A critical assessment of fluorescent probes as cellular reporters for thioredoxin system enzyme activities

Fluorescent probes reporting on the activity of thioredoxin (TXN) and thioredoxin reductase (TXNRD) have been at the forefront of methodological development for TXN-related research in recent years. Here, we summarize and critically evaluate such probes targeting the TXN system. Additionally, we discuss key challenges in designing selective chemical probes for intracellular dithiol-type reductases, their mechanisms of action, selectivity, and cellular performance, focusing on the experimental validation of individual probes. We thus highlight concerns regarding the reliability of some probes claimed to be selective while they are evidently not, which if used indiscriminately may undermine trust in chemical redox probes in general. The absence of standardized validation protocols complicates the assessment of probe specificity in several of the cases reported in the literature, underscoring the need for rigorous and consistent evaluation criteria, for which we present an experimental pipeline.