Targeting dihydroorotate dehydrogenase (hDHODH) beyond the barrier: discovery of MEDS700 as blood-brain barrier permeable hDHODH inhibitor

: Targeting cancer metabolism, particularly de novo nucleotide biosynthesis, has emerged as a promising and innovative therapeutic strategy for both hematologic and solid malignancies, including those of the Central Nervous System (CNS). Glioblastoma cancer stem-like cells are especially vulnerable to pyrimidine synthesis inhibition, highlighting human dihydroorotate dehydrogenase (hDHODH), a rate-limiting enzyme in the de novo pathway, as a potential therapeutic target. MEDS433 is a best-in-class hDHODH inhibitor, that shows efficacy in vivo after oral administration but lacks efficient penetration of the blood-brain barrier (BBB), limiting its utility against CNS tumors. Its lipophilic analogue MEDS613 showed enhanced cellular potency, but its poor metabolic stability and rapid conversion to hydroxylated metabolites precluded its future clinical development. In this study, we aimed to design a novel BBB-permeable hDHODH inhibitor, capable of effectively targeting CNS-localized hDHODH. We began by identifying the metabolic soft spots present in the propyloxy side chain of MEDS613 using this information to develop a metabolically stable analogue, MEDS700 (compound 3, as named in the manuscript), that was shown to inhibit hDHODH in the low nanomolar range (IC50hDHODH 1.5 nM). Subsequently, MEDS700 was fully profiled, including detailed analysis of its crystallographic binding mode, pan-antitumor activities in cell-based assays and in vitro cytotoxicity on Peripheral Blood Mononuclear Cells (PBMC). An in vivo pharmacokinetic experiment demonstrated that MEDS700 was able to cross the blood-brain barrier, maintaining therapeutically relevant intracerebral concentrations for up to 24 h after oral administration. Our findings establish MEDS700 as a potent, safe, metabolically stable hDHODH inhibitor, indicating it as a promising candidate for the treatment of hard-to-reach brain tumors.