A Course of Proteolytic Stability Experiment for Undergraduates: Drug Affinity Responsive Target Stability (DARTS)

Proteins are key executors of biological functions in living organisms and serve as important targets in modern drug development. Protein stability is a critical physicochemical property that can be significantly altered by interactions with drugs or other ligands. Understanding the changes in protein stability and the factors that influence these changes is essential for undergraduates in the field of pharmacy and related subjects. Drug affinity responsive target stability (DARTS) technology, which is based on changes in proteolytic stability, is widely used to identify drug targets and validate drug–protein interactions. To enhance student learning about protein stability, we designed a four-week experimental course based on DARTS for upper-division undergraduates. In this course, students used DARTS, SDS–PAGE, and computational tools to study the effect of ibuprofen binding on the proteolytic stability of human serum albumin (HSA). Students reviewed the relevant literature to obtain foundational knowledge, optimized Pronase hydrolysis conditions, evaluated the impact of ibuprofen on HSA proteolysis, and characterized the samples using SDS–PAGE. Additionally, the students used PyMOL to visualize molecular interactions, gaining a molecular-level understanding of how drug binding affects protein stability. This hands-on course combines wet and dry experiments, helps students better understand protein stability and its importance in drug development, develops skills in literature review, experimental techniques, and computational visualization, and provides essential theoretical knowledge and practical experience for careers in drug research.