Selective inhibition of indoleamine and tryptophan 2,3-dioxygenases: Comparative study on kynurenine pathway in cell lines via LC-MS/MS-based targeted metabolomics

by Selleckchem | Dec 06 2023

In the last decade, the kynurenine pathway, which is the primary metabolic route for tryptophan (TRP) catabolism, has sparked great interest in the pharmaceutical sciences due to its role in immune regulation and cancer immunoediting. In this context, the development of cell-based assays might represent a tool to: i) characterize the cell secretome according to cell types; ii) gain more insight into the role of kynurenines in different disease scenarios; iii) screen hIDO1 (human indoleamine 2,3-dioxygenase) inhibitors and evaluate their effect on downstream TRP-catabolizing enzymes. This paper reports a validated Liquid Chromatography with tandem mass spectrometry (LC-MS/MS) method to simultaneously quantify TRP, L-kynurenine (KYN), xanthurenic acid (XA), 3-hydroxykynurenine (3OHKYN), kynurenic acid (KA), 3-hydroxyanthranilic acid (3OHAA), anthranilic acid (AA), 5-hydroxytryptamine (serotonin, 5HT) and tryptamine (TRYP) in Dulbecco's Modified Eagle and Eagle's Minimum Essential Media (DMEM and EMEM, respectively). The quantitative method was validated according to FDA, ICH and EMA guidelines, later applied: i) to assess the impact of selective inhibition of hIDO1 or hTDO (human tryptophan 2,3-dioxygenase) on the kynurenine pathway in A375 (melanoma), MDA-MB-231 (breast cancer), and U87 (glioblastoma) cell lines using multivariate analysis (MVA); ii) to determine the IC50 values of both well-known (i.e., epacadostat, linrodostat) and the novel hIDO1 inhibitor (i.e., BL5) in the aforementioned cell lines. The proposed LC-MS/MS method is reliable and robust. Furthermore, it is highly versatile and suitable for applications in the preclinical drug research and in vitro assays.

Comments:

The paper you're referring to discusses a comprehensive study involving the kynurenine pathway, a crucial metabolic route for tryptophan catabolism. The authors have developed a Liquid Chromatography with tandem mass spectrometry (LC-MS/MS) method to measure various metabolites involved in this pathway, such as tryptophan, L-kynurenine, xanthurenic acid, 3-hydroxykynurenine, kynurenic acid, 3-hydroxyanthranilic acid, anthranilic acid, serotonin, and tryptamine in specific cell culture media. The study has several important aspects and implications:

1. **Disease Understanding and Therapy Development:** By analyzing the kynurenine pathway metabolites in different disease scenarios (melanoma, breast cancer, and glioblastoma), researchers can gain valuable insights into the role of kynurenines in these diseases. Understanding the metabolism of tryptophan in these contexts is critical for developing targeted therapies.

2. **Assessment of Enzyme Inhibitors:** The study assesses the impact of selective inhibition of hIDO1 and hTDO on the kynurenine pathway in various cancer cell lines. This is essential for evaluating the effectiveness of potential inhibitors in modulating the pathway, which is crucial for cancer immunotherapy.

3. **Development of New Inhibitors:** The research also determines the IC50 values of both well-known inhibitors like epacadostat and linrodostat, as well as a novel hIDO1 inhibitor (BL5). This information is vital for the development of new drugs or improving existing ones in the context of cancer treatment.

4. **Methodology Advancement:** The development and validation of a robust LC-MS/MS method for simultaneous quantification of multiple metabolites provide a reliable tool for future studies. Such methods are crucial in understanding complex metabolic pathways and developing targeted therapies.

5. **Multivariate Analysis (MVA):** The use of multivariate analysis in this context is noteworthy. MVA techniques can unravel intricate relationships within data sets, providing a deeper understanding of the interplay between different variables. This analytical approach enhances the interpretation of the experimental results.

6. **Regulatory Compliance:** The validation of the method according to FDA, ICH, and EMA guidelines ensures that the results obtained are reliable and meet international regulatory standards. This is vital for the credibility and acceptance of the study in the scientific community and pharmaceutical industry.

In summary, this research significantly contributes to our understanding of the kynurenine pathway in different diseases and provides a foundation for the development and evaluation of therapeutic interventions targeting this pathway, especially in the context of cancer immunotherapy. The robust methodology and analytical techniques employed in this study enhance the reliability and applicability of the findings in both research and pharmaceutical applications.