Sodium-Dependent Multivitamin Transporter Facilitates Brain and Neuronal Uptake of a Creatine Analogue

Abstract Creatine transporter 1 (CRT1, SLC6A8) is a membrane transporter essential for importing creatine across the blood–brain barrier (BBB) into neurons and glial cells. Loss-of-function mutations in CRT1 result in creatine transporter deficiency syndrome (CTDS), a severe X-linked neurological disorder with no effective treatment. To explore alternative uptake pathways for creatine, a carrier-mediated delivery system utilizing the sodium-dependent multivitamin transporter (SMVT), a potential neuronal target to facilitate creatine brain uptake, was employed. In this study, SMVT was investigated in silico and in vitro to identify key substrate-binding residues and to functionally characterize the transporter in human glioblastoma (U-87MG) and mouse neuronal cells. Furthermore, four novel SMVT-utilizing creatine derivatives were designed, synthesized, and assessed in vitro. The most promising derivative was studied in vivo and in a creatine kinase (CK) enzymatic assay and molecular dynamics (MD) simulations to determine its interactions with the ATP/ADP system. The studies confirmed SMVT expression in human glioblastoma cells and mouse neurons, and functional assays demonstrated pH- and concentration-dependent transporter activity in both cell lines. The creatine derivatives were taken up by human glioblastoma cells, and the lead derivative, compound 1 (COMP 1), demonstrated SMVT-mediated uptake at concentrations below 100 μM and was also taken up by mouse neurons. Finally, COMP 1 accumulated in the mouse brain and showed interactions with the CK system. Collectively, these findings identify SMVT as a potential route for brain uptake of creatine derivatives and support further investigation of SMVT-targeting derivatives in relevant CTDS animal models.