Kinetic Analysis of Human GGT1 Substrate Specificity for the Cysteamine Prodrug “CF10” using a HPLC-based Assay Method. Kinetic Analysis of Human GGT1 Substrate Specificity for the Cysteamine Prodrug “CF10” using a HPLC-based Assay Method.
Summer Studentship Report 2025.
By Myungkyun Choi and Dr Stephanie Myers and Dr Stephen Childs at the University of Sunderland.
CF10 is a novel prodrug of cysteamine, identified at the University of Sunderland, and currently undergoing preclinical evaluation for its use as a therapeutic agent for nephropathic symptoms of cystinosis. (1)
The project objectives were to develop and validate a fully quantitative HPLC-based kinetic assay for the analysis of the intermediate compound CF104 as a substrate of gamma-glutamyl transpeptidase 1 (GGT1) and the hydrolysis product cysteamine to determine the enzyme kinetics of this reaction, to support further development of CF10 into eyedrop formulations for the treatment of ocular symptoms of this condition.
Owing to the lack of chromophores, CF104 and cysteamine were derivatized using Ellman’s Reagent (ESSE) to develop a quantitative in-house assay. Method validation measures included purity testing, external standard use, precision and robustness testing. A kinetic experiment was subsequently devised which enabled determination of Vmax (9.83 µM/min) and Km (0.90 mM) of CF104 for GGT1 compared with glutathione (GSH) Vmax (22.9 µM/min) and Km (0.81 mM).
ChromSoc/BMSS Summer Studentship program information.
