title: Reporter assay for endo/lysosomal escape of toxin-based therapeutics.
abstract: 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, (Alexa)saporin 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 (Alexa)saporin 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.
authors.affiliation: Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité-Universitätsmedizin Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, Berlin D-13353, Germany. roger.gilabert-oriol@charite.de.
authors.forename: Roger
authors.initials: R
authors.lastname: Gilabert-Oriol
authors.affiliation: Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité-Universitätsmedizin Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, Berlin D-13353, Germany. mayank.thakur@charite.de.
authors.forename: Mayank
authors.initials: M
authors.lastname: Thakur
authors.affiliation: Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité-Universitätsmedizin Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, Berlin D-13353, Germany. benedicta.von-mallinckrodt@charite.de.
authors.forename: Benedicta
authors.initials: B
authors.lastname: von Mallinckrodt
authors.affiliation: Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité-Universitätsmedizin Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, Berlin D-13353, Germany. cheenu.bhargava@charite.de.
authors.forename: Cheenu
authors.initials: C
authors.lastname: Bhargava
authors.affiliation: Leibnizinstitut für Molekulare Pharmakologie (FMP), Berlin D-13125, Germany. wiesner@fmp-berlin.de.
authors.forename: Burkhard
authors.initials: B
authors.lastname: Wiesner
authors.affiliation: Leibnizinstitut für Molekulare Pharmakologie (FMP), Berlin D-13125, Germany. eichhorst@fmp-berlin.de.
authors.forename: Jenny
authors.initials: J
authors.lastname: Eichhorst
authors.affiliation: Institute of Pharmacy, Freie Universität Berlin, Königin-Luise-Straße 2 + 4, Berlin D-14195, Germany. melzig@zedat.fu-berlin.de.
authors.forename: Matthias F
authors.initials: MF
authors.lastname: Melzig
authors.affiliation: Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité-Universitätsmedizin Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, Berlin D-13353, Germany. hendrik.fuchs@charite.de.
authors.forename: Hendrik
authors.initials: H
authors.lastname: Fuchs
authors.affiliation: Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité-Universitätsmedizin Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, Berlin D-13353, Germany. a.weng@ucl.ac.uk.
authors.forename: Alexander
authors.initials: A
authors.lastname: Weng
mesh_terms: D002460:Cell Line; D011992:Endosomes / Q000187:drug effects* / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000378:metabolism; D006801:Humans; D008247:Lysosomes / Q000187:drug effects* / Q000378:metabolism; D014118:Toxins, Biological / Q000494:pharmacology* / Q000627:therapeutic use
chemical_list: D014118:Toxins, Biological; D006735:Horseradish Peroxidase