10.1002/anie.201804072
Angewandte Chemie International Edition
COMMUNICATION
To test whether PSP-2 can be used to selectively target
copper-dependent pathways within a complex biological environ-
ment, we further explored its activity both in cell culture and in
mouse hippocampal brain tissue. At the cellular level, we
employed HeLa cells to explore the effect of PSP-2 on trafficking
of the Menkes protein, a copper-transporting P-type ATPase
involved in metalation of cuproenzymes in the Golgi apparatus.[17]
In the presence of excess copper, the Menkes protein relocates
from the trans-Golgi network to the plasma membrane to mediate
copper efflux.[18] Consistent with previous reports,[8a,18] under
basal growth conditions the Menkes ATPase is mostly localized
to the Golgi network near the nucleus, but relocates to the
cytoplasm upon supplementation of the medium with CuCl2
(Figure 4A). After replacement of the copper-supplemented
incubation buffer with 100 μM PSP-2, the cytoplasmic localization
of the Menkes protein is reversed back to the Golgi (Figure 4A,
right panel). In contrast, a control experiment with buffer lacking
PSP-2 showed insignificant changes (Figure S12). Likewise,
electrophysiological studies with hippocampal brain tissue slices
revealed that PSP-2 suppresses long-term potentiation (LTP).
The importance of copper in the central nervous system is well
established,[19] and increasing evidence suggests that copper
may be directly involved in modulating neuronal activity.[20]
Induced by high-frequency stimulation of synapse inputs, LTP
results in an increase of synaptic strength and is thought to play
a critical role in learning and memory. As illustrated with Figure
4B, we induced LTP with two trains of 100 Hz stimulation in
hippocampal neurons of brain slices from 4-5 week-old C57BL/6
mice and recorded field potentials (fEPSPs) in CA1 neurons.
Upon application of 20 μM PSP-2 for 20 mins, LTP was
reproducibly blocked (P<0.05, n=6), as indicated by return to
baseline at 100%. Compared to control, LTP was thus
significantly suppressed in PSP-2 treated slices while a paired-
pulse test did not show significant difference between the two
conditions. In contrast, [(PSP-2)CuI]Cl had no effect on LTP
induction under identical conditions. Altogether, these data
demonstrate that PSP-2 can affect copper-dependent activities
within complex biological systems, likely through competitive
chelation of the metal ion from endogenous cuproproteins and
ligands.
Acknowledgements
Financial support from the National Institutes of Health (GM67169
to CJF, MH106027 and EY023173 to CRF) and the Parker H.
Petit Institute for Bioengineering and Bioscience, including the
Neuro Design Suite core facility, is gratefully acknowledged. We
thank Dr. John Bacsa, Emory X-ray Crystallography Facility,
for the X-ray structural analysis. We also acknowledge the use
of the Rigaku SYNERGY diffractometer, supported by
the National Science Foundation under grant CHE-1626172.
Keywords: copper • chelation • metal homeostasis • signaling •
electrophysiology
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In summary, the presented ligand design approach using
phosphine sulfide-stabilized phosphine (PSP) donors yielded a
membrane-permeant CuI-chelator, PSP-2, with an unpreceden-
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resistant towards oxidation by disulfides and air under
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