Investigating the utilization mechanism and kinetics of sialic acid mimetics in mammalian cell lines

Sialic acid mimetics (SAMs) are chemically modified derivatives of sialic acids that can act as metabolic inhibitors or as sugar donors for sialyltransferases. This makes SAMs highly useful research tools to study and manipulate the biosynthesis of sialic acid-carrying glycans (sialoglycans). Moreover, SAMs that inhibit aberrant sialylation in cancer cells are emerging as potential therapeutics. Despite the wide use of SAMs, many aspects regarding their cellular uptake and metabolic fate are unknown. Here, we investigated the metabolic fate of an inhibitory SAM (P-SiaFNEtoc) and an incorporative SAM (P-SiaNPoc) in various mammalian cell lines. Using kinetic experiments and read-outs based on sialic acid-binding lectins, click chemistry, and nucleotide sugar analysis, we monitored the key steps of cellular SAM utilization. We found differences in the metabolism of SAMs that determine their potency in different mammalian cell lines. By identifying a murine macrophage cell line that is insensitive to SAMs, we have identified esterase activity as a bottleneck for the cellular utilization of SAMs. This study contributes to the understanding of the mechanisms underlying SAMs utilization in mammalian cell lines and provide relevant considerations for the future chemical design of SAMs and their application in mammalian systems.