Abstract
The biophysical characterization of drug binding to proteins plays a key role in structural biology and in the discovery and optimization of drug discovery processes. The search for optimal combinations of biophysical techniques that can correctly and efficiently identify and quantify binding of metal-based drugs to their final target is challenging, due to the physicochemical properties of these agents. Different cisplatin derivatives have shown different citotoxicities in most common cancer lines, suggesting that they exert their biological activity via different mechanisms of action. Here we carried out a comparative analysis, by studying the behaviours of three Pt-compounds under the same experimental conditions and binding assays to properly deepen the determinants of the different MAOs. Indeed we compared the results obtained using surface plasmon resonance, isothermal titration calorimetry, fluorescence spectroscopy and thermal shift assays based on circular dichroism experiments in the characterization of the formation of adducts obtained upon reaction of cisplatin, carboplatin and iodinated analogue of cisplatin, cis-Pt (NH3)2I2, with the model protein hen egg white lysozyme, both at neutral and acid pHs. Further we reasoned on the applicability of employed techniques for the study the thermodynamics and kinetics of the reaction of a metallodrug with a protein and to reveal which information can be obtained using a combination of these analyses. Data were discussed on the light of the existing structural data collected on the platinated protein.
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Abbreviations
- CD:
-
Circular dichroism
- DMSO:
-
Dimethyl sulfoxide
- EDC/NHS:
-
Ethyl(dimethylaminopropyl) carbodiimide/N-Hydroxysuccinimide
- EDTA:
-
Ethylenediaminetetraacetic acid
- FBDD:
-
Fragment-based drug-discovery
- HBS:
-
Hepes buffered saline
- HEPES:
-
N-(2-Hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid)
- HEWL:
-
Hen egg white lysozyme
- HSA:
-
Human serum albumin
- ITC:
-
Isothermal titration calorimetry
- MIS:
-
Multiple independent site
- NMR:
-
Nuclear magnetic resonance
- PMT:
-
Photomultiplier
- RU:
-
Resonance unit
- SPR:
-
Surface plasmon resonance
- TSAs:
-
Thermal shift assays
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Acknowledgements
This work was partially supported by the University of Naples “Federico II” (“000005–ALTRI_DR_409_2017_Rec_Ateneo_prof_MARASCO) to D. M.
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Ferraro, G., De Benedictis, I., Malfitano, A. et al. Interactions of cisplatin analogues with lysozyme: a comparative analysis. Biometals 30, 733–746 (2017). https://doi.org/10.1007/s10534-017-0041-y
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DOI: https://doi.org/10.1007/s10534-017-0041-y