Investigation of novel ATX inhibitor metabolites by UHPLC-orbitrap-MS/MS and molecular docking studies
Hongjin Wang1, Yuxiang Chen2, Xinjie Qi1
1School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China.
Abstract:
Lysophosphatidic acid (LPA) mediated signaling is relevant to fibrosis and the proliferation of pulmonary cells, such as the promotion of fibroblast accumulation and stimulation of the TGF-β signal. One of the essential mechanisms to reduce lysophosphatidic acid production is inhibiting autotaxin activity, which can block the hydrolysis of a lysophosphatidylcholine (LPC) into LPA, leading to the alleviation of the development of pulmonary fibrosis. Recently, 3,4-difluorobenzyl(1-ethyl-5-(4-((4-hydroxypiperidin-1-yl)-methyl)-thiazol-2-yl)-1H-indol-3-yl)carbamate (NAI59), a novel autotaxin inhibitor, showed outstanding anti-pulmonary fibrosis therapeutic effectiveness; however, the metabolic information of NAI59 has not been comprehensively expounded yet. Therefore, in this study, we aim to identify the metabolites of NAI59 in rats using ultra-high-performance liquid chromatography-high-resolution tandem mass spectrometry, and we analyze the potential anti-fibrosis of metabolites in plasma using molecular docking with the crystal structure autotaxin. As a result, a total of 24 metabolites, namely, 15 phase I metabolites and 9 phase II metabolites, were identified, all of which are the novel discovered metabolites. The metabolic pathways of NAI59 were primarily hydroxylation, oxidation, hydrogenation, glucuronic acid conjugation, sulfate conjugation, acetylation, and glucosylation. The molecular docking result showed that the identified metabolites in plasma had great affinities upon docking into the active site of autotaxin, indicating they may have potential anti-pulmonary fibrosis activity. The interaction plots revealed the importance of fluorophenyl and the hydroxyl groups on piperidine that contribute toward the formation of hydrogen bonds with autotaxin. Additionally, we found that the metabolite possessed a better docking result when the structure contained a glucuronic acid group. To the best of our knowledge, this study is the first research into the metabolic fate of NAI59 in rats, and we analyze the potential anti-fibrosis aspects of metabolites using molecular docking. This study provides a metabolic rationale for further preclinical research and imparts new insights for developing improved ATX inhibitors.
Insights
This study identifies 24 novel metabolites of the autotaxin inhibitor NAI59 in rats. These metabolites show potential anti-pulmonary fibrosis activity by interacting with autotaxin, offering insights for new drug development.
Area of Science:
- Pharmacology
- Medicinal Chemistry
- Biochemistry
Background:
- Lysophosphatidic acid (LPA) signaling drives pulmonary fibrosis by promoting fibroblast accumulation and TGF-β signaling.
- Inhibiting autotaxin (ATX), an enzyme that produces LPA, is a key strategy to combat pulmonary fibrosis.
- NAI59 is a novel ATX inhibitor demonstrating significant anti-pulmonary fibrosis effects, but its metabolic profile is unknown.
Purpose of the Study:
- To identify and characterize the metabolites of NAI59 in rats.
- To evaluate the potential anti-fibrotic activity of NAI59 metabolites using molecular docking.
- To elucidate the metabolic pathways and structure-activity relationships of NAI59 metabolites.
Main Methods:
- Ultra-high-performance liquid chromatography-high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) was used to identify NAI59 metabolites in rat plasma.
- Molecular docking simulations were performed to assess the binding affinity of identified metabolites to the autotaxin active site.
- Metabolic pathways were analyzed based on the identified phase I and phase II metabolites.
Main Results:
- A total of 24 novel metabolites were identified: 15 phase I (e.g., hydroxylation, oxidation) and 9 phase II (e.g., glucuronidation, sulfation).
- Molecular docking revealed that plasma metabolites of NAI59 exhibit strong binding affinities to the autotaxin active site.
- Specific structural features, like the fluorophenyl and piperidine hydroxyl groups, and glucuronic acid conjugation, enhance binding to autotaxin.
Conclusions:
- This study provides the first comprehensive metabolic profile of NAI59 in rats.
- Identified NAI59 metabolites possess potential anti-pulmonary fibrosis activity, suggesting they may contribute to the drug's therapeutic efficacy.
- The findings offer a metabolic rationale for NAI59's preclinical development and guide the design of next-generation autotaxin inhibitors.
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