Therapeutic Targeting of Fumaryl Acetoacetate Hydrolase in Hereditary Tyrosinemia Type I
Abstract
:1. Introduction
2. Results
2.1. The Dimerization Interface Is Druggable
2.2. Small Molecules to Stabilize the Dimer Conformation of FAH
2.3. Functional Screening to Improve Intracellular FAH Homeostasis
2.4. Organic Molecules Modulate FAH Catalytic Activity In Vitro
2.5. The Compound G1.30.B10 Stabilizes the Dimeric Species of FAH
2.6. Structural Design of a Second Generation of FAH Activators
2.7. Inhibitors Restore the Normal Phenotype in a CRISPR/Cas9 Cellular Model of HT1
3. Discussion
4. Materials and Methods
4.1. Computational Analysis
4.2. Protein Production and Purification
4.3. Chemical Libraries
4.4. Protein Thermal Shift Assay
4.5. FAA Synthesis and Purification
4.6. Enzymatic Assay
4.7. NMR Spectroscopy Experiments
4.8. Mammalian Cell Culture and Transfection
4.9. GFP Fluorescence Detection
4.10. Cell Viability Assays (MTT Assay)
4.11. Generation of FAH-G337S Mutant HEK Cells by CRISPR/Cas9-Mediated Genomic Edition
4.12. Metabolite Extraction for NMR
4.13. Statistical Analyses
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Gil-Martínez, J.; Macias, I.; Unione, L.; Bernardo-Seisdedos, G.; Lopitz-Otsoa, F.; Fernandez-Ramos, D.; Lain, A.; Sanz-Parra, A.; Mato, J.M.; Millet, O. Therapeutic Targeting of Fumaryl Acetoacetate Hydrolase in Hereditary Tyrosinemia Type I. Int. J. Mol. Sci. 2021, 22, 1789. https://doi.org/10.3390/ijms22041789
Gil-Martínez J, Macias I, Unione L, Bernardo-Seisdedos G, Lopitz-Otsoa F, Fernandez-Ramos D, Lain A, Sanz-Parra A, Mato JM, Millet O. Therapeutic Targeting of Fumaryl Acetoacetate Hydrolase in Hereditary Tyrosinemia Type I. International Journal of Molecular Sciences. 2021; 22(4):1789. https://doi.org/10.3390/ijms22041789
Chicago/Turabian StyleGil-Martínez, Jon, Iratxe Macias, Luca Unione, Ganeko Bernardo-Seisdedos, Fernando Lopitz-Otsoa, David Fernandez-Ramos, Ana Lain, Arantza Sanz-Parra, José M Mato, and Oscar Millet. 2021. "Therapeutic Targeting of Fumaryl Acetoacetate Hydrolase in Hereditary Tyrosinemia Type I" International Journal of Molecular Sciences 22, no. 4: 1789. https://doi.org/10.3390/ijms22041789