96-Well Plate Colorimetric Assay for Ki Determination of (+/-)-2-Benzylsuccinic Acid, an Inhibitor of Carboxypeptidase A: A Laboratory Experiment in Drug Discovery

A new 96-well microplate bioassay has been developed for students to learn and practice biotechnologies that researchers use to discover therapeutics. This experiment and others are part of the recently initiated course, Drug Discovery (CHEM-4330) at Rensselaer Polytechnic Institute. In this experiment, students determine the dissociation constant for inhibitor binding (K_i) of the carboxypeptidase A inhibitor, (±)-2-benzylsuccinic acid, in kinetic mode. While carboxypeptidase A and 2-benzylsuccinic acid have no direct association with human drug action, pioneering research in rational drug design in the 1970s used both as a crucial springboard to identify captopril, the first clinically useful antihypertensive agent of the ACE inhibitor class. To relate lab to lecture, a case study in captopril discovery is presented in the lecture part of the course; other lecture topics include molecular target validation, high throughput screening–assay development, and combinatorial chemistry. In this experiment, students learn principles and techniques used by drug discovery researchers, including the use of microplate readers, programming plate reader software, layout of bioassays, Lineweaver–Burk plots, and multichannel–repeater pipetting.