Electrochemical assay for the quantification of anticancer drugs and their inhibition mechanism

Ricardo Jose Branco Leote; Caroline G Sanz; Victor C Diculescu; Madalina Maria Barsan

doi:10.1016/j.ymeth.2025.05.002

Electrochemical assay for the quantification of anticancer drugs and their inhibition mechanism

Methods. 2025 May 8:241:13-23. doi: 10.1016/j.ymeth.2025.05.002. Online ahead of print.

Authors

Ricardo Jose Branco Leote¹, Caroline G Sanz², Victor C Diculescu², Madalina Maria Barsan³

Affiliations

¹ National Institute of Materials Physics (NIMP), Str. Atomistilor 405A, 077125 Măgurele, Romania; Faculty of Physics, University of Bucharest, Atomistilor 405, 077125 Măgurele, Romania.
² National Institute of Materials Physics (NIMP), Str. Atomistilor 405A, 077125 Măgurele, Romania.
³ National Institute of Materials Physics (NIMP), Str. Atomistilor 405A, 077125 Măgurele, Romania. Electronic address: madalina.barsan@infim.ro.

PMID: 40345605
DOI: 10.1016/j.ymeth.2025.05.002

Abstract

Overexpression of pyruvate kinase (PyK) is linked to many kinds of malignant tumors, representing therefore one of the most promising therapeutic targets for cancer treatment. Inhibition of PyK slows down tumor growth or causes tumor cell death, minimizing cancer cell proliferation, and understanding inhibitor mechanism of action can significantly improve cancer therapy. The present work describes the use of an amperometric bienzymatic biosensor, based on PyK and pyruvate oxidase (PyOx), in enzyme inhibition studies of four kinase inhibitors, CPG77675, Nilotinib, Ruxolitinib, Cerdulatinib. Their inhibition mechanism is studied and discussed in detail, with a thorough evaluation of their enzyme-inhibitor complex binding constants (K_i) and the inhibitor concentration required for 50% inhibition (IC₅₀), employing standard inhibition procedure graphical methods. The biosensor is successfully applied for the quantification of the inhibitors by fixed potential amperometry, with excellent detection limit values in the pM range. It is the first detection method reported for the anticancer drugs CPG77675 and Cerdulatinib. The electrochemical assay based on the biosensor brings several advantages over the available assay kits for high-throughput screening (HTS) of kinase inhibitors, namely: low cost, easy operability and robustness demonstrated by biosensor high reproducibility and both operational and storage stability, offering an opportunity to discover new inhibitors and optimize their therapeutic index.

Keywords: Electrochemical assay; Inhibition mechanism; Inhibitor detection; Pyruvate kinase.