Transketolase electrochemical bioassay for the screening of inhibitors in microplates – TRANSBIOSCREEN

In the cell metabolism, transketolase (TK) is the most critical enzyme of the non-oxidative branch of the pentose phosphate pathway (PPP) leading to the biosynthesis of D-ribose-5-phosphate (D-R-5-P) required for nucleic acid synthesis. Activation of the PPP was evidenced in the abnormal proliferation of tumour cells. Besides, the deficiency in transketolase activity was shown for patients showing some neurodegenerative diseases such as Alzheimer’s disease.
In this context, TK has been suggested as a therapeutic target for inhibition in the treatment of cancer. Here we propose to develop a 96-well electrochemical TK assay for the High Throughput Screening (HTS) of putative TK inhibitors. As a rule, the rapid electrochemical screening of enzyme activities in bioelectronics is still a challenging issue.
Our system will be composed of 96 screen-printed electrodes on a printed circuit board adapted from a commercial system (carbon is used as the working electrode and silver chloride as the counter/reference electrode). The associated device allows performing the measurements on the 96 microelectrodes within a few seconds. To note, as low as 30 to 50 µL of sample volume are required. Moreover, this method is reagentless decreasing highly the costs of the assay, in particular if one considers that the printed circuit board could be easily re-used.
The originality of this work is related also with the immobilization of TK on its apoenzyme form. To date, the most active TK inhibitors reported on are thiamine diphosphate analogs, i.e competitive inhibitors of thiamine diphosphate (ThDP, TK natural cofactor).
The first challenge of this project consists in the chose an adequate multienzymatic system involving one (or more) auxiliary enzyme(s) to convert TK enzymatic activity into a measurable electrical signal. The development of these biosensing devices requires the immobilization of these enzymes on the transductor (electrode). The approach will be realized by encapsulation of these enzymes within lamellar inorganic materials, so-called Layered double hydroxides (LDH) or cationic clays. Those materials are known to be particularly biocompatible for enzyme immobilization. Different transduction mode will be envisaged, using mediated electron transfer. Modulation of enzymatic response with various referenced and putative inhibitors will be carried out using 96 screen-printed electrodes to ensure sufficient throughput in small scale of time. The ultimate goal would be to render our system ready-to-use for the screening of libraries of synthetic compounds in parallel.
The multidisciplinary nature of the project requires a combination of specific skills and expertise. F. Charmantray, the coordinator of this project, will bring his skills in the field of biocatalysis and more precisely in the use of enzyme in cascade reactions. Moreover, he is also in charge of the robotic automated platform of the ICCF laboratory. This platform is dedicated to enzyme screening in miniaturized devices. F. Charmantray proposes to build-up a team around young researchers from ICCF UMR CNRS 6296 (V. Prévot, Aubière) and ICBMS UMR CNRS 5246 (B. Doumèche, Lyon). V. Prévot is an expert in the field of chemistry of biohybrid materials based on LDH, while B. Doumèche will bring is competences in the field of electrochemical transduction signal in miniaturization devices. L. Hecquet and C. Mousty (ICCF-Aubière) will bring their knowledge in the fields of Biocatalysis and Electrochemistry applied in biosensors development, respectively.