Resurrecting the phoenix: When an assay fails

Understanding protein-small-molecule interactions is a critical component of rational drug-design. Structure-activity relationship (SAR)-guided medicinal chemistry is informed by the biological outcome, as assessed by biochemical activity or cellular effect, of chemical modifications on small molecules. The effectiveness of SAR is reliant on the sturdiness and durability of assay design and the quality of information garnered from assays. Lack of quality data at this step can lead to obstruction of the drug discovery pipeline with profound implications for the timelines of introducing a drug into the market. Hence, it would not be an overstatement to consider biochemical/biological assays as the backbone of drug-discovery. Enzyme assays can fail for many different reasons, with the enzyme and the substrate being the principal players. Lack of clarity can hamper progress and can lead to mounting costs and potentially losing competitive advantage. Although each assay is unique and requires a specific approach to troubleshoot the problem at hand, there are general guidelines that can be followed to maximize the chances of success. This review is a step-by-step attempt at reintroducing fundamental biochemical concepts within the context of an enzyme assay, delineating probable causes for failure and potential approaches to get an assay back up and running.