High throughput screening has become the dominant tool in the drug discovery process. In its earliest form, drug screening typically involved antimicrobial formats, however rapid progress in the fields of genomics, proteomics and molecular biology has expanded the target landscape beyond microbial targets, to both increase the numbers of potential drug targets, and facilitate development of assays to screen these targets. In parallel with these changes, developments in robotics and combinatorial chemical synthesis have driven the production of very large numbers of compounds with potential for pharmacological activity. The need to screen large libraries of chemical compounds against multiple targets has stimulated improvements in assay technology, instrumentation, and automation that evolved into the field of HTS and has revolutionized the field of drug discovery. In recent years, advances in miniaturization, parallel processing and data management have enhanced efficiency by controlling costs and increasing throughput thereby supporting the evolution of multiple platforms for lead generation.
Upon selection of a target, a lead generation strategy is defined which typically includes HTS as the first step – the “primary” assay, followed by hit confirmation – “secondary” assays, which precede lead validation and optimization. Many factors can influence the format of the assays employed and their positioning in the screening paradigm; the type of pharmacological information sought, the target class, throughput, cost, and other logistical or practical considerations. Cell based screens and biochemical or isolated target screens each have their merits. Biochemical assays which utilize isolated enzymes, proteins or receptor preparations are generally amenable to homogeneous assay formats and miniaturization, potentially identify the majority of chemotypes that interact with the target, and can provide target specificity information. In contrast, issues like cell membrane permeability and compound cytotoxicity may limit the diversity of chemotypes and pharmacophoric information obtained from cell based screens, and cellular assays are more challenging to miniaturize or automate.
The purpose of HTS is the interrogation of large chemical collections in the context of a biological target to accurately identify active chemotypes. To achieve this purpose, screens must be configured to provide a robust, reproducible signal with adequate throughput to screen large compound libraries. Since the activity or inactivity of any given compound in an HTS will typically be determined in a single well at one concentration, the assay signal window (dynamic range) must be sufficiently rugged to provide adequate separation between the maximum and minimum responses, and should enable the response to active compounds to be discriminated from the background variability (noise) associated with the top and bottom of the signal window. In addition to studies designed to validate the kinetics and pharmacology of the assay, efforts are made to optimize the signal window and/or variability of the assay in the context of a number of variables dictated by the automated process; DMSO tolerance, reagent stability, and signal stability. Superimposed upon assay development parameters associated with biochemical HTS formats, the implementation of cell-based screens present additional challenges; generation and/or characterization of an appropriate cell model, production of sufficient cells for HTS, plating cells for the assay, effects of compound exposure, and capture of the assay signal.