Design, Synthesis, and Mechanistic Studies of (R)‑3-Amino-5,5-difluorocyclohex-1-ene-1-carboxylic Acid as an Inactivator of Human Ornithine Aminotransferase

Human ornithine aminotransferase (hOAT), a pyridoxal 5′-phosphate (PLP)-dependent enzyme, has been shown to play an essential role in the metabolic reprogramming and progression of hepatocellular carcinoma (HCC). HCC accounts for approximately 75% of primary liver cancers and is within the top three...

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Published in:ACS chemical biology 2024-05, Vol.19 (5), p.1066-1081
Main Authors: Devitt, Allison N., Vargas, Abigail L., Zhu, Wei, Des Soye, Benjamin James, Butun, Fatma Ayaloglu, Alt, Tyler, Kaley, Nicholas, Ferreira, Glaucio M., Moran, Graham R., Kelleher, Neil L., Liu, Dali, Silverman, Richard B.
Format: Article
Language:English
Subjects:
Carboxylic Acids - chemical synthesis
Carboxylic Acids - chemistry
Carboxylic Acids - pharmacology
Carcinoma, Hepatocellular - drug therapy
Carcinoma, Hepatocellular - metabolism
Crystallography, X-Ray
Cyclohexenes - chemical synthesis
Cyclohexenes - chemistry
Cyclohexenes - metabolism
Cyclohexenes - pharmacology
Drug Design
Enzyme Inhibitors - chemical synthesis
Enzyme Inhibitors - chemistry
Enzyme Inhibitors - pharmacology
Humans
Models, Molecular
Ornithine-Oxo-Acid Transaminase - antagonists & inhibitors
Ornithine-Oxo-Acid Transaminase - chemistry
Ornithine-Oxo-Acid Transaminase - metabolism
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Summary:Human ornithine aminotransferase (hOAT), a pyridoxal 5′-phosphate (PLP)-dependent enzyme, has been shown to play an essential role in the metabolic reprogramming and progression of hepatocellular carcinoma (HCC). HCC accounts for approximately 75% of primary liver cancers and is within the top three causes of cancer death worldwide. As a result of treatment limitations, the overall 5-year survival rate for all patients with HCC is under 20%. The prevalence of HCC necessitates continued development of novel and effective treatment methods. In recent years, the therapeutic potential of selective inactivation of hOAT has been demonstrated for the treatment of HCC. Inspired by previous increased selectivity for hOAT by the expansion of the cyclopentene ring scaffold to a cyclohexene, we designed, synthesized, and evaluated a series of novel fluorinated cyclohexene analogues and identified (R)-3-amino-5,5-difluorocyclohex-1-ene-1-carboxylic acid as a time-dependent inhibitor of hOAT. Structural and mechanistic studies have elucidated the mechanism of inactivation of hOAT by 5, resulting in a PLP-inactivator adduct tightly bound to the active site of the enzyme. Intact protein mass spectrometry, 19F NMR spectroscopy, transient state kinetic studies, and X-ray crystallography were used to determine the structure of the final adduct and elucidate the mechanisms of inactivation. Interestingly, despite the highly electrophilic intermediate species conferred by fluorine and structural evidence of solvent accessibility in the hOAT active site, Lys292 and water did not participate in nucleophilic addition during the inactivation mechanism of hOAT by 5. Instead, rapid aromatization to yield the final adduct was favored.
ISSN:1554-8929
1554-8937
1554-8937
DOI:10.1021/acschembio.4c00022