The Phenotypic Hit-to-Probe Challenge

Phenotypic drug discovery (PDD) fuels the clinical pipelines with first-in-class therapeutic modalities. Success rates depend on the quality of the assay models and can be hampered by resource-intensive processes of target and mode-of-action deconvolution. A critical part of this practice is generating focused sets of phenotypic probes to accelerate in vivo validation and to support target/concept tractability and translatability, raising the question of which phenotypic hit analogues to synthesize as high-quality chemical probes for a given campaign. Here, we outline recommendations and discuss criteria tailored to the specific demands of phenotypic probes. With examples of successful PDD programs, some of which have yielded approved first-in-class drugs, we illustrate common design principles. These underscore that phenotypic probes benefit from (a) availability of matched pairs of active/inactive stereoisomers, (b) ligand topology-guided optimization, and (c) exit vectors for ADME- and target deconvolution-driven acceleration of early in vivo proof-of-concept and advancement towards clinical candidates.