?

High-throughput screening (HTS) has become an integral part of early drug discovery in the pharmaceutical industry and academia and has enabled the discovery and insight into the biological actions of numerous reagents. Here, we focus on those small molecules in a screening collection that have shown no biological activity despite having been exhaustively tested in numerous HTS assays. These compounds are referred to as “dark chemical matter” (DCM). For the first time, we quantify DCM, validate it in quality control experiments, describe its physicochemical properties, and map it into the chemical space relative to the other compounds in the screening collection. Through analysis of prospective reporter gene assay, gene expression, and yeast chemogenomics experiments, we evaluate the potential of dark matter compounds to show biological activity in future screens. We demonstrate that despite the apparent lack of activity of DCM, occasionally these compounds can result in potent hits with unique activity profiles. Among the identified DCM hits is a new anti-fungal chemotype with strong activity against the pathogen Cryptococcus neoformans, but little activity at human host targets. Testing DCM for biological activity may be a widely applicable strategy to finding hits with reduced promiscuity and clean safety profiles, which makes these compounds valuable starting points for small molecule lead optimization efforts.