FGF2 Dual Warhead Conjugate with Monomethyl Auristatin E and α-Amanitin Displays a Cytotoxic Effect towards Cancer Cells Overproducing FGF Receptor 1
Abstract
:1. Introduction
2. Results
2.1. Dual Conjugation of α-Amanitin and Monomethyl Auristatin E to Fibroblast Growth Factor 2 (FGF2)
2.2. Characterization of α-Amainitin/Monomethyl Auristatin E (MMAE)-FGF2 Conjugate
2.3. FGF2 Conjugates Are Selectively Internalized into Cancer Cells in the Fibroblast Growth Factor Receptor 1 (FGFR1)-Dependent Manner
2.4. Cytotoxicity of α-Amainitin/MMAE-FGF2 Conjugate
3. Discussion
4. Materials and Methods
4.1. FGF2 Cys96PrK Variant Expression and Purification
4.2. FGF2 Cys78-α-Amanitin Conjugate Preparation
4.3. FGF2 PrK96-MMAE Conjugate Preparation
4.4. FGF2 Cys78-α-Amanitin/PrK96-MMAE Conjugate Preparation
4.5. Mass Spectrometry
4.6. Circular Dichroism Spectroscopy
4.7. BLI Assays
4.8. The Stability of α-Amanitin/MMAE-FGF2 Conjugate in Human Serum
4.9. Western Blotting
4.10. Fluorescence Microscopy
4.11. Cell Lines
4.12. Cytotoxicity Assay
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
α-Amanitin-FGF2 | FGF2 single conjugate with α-amanitin incorporated at Cys78 position |
α-Amanitin/MMAE-FGF2 | FGF2 dual warheads conjugate with α-amanitin incorporated at Cys78 position and MMAE incorporated at PrK96 position |
ADCs | Antibody drug conjugates |
AR2G | Amine reactive second-generation biosensor |
ATCC | American type culture collection |
BLI | Bio-layer Interferometry technique |
CuAAC | Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition |
FBS | Fetal bovine serum |
FGF2 | Fibroblast growth factor 2 |
FGFR1 | Fibroblast growth factor receptor 1 |
MMAD | Monomethyl auristatin D |
MMAE | Monomethyl auristatin E |
MMAE-FGF2 | FGF2 single conjugate with MMAE incorporated at PrK96 position |
NBCS | Newborn bovine calf serum |
PrK | N-ε-Propargyloxycarbonyl-l-lysine |
THPTA | Tris(3-hydroxypropyltriazolylmethyl)amine |
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Świderska, K.W.; Szlachcic, A.; Opaliński, Ł.; Zakrzewska, M.; Otlewski, J. FGF2 Dual Warhead Conjugate with Monomethyl Auristatin E and α-Amanitin Displays a Cytotoxic Effect towards Cancer Cells Overproducing FGF Receptor 1. Int. J. Mol. Sci. 2018, 19, 2098. https://doi.org/10.3390/ijms19072098
Świderska KW, Szlachcic A, Opaliński Ł, Zakrzewska M, Otlewski J. FGF2 Dual Warhead Conjugate with Monomethyl Auristatin E and α-Amanitin Displays a Cytotoxic Effect towards Cancer Cells Overproducing FGF Receptor 1. International Journal of Molecular Sciences. 2018; 19(7):2098. https://doi.org/10.3390/ijms19072098
Chicago/Turabian StyleŚwiderska, Karolina Weronika, Anna Szlachcic, Łukasz Opaliński, Małgorzata Zakrzewska, and Jacek Otlewski. 2018. "FGF2 Dual Warhead Conjugate with Monomethyl Auristatin E and α-Amanitin Displays a Cytotoxic Effect towards Cancer Cells Overproducing FGF Receptor 1" International Journal of Molecular Sciences 19, no. 7: 2098. https://doi.org/10.3390/ijms19072098