Reporter Assay for Endo/Lysosomal Escape of Toxin-Based Therapeutics

Toxins

Roger Gilabert-Oriol¹  Mayank Thakur¹  Benedicta von Mallinckrodt¹  Cheenu Bhargava¹  Burkhard Wiesner³  Jenny Eichhorst³  Matthias F. Melzig²  Hendrik Fuchs¹ 

[1]Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité–Universitätsmedizin Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, Berlin D-13353, Germany

[2] E-Mails:

[3]Institute of Pharmacy, Freie Universität Berlin, Königin-Luise-Straße 2 + 4, Berlin D-14195, Germany

[4] E-Mail:

[5]Leibnizinstitut für Molekulare Pharmakologie (FMP), Berlin D-13125, Germany

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关键词: endosomal escape;    reporter assay;    protein therapeutics;    ribosome inactivating proteins;    horseradish peroxidase;    Alexa Fluor 488;    ricin A-chain;    saporin;    triterpenoidal saponins;   

DOI  :  10.3390/toxins6051644

来源: mdpi

【 摘 要 】

Protein-based therapeutics with cytosolic targets are capable of exhibiting their therapeutic effect once they have escaped from the endosomes or lysosomes. In this study, the reporters—horseradish peroxidase (HRP), Alexa Fluor 488 (^Alexa) and ricin A-chain (RTA)—were investigated for their capacity to monitor the endo/lysosomal escape of the ribosome-inactivating protein, saporin. The conjugates—saporin-HRP, ^Alexasaporin and saporin-KQ-RTA—were constructed, and the endo/lysosomal escape of these conjugates alone (lack of endo/lysosomal release) or in combination with certain structurally-specific triterpenoidal saponins (efficient endo/lysosomal escape) was characterized. HRP failed in reporting the endo/lysosomal escape of saporin. Contrastingly, Alexa Fluor 488 successfully allowed the report of the process at a toxin concentration of 1000 nM. In addition, single endo/lysosome analysis facilitated the determination of the amount of ^Alexasaporin released from each vesicle. RTA was also successful in reporting the endo/lysosomal escape of the enzymatically inactive mutant, saporin-KQ, but in this case, the sensitivity of the method reached a toxin concentration of 10 nM. In conclusion, the simultaneous usage of Alexa Fluor 488 and RTA as reporters may provide the possibility of monitoring the endo/lysosomal escape of protein-based therapeutics in the concentration range of 10–1000 nM.

【 授权许可】

CC BY   
© 2014 by the authors; licensee MDPI, Basel, Switzerland.