Communication
ChemComm
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non-toxic. Otherwise, one must accept a trifecta of implausible
conclusions: (i) 2 is merely a carrier of DMNB, (ii) 1 is actually
nontoxic, and (iii) DMNB is as toxic as a-amanitin!
Just how inhibition of Pol II leads to its degradation and cell
death remains a mystery that Ama-Flash 2 may help solve. As cell
uptake can now be decoupled from cell death, synchronized cells
could be loaded with 2 while photolysis could then be used to
trigger transcriptional arrest, Pol II ubiquitination7 and
degradation,8 in a cell-cycle dependent fashion to probe the
complex cascade by which Pol II inhibition leads to cell death.
Other effects e.g. ROS stimulation that amanitin induces may be
probed. While amanitin inhibits all mammalian RNA Pol IIs and
is cytotoxic to all mammalian cells, it will show different IC50
values depending on cell line. Hence for use, we caution that
different doses of 2 along with recalibrating for photosensitivity
may be needed depending on the cell line of interest. In addition,
we note the potential for using other masking agents for near-IR
photo-release that might find use in phototherapeutic applica-
tions. It is also appreciated that 2 or other masked amanitin could
be conjugated to an antibody or other targeting agent to potenti-
ate uptake or to increase its specific cytotoxicity.
In conclusion, Ama-Flash 2 is the first example of a caged
amanitin for targeting RNA Pol II. This is enabled by grafting a
well-known photo-labile 6-nitroveratryl group to the Hyp that is
critical for binding RNA Pol II. Cellular uptake of Ama-Flash
followed by washing and irradiation demonstrate that the intra-
cellularly released dideoxy-amanitin (1) is nearly as toxic as
a-amanitin. Our synthetic route enables one to develop new
tools to study transcriptional inhibition as related to cell death.
We acknowledge grants from the Can. Inst. Health Res. #220656
and the Can. Cancer Soc. Research Initiative #703374. We thank
Dr. E. Polishchuk and Ms. J. Chen for help with MTT assays.
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Conflicts of interest
There are no conflicts to declare.
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