Off-Target Inhibition of Human Dihydroorotate Dehydrogenase (<i>h</i>DHODH) Highlights Challenges in the Development of Fat Mass and Obesity-Associated Protein (FTO) Inhibitors

FTO, an <i>N</i> <sup>6</sup>-methyladenosine (m<sup>6</sup>A) and <i>N</i> <sup>6</sup>,2'-<i>O</i>-dimethyladenosine (m<sup>6</sup>A<sub>m</sub>) 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 (<i>h</i>DHODH). Notably, structural similarities between the catalytic pockets of FTO and hDHODH enabled FB23-2 to inhibit both enzymes. In contrast, the <i>h</i>DHODH-inactive FB23-2 analog, ZLD115, required FTO for its antiproliferative activity. Similarly, the FTO inhibitor CS2 (brequinar), known as one of the most potent <i>h</i>DHODH 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 <i>h</i>DHODH in the development of FTO inhibitors to optimize their therapeutic potential and minimize unintended consequences.

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Journal
ACS Pharmacology & Translational Science
Published
2024-11-26
DOI
https://doi.org/10.1021/acsptsci.4c00533
Citations
8
Primary Topic
RNA modifications and cancer
Type
article
Field-Weighted Citation Impact
1.87

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article

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

Serena Rosignoli, Alessio Incocciati, Silvia Masciarelli, Dante Rotili et al.
8 citations
ACS Pharmacology & Translational Science
RNA modifications and cancer
1.87
article

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

Serena Rosignoli, Alessio Incocciati, Silvia Masciarelli, Dante Rotili, Antonello Mai, Guillermo Fernandez Rodriguez, Francesco Fazi, Marco Tarullo, Amedeo Caflisch, Alessandro Fatica, Alessia Iaiza, Alessandro Paiardini, Marta Giorgis, Marco L. Lolli, Elena Bochenkova, Marcella Marchioni, Ludovica Proietti, Alberto Macone, Florian Grebien, Federico Fornaseri, Sara Venezia
article en
8 citations

Abstract

FTO, an <i>N</i> <sup>6</sup>-methyladenosine (m<sup>6</sup>A) and <i>N</i> <sup>6</sup>,2'-<i>O</i>-dimethyladenosine (m<sup>6</sup>A<sub>m</sub>) 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 (<i>h</i>DHODH). Notably, structural similarities between the catalytic pockets of FTO and hDHODH enabled FB23-2 to inhibit both enzymes. In contrast, the <i>h</i>DHODH-inactive FB23-2 analog, ZLD115, required FTO for its antiproliferative activity. Similarly, the FTO inhibitor CS2 (brequinar), known as one of the most potent <i>h</i>DHODH 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 <i>h</i>DHODH in the development of FTO inhibitors to optimize their therapeutic potential and minimize unintended consequences.

ACS Pharmacology & Translational ScienceVol. 7(12)
Roma Tre University (IT), Austrian Academy of Sciences (AT), University of Veterinary Medicine Vienna (AT), University of Zurich (CH), St Anna Children's Hospital (AT), CeMM Research Center for Molecular Medicine (AT), St. Anna Children's Cancer Research Institute, University of Turin (IT), Sapienza University of Rome (IT)
Cancer Research Institute, North Atlantic Treaty Organization, Università degli Studi Roma Tre, European Commission, Österreichischen Akademie der Wissenschaften, Austrian Science Fund, Ministero dell’Istruzione, dell’Università e della Ricerca, Sapienza Università di Roma, Consiglio Nazionale delle Ricerche, Universität Zürich, Horizon 2020 Framework Programme, HORIZON EUROPE Marie Sklodowska-Curie Actions
Zero hunger
Openalex Percentile: Top 14%
RNA modifications and cancer
1.87
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