Development
of a Cysteine-Conjugatable Disulfide FRET
Probe: Influence of Charge on Linker Cleavage and Payload Trafficking
for an Anti-HER2 Antibody Conjugate
Suzie
J. Scales
Siao Ping Tsai
Neelie Zacharias
Josefa dela Cruz-Chuh
Gordy Bullen
Erick Velasquez
Julie Chang
Elise Bruguera
Katherine R. Kozak
Jack Sadowsky
10.1021/acs.bioconjchem.9b00678.s001
https://acs.figshare.com/articles/journal_contribution/Development_of_a_Cysteine-Conjugatable_Disulfide_FRET_Probe_Influence_of_Charge_on_Linker_Cleavage_and_Payload_Trafficking_for_an_Anti-HER2_Antibody_Conjugate/11310506
Disulfide-linked bioconjugates allow the delivery of
pharmacologically
active or other cargo to specific tissues in a redox-sensitive fashion.
However, an understanding of the kinetics, subcellular distribution,
and mechanism of disulfide cleavage in such bioconjugates is generally
lacking. Here, we report a modular disulfide-linked TAMRA-BODIPY based
FRET probe that can be readily synthesized, modified, and conjugated
to a cysteine-containing biomolecule to enable real-time monitoring
of disulfide cleavage during receptor-mediated endocytosis in cells.
We demonstrate the utility of this probe to study disulfide reduction
during HER2 receptor-mediated uptake of a Cys-engineered anti-HER2
THIOMAB antibody. We found that introduction of positive, but not
negative, charges in the probe improved retention of the BODIPY catabolite.
This permitted the observation of significant disulfide cleavage in
endosomes or lysosomes on par with proteolytic cleavage of a similarly
charged valine–citrulline peptide-based probe. In general,
the FRET probe we describe should enable real-time cellular monitoring
of disulfide cleavage in other targeted delivery systems for mechanistic
or diagnostic applications. Furthermore, modifications to the released
BODIPY moiety permit evaluation of physicochemical properties that
govern lysosomal egress or retention, which may have implications
for the development of next-generation antibody–drug conjugates.
2019-12-03 15:24:43
disulfide cleavage
retention
FRET probe
BODIPY
Cysteine-Conjugatable Disulfide FRET Probe
HER 2 receptor-mediated uptake
TAMRA-BODIPY
study disulfide reduction
monitoring
Anti-HER 2 Antibody Conjugate Disulfide-linked bioconjugates
Cys-engineered anti-HER 2 THIOMAB antibody