In Vitro Resistance-Predicting Studies and In Vitro Resistance-Related Parameters-A Hit-to-Lead Perspective

Despite the urgent need for new antibiotics, very few innovative antibiotics have recently entered clinics or clinical trials. To provide a constant supply of new drug candidates optimized in terms of their potential to select for resistance in natural settings, in vitro resistance-predicting studies need to be improved and scaled up. In this review, the following in vitro parameters are presented: frequency of spontaneous mutant selection (FSMS), mutant prevention concentration (MPC), dominant mutant prevention concentration (MPC-D), inferior-mutant prevention concentration (MPC-F), and minimal selective concentration (MSC). The utility of various adaptive laboratory evolution (ALE) approaches (serial transfer, continuous culture, and evolution in spatiotemporal microenvironments) for comparing hits in terms of the level and time required for multistep resistance to emerge is discussed. We also consider how the hit-to-lead stage can benefit from high-throughput ALE setups based on robotic workstations, do-it-yourself (DIY) continuous cultivation systems, microbial evolution and growth arena (MEGA) plates, soft agar gradient evolution (SAGE) plates, microfluidic chips, or microdroplet technology. Finally, approaches for evaluating the fitness of in vitro-generated resistant mutants are presented. This review aims to draw attention to newly emerged ideas on how to improve the in vitro forecasting of the potential of compounds to select for resistance in natural settings.