Off-Target Inhibition of Human Dihydroorotate Dehydrogenase (hDHODH) Highlights Challenges in the Development of Fat Mass and Obesity-Associated Protein (FTO) Inhibitors.

FTO, an N ⁶-methyladenosine (m⁶A) and N ⁶,2'-O-dimethyladenosine (m⁶A_m) RNA demethylase, is a promising target for treating acute myeloid leukemia (AML) due to the significant anticancer activity of its inhibitors in preclinical models. Here, we demonstrate that the FTO inhibitor FB23-2 suppresses proliferation across both AML and CML cell lines, irrespective of FTO dependency, indicating an alternative mechanism of action. Metabolomic analysis revealed that FB23-2 induces the accumulation of dihydroorotate (DHO), a key intermediate in pyrimidine nucleotide synthesis catalyzed by human dihydroorotate dehydrogenase (hDHODH). Notably, structural similarities between the catalytic pockets of FTO and hDHODH enabled FB23-2 to inhibit both enzymes. In contrast, the hDHODH-inactive FB23-2 analog, ZLD115, required FTO for its antiproliferative activity. Similarly, the FTO inhibitor CS2 (brequinar), known as one of the most potent hDHODH inhibitors, exhibited FTO-independent antileukemic effects. Uridine supplementation fully rescued leukemia cells from FB23-2 and CS2-induced growth inhibition, but not ZLD115, confirming the inhibition of pyrimidine synthesis as the primary mechanism of action underlying their antileukemic activity. These findings underscore the importance of considering off-target effects on hDHODH in the development of FTO inhibitors to optimize their therapeutic potential and minimize unintended consequences.