Innovative Electrochemical Screening Allows Transketolase Inhibitors to Be Identified

Abstract

Transketolases (TKs) are ubiquitous thiamine pyrophosphate (TPP)-dependent enzymes of the nonoxidative branch of the pentose phosphate pathway. They are considered as interesting therapeutic targets in numerous diseases and infections (e.g., cancer, tuberculosis, malaria), for which it is important to find specific and efficient inhibitors. Current TK assays require important amounts of enzyme, are time-consuming, and are not specific. Here, we report a new high throughput electrochemical assay based on the oxidative trapping of the TK-TPP intermediate. After electrode characterization, the enzyme loading, electrochemical protocol, and substrate concentration were optimized. Finally, 96 electrochemical assays could be performed in parallel in only 7 min, which allows a rapid screening of TK inhibitors. Then, 1360 molecules of an in-house chemical library were screened and one early lead compound was identified to inhibit TK from E. coli with an IC of 63 μM and an inhibition constant ( K) of 3.4 μM. The electrochemical assay was also used to propose an inhibition mechanism.

Layered double hydroxide-based nanomaterials for biomedical applications

“…[357][358][359] Therefore, nanomaterial-based biosensors have sparked a wealth of interest to researchers. [360][361][362][363] LDHs have great potential in the construction of highperformance biosensors due to the following characteristics:…”

“…357–359 Therefore, nanomaterial-based biosensors have sparked a wealth of interest to researchers. 360–363…”

High-Throughput Electrochemistry: State of the Art, Challenges, and Perspective

“…In addition to biomolecule detection, screen-printed multielectrode arrays have been adapted for high-throughput screening of enzyme libraries generated from directed evolution. , In these systems, the electrodes in each well are covered with 35–200 μL of electrolyte, and the enzymatic activity is determined using intermittent pulse amperometry. This many electrodes, many electrolytes approach has been used to select enzymes for biofuel cells and synthetic biology as well as the screening of enzyme inhibitors . Screen-printed multielectrode arrays have also been developed in a three-electrode format to allow more precise control over the electrode potential .…”

Tailored Biosensors for Drug Screening, Efficacy Assessment, and Toxicity Evaluation

“…To overcome this hurdle, a new high-throughput electrochemical assay was created by the oxidative trapping of the transketolases−thiamine pyrophosphate intermediate. 106 Of note, 96 electrochemical assays were accomplished in parallel in only 7 min. It was applied to screen against an in-house chemical library of 1360 molecules.…”