Identification of Putative Non-Substrate-Based XT-I Inhibitors by Natural Product Library Screening
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Expression of Recombinant Human XT-I in pgsA-745 Chinese Hamster Ovary Cells
2.3. Bicinchonic Acid Assay
2.4. Determination of XT-I Activity by Mass Spectrometry
2.5. Inhibitor Screening Assay and Determination of IC50 Values
2.6. Enzyme and Inhibition Kinetics
2.7. Displaying Putative Inhibitor Binding Sites by Molecular Docking
2.8. Primary Cell Culture, Treatment and Sample Preparation
2.9. Cell Proliferation Assay
2.10. Nucleic Acid Extraction and Synthesis of Complementary DNA
2.11. mRNA and miRNA Expression Analyses
2.12. Statistical Analysis
3. Results
3.1. Identification of Putative Non-Substrate-Derived XT-I Inhibitors Celastrol and Amphotericin B
3.2. Celastrol and Amphotericin B Inhibit Cellular Proliferation Dose-Dependently
3.3. Dual Effect of Putative XT-I Inhibitors on XYLT1 mRNA Expression and XT Activity of NHDF
3.4. Inhibitor-Induced mRNA Expression Changes Lead to Decreased XT-I Protein Expression in NHDF
3.5. Celastrol-Induced XYLT1 Suppression Might Be Mediated by the miRNA-21 Pathway
3.6. Amphotericin B Mediates XYLT1 Suppression by Interfering with TGF-β Pathway Components
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Gene | Primers | TA [°C] | Product Size [bp] |
---|---|---|---|
COL1A1 | 5′-GATGTGCCACTCTGACT-3′ 5′-GGGTTCTTGCTGATG-3′ | 63 | 151 |
Compound | Name | XT-I Activity [%] |
---|---|---|
17 | Amphotericin B | 25 |
68 | Celastrol | 38 |
74 | Oxytetracycline (Terramycin) | 17 |
86 | Curcumin | 43 |
0 | Dimethyl sulfoxide | 100 |
Compound | Score | Chimera Models | H Bond Involvement | Inhibitor Binding |
---|---|---|---|---|
17 | −11.1 | Amphotericin B #1 | yes | Xylo_C/GT-A domain |
17 | −10.1 | Amphotericin B #2 | yes | Xylo_C/GT-A domain |
68 | −9.7 | Celastrol #1 | no | Xylo_C domain |
68 | −9.1 | Celastrol #2 | no | GT-A domain |
74 | −8.6 | Oxytetracycline #1 | yes | Xylo_C domain |
74 | −8.5 | Oxytetracycline #2 | yes | Xylo_C/GT-A domain |
86 | −8.1 | Curcumin #1 | yes | GT-A domain |
86 | −7.4 | Curcumin #2 | yes | Xylo_C domain |
22 | −6.6 | Sulfamethoxazole #1 | yes | Xylo_C domain |
22 | −6.5 | Sulfamethoxazole #2 | no | Xylo_C domain |
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Ly, T.-D.; Kleine, A.; Fischer, B.; Schmidt, V.; Hendig, D.; Kuhn, J.; Knabbe, C.; Faust, I. Identification of Putative Non-Substrate-Based XT-I Inhibitors by Natural Product Library Screening. Biomolecules 2020, 10, 1467. https://doi.org/10.3390/biom10101467
Ly T-D, Kleine A, Fischer B, Schmidt V, Hendig D, Kuhn J, Knabbe C, Faust I. Identification of Putative Non-Substrate-Based XT-I Inhibitors by Natural Product Library Screening. Biomolecules. 2020; 10(10):1467. https://doi.org/10.3390/biom10101467
Chicago/Turabian StyleLy, Thanh-Diep, Anika Kleine, Bastian Fischer, Vanessa Schmidt, Doris Hendig, Joachim Kuhn, Cornelius Knabbe, and Isabel Faust. 2020. "Identification of Putative Non-Substrate-Based XT-I Inhibitors by Natural Product Library Screening" Biomolecules 10, no. 10: 1467. https://doi.org/10.3390/biom10101467