Electrochemical screening as a tool for identification of transketolase inhibitors

This chapter describes the development and application of a 96-well electrochemical screening plate for the identification of inhibitors of transketolase, a key enzyme of the pentose phosphate pathway, involved in multiple diseases (cancers, neurodegenerative disorders, and infections). This work details the design and manufacturing of printed circuit board (PCB) plates with screen-printed electrodes, optimized for independent electrochemical readout in each well. It describes the electrochemical assay, including electrode quality control, as well as the high-throughput screening of a chemical library of 1360 molecules using an affordable substrate (L-erythrulose). This proof-of-concept is validated with the transketolase of Escherichia coli, as model of bacterial enzyme. Key advantages include speed, universality, and not requiring chromogenic reagents, offset by the need for specialized equipment and strict electrode quality control. Hits are validated by complementary colorimetric assays to confirm results and determine kinetic parameters.