X-Ray fluorescence microscopy reveals that rhenium(i) tricarbonyl isonitrile complexes remain intact: In vitro

Article abstract of DOI:10.1039/d0cc02451a

The complex fac-[Re(CO)3(dmphen)(para-tolylisonitrile)]+ (TRIP), where dmphen = 2,9-dimethyl-1,10-phenanthroline, is an endoplasmic reticulum stress-inducing anticancer agent (A. P. King, S. C. Marker, R. V. Swanda, J. J. Woods, S.-B. Qian and J. J. Wilson, Chem.-Eur. J., 2019, 25, 9206-9210). A second-generation compound fac-[Re(CO)3(dmphen)(para-iodobenzeneisonitrile)]+ (I-TRIP) was synthesized, and its intracellular distribution was investigated using X-ray fluorescence microscopy to show that these complexes are highly stable in vitro. This journal is

Full text of DOI:10.1039/d0cc02451a

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X-Ray fluorescence microscopy reveals that
rhenium(I) tricarbonyl isonitrile complexes remain
Cite this:DOI: 10.1039/d0cc02451a
intact in vitro†
Received 4th April 2020,
Accepted 5th May 2020
a
b
b
Chilaluck C. Konkankit,
James Lovett,
Hugh H. Harris
and
a
Justin J. Wilson
*
DOI: 10.1039/d0cc02451a
rsc.li/chemcomm
+
3
The complex fac-[Re(CO) (dmphen)(para-tolylisonitrile)] (TRIP),
where dmphen = 2,9-dimethyl-1,10-phenanthroline, is an endo-
plasmic reticulum stress-inducing anticancer agent (A. P. King,
S. C. Marker, R. V. Swanda, J. J. Woods, S.-B. Qian and J. J. Wilson,
Chem. – Eur. J., 2019, 25, 9206–9210). A second-generation
+
compound
3
fac-[Re(CO) (dmphen)(para-iodobenzeneisonitrile)]
(I-TRIP) was synthesized, and its intracellular distribution was inves-
tigated using X-ray fluorescence microscopy to show that these
Fig. 1 Structures of the complex cations TRIP and I-TRIP.
complexes are highly stable in vitro.
There is an increasing number of reports on the promising causing cancer cell death. Additionally, TRIP induces the accu-
in vitro and in vivo anticancer activity of Re-based compounds mulation of misfolded proteins, thereby giving rise to endoplas-
due to their high stability, structural diversity, amenability for mic reticulum (ER) stress, the unfolded protein response (UPR)
1
–19
real-time imaging, and lack of off-site toxicity in vivo.
Among these Re-based drug candidates, complexes containing pathway, which is a consequence of ER stress, usually operates
the Re(I) tricarbonyl (Re(CO) ) core are the most commonly in a cytoprotective manner that may result in resistant and more
pathway, and intrinsic apoptotic cell death. Although the UPR
3
25
explored for this application. Within this research area, our lab has lethal forms of cancer, acute levels of ER stress can overburden the
led several efforts to design and identify Re(CO) compounds as UPR pathway, leading to apoptotic cell death. This cell death
3
20–23
potent chemotherapeutic drug candidates.
strated their ability to operate in vivo without inducing toxic side activity of several transition metal polypyridyl complexes.
effects, demonstrating the potential of this class of compounds for Because TRIP induces this somewhat unusual form of UPR-
We have also illu- pathway has been demonstrated to mediate the in vitro cytotoxic
2
6–31
2
4
cancer treatment. Recently, we have identified a tricarbonyl Re mediated cancer cell death, we sought to investigate the speciation,
isonitrile polypyridyl complex fac-[Re(CO) (dmphen)(para-tolyl- namely the stability of the axial ligand, of this compound in vitro
isonitrile)] , where dmphen = 2,9-dimethyl-1,10-phenanthro- using synchrotron X-ray fluorescence microscopy (XFM).
line, called TRIP, with promising in vitro cytotoxic activity XFM is an effective intracellular characterization method
Fig. 1). TRIP was imaged via confocal fluorescence micro- that relies on element-specific X-ray fluorescence emissions from
3
+
2
2
(
scopy using its photoluminescent triplet metal-to-ligand charge core–shell transitions. This technique is highly sensitive and can
3
transfer ( MLCT) excited state. These results are similar to the effectively probe for cellular uptake and intracellular distribution
+
first-generation complex, fac-[Re(CO)
3
(dmphen)(H
2
O)] , which of a wide range of complexes, including metal-based anticancer
2
0
32–40
was also imaged in a similar manner. The ability to detect the agents.
emissive MLCT state of these complexes in cells suggests works best with an exogenous element present in the molecule
When imaging compounds in cells, this method
3
that the dmphen ligand remains bound to the Re center while of interest. To investigate the axial ligand stability of TRIP using
this method, we utilized a second-generation Re(CO) complex of
3
+
the formula fac-[Re(CO) (dmphen)(I-ICN)] , where I-ICN is para-
iodobenzeneisonitrile (I-TRIP) (Fig. 1). In comparison to TRIP,
3
a
Department of Chemistry and Chemical Biology, Cornell University, Ithaca,
New York 14853, USA. E-mail: jjw275@cornell.edu
b
this compound bears an iodo substituent in place of the methyl
group on the axial isonitrile ligand. Because I is of low abundance
Department of Chemistry, The University of Adelaide, South Australia 5005,
Australia
4
1
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/d0cc02451a
in most in vitro cellular systems, the iodo group on I-TRIP
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provides an additional XFM spectroscopic handle. Using this
complex, the axial ligand stability of this class of compounds
can be determined by directly comparing the elemental distributions
of I and Re in cells. Prior to carrying out XFM analysis, we first
sought to characterize the biological activity of I-TRIP to deter-
mine if the complex operates via a similar mechanism of action
as the parent compound, TRIP.
TRIP was synthesized following previously reported methods
1
and verified to be 495% pure by H NMR spectroscopy and
reversed-phase HPLC (RP-HPLC). I-TRIP was similarly synthe-
sized using the I-bearing isonitrile ligand (Fig. S1 and S2, ESI†)
1
and was fully characterized with H NMR spectroscopy (Fig. S3,
ESI†), UV-Vis spectroscopy (Fig. S4, ESI†), Fourier transform
infrared (FTIR) spectroscopy (Fig. S5, ESI†), and electrospray
ionization mass spectrometry (ESI-MS) (Fig. S6, ESI†). The
purity of this compound was verified to be greater than 95%
via RP-HPLC (Fig. S7, ESI†) and elemental analysis. With I-TRIP
in hand, its cytotoxicity was evaluated and compared to that of Fig. 2 Compound I-TRIP was examined for its biological activity. Dose–
response curves in the presence of (a) the pan-caspase inhibitor, Z-VAD-FMK,
TRIP in HeLa cervical cancer cells via the thiazolyl blue tetrazolium
and (b) the ER stress modulator, salubrinal. (c) Western blot for CHOP
bromide (MTT) assay (Table S1 and Fig. S8, ESI†). The 50% growth
expression of HeLa cell lysates treated with DMSO (0.3% v/v), TRIP (10 mM),
inhibitory concentration (IC ) value of I-TRIP in this this cell line is
50
or I-TRIP (10 mM) for 24 h. (d) Western blot for puromycin incorporation of
.3 mM, whereas this value is 1.8 mM for the parent compound, HeLa cell lysates treated with DMSO (0.3% v/v), TRIP (5 and 10 mM), or
3
TRIP. Accounting for experimental error in each of these measure- I-TRIP (5 and 10 mM) for 24 h. After the 24 h incubation, puromycin (60 mM)
was added for an additional 10 min.
ments, the difference between these values is not statistically
significant (p 4 0.05), thus indicating that both compounds exhibit
similar cytotoxic activities in HeLa cells. Having demonstrated that
these complexes possess comparable potencies, we next evaluated specific high-affinity antibodies via western blot.
48,49
As shown in
whether they operate via similar mechanisms of action. Fig. 2d, both TRIP and I-TRIP significantly reduce incorporation of
TRIP induces cancer cell death in a manner that is accom- puromycin into the proteome, indicating that these agents effectively
panied by ER stress, the UPR pathway, and inhibition of protein inhibit protein translation. Taken together, these biological studies
22
translation. To verify that I-TRIP is an appropriate model for demonstrate that I-TRIP causes similar in vitro biological effects as
TRIP that causes cell death in a similar fashion, we carried out TRIP, thus indicating that alteration of the isonitrile substituents
detailed biological mechanistic studies for this compound. Similar within this class of compounds does not change their mechanisms
to TRIP, I-TRIP kills cancer cells via caspase-dependent apoptosis. of action.
This conclusion is supported by the fact that the cytotoxic activity
Having demonstrated that I-TRIP operates in a similar
of I-TRIP is diminished in the presence of the pan-caspase manner as TRIP, we next sought to use the elemental signature
inhibitor, Z-VAD-FMK (Fig. 2a). Furthermore, the cytotoxicity of of the I in its axial ligand to probe its stability in cells via XFM.
I-TRIP is enhanced in the presence of the compound salubrinal Our approach follows that of a previously reported study involving a
1
0
(
Fig. 2b), marking another similarity to TRIP. Salubrinal inhibits Re(CO)
dephosphorylation of the protein eukaryotic translation initiation In this previous study, the compound, fac-[Re(CO)
factor 2a (eIF2a), which is intimately involved in the UPR pathway phen = 1,10-phenanthroline and L = 5-(4-iodophenyl)-tetrazolate,
3
complex bearing an axial I-containing tetrazolate ligand.
3
(phen)L], where
42–44
and ER stress.
The synergistic effects of I-TRIP with salubrinal was imaged in cells by XFM to show that Re and I elemental maps
are consistent with previous results for TRIP, and they indicate were coincident with one another, signifying that the axial ligand
that this new Re complex also induces ER stress. In further support remains bound under these conditions. Inspired by this study, we
of this mechanistic similarity between TRIP and I-TRIP, western explored the elemental distributions of Re and I in HeLa cells
blotting was performed to determine the expression level of the treated with TRIP and I-TRIP using XFM (Fig. 3a–c). Both TRIP and
45,46
proapoptotic protein, CHOP, which is induced via ER stress.
I-TRIP give rise to detectable Re elemental maps, demonstrating
The blot depicts the induced expression of CHOP after treating that these compounds can effectively enter cells. In comparison to
with the Re complexes, verifying that I-TRIP induces apoptotic cell both vehicle-treated and TRIP-treated samples, cells exposed to
death via ER stress in a similar fashion to TRIP (Fig. 2c). As a final I-TRIP displayed detectable X-ray emissions matching the edge
validation that TRIP and I-TRIP follow similar biological mechan- energy of I. We note that the I distribution maps appear coarser
isms of action, a puromycin incorporation assay was used to and less bright than the Re maps. This feature is due to the
investigate the abilities of these compounds to inhibit protein lower energy L edge lines (4.5 keV) of I compared to Re (10 keV),
47
translation. Puromycin is a tyrosine and phenylalanine mimic which gives rise to photons with less penetrating capability and
that is incorporated into proteins during translation, providing thus poorer detection efficiency. Despite the difference in L
a quantifiable marker for this process which can be detected by edge intensities, the colocalization of the Re and I signal is
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Office Cornell Technology Acceleration and Maturation (CTAM)
fund, and by the Office of the Assistant Secretary of Defense for
Health Affairs through the Ovarian Cancer Research Program
under award no. W81XWH-17-1-0097. This work made use of
the NMR facility at Cornell University, which is in-part supported
by the NSF under award number CHE-1531632. This research used
resources of the Advanced Photon Source, a U.S. Department of
Energy (DOE) Office of Science User Facility operated for the DOE
Office of Science by Argonne National Laboratory under Contract
No. DE-AC02-06CH11357. Travel funding for Prof. Hugh Harris
and Mr. James Lovett to perform experiments at the Advanced
Photon Source was provided by the International Synchrotron
Access Program (ISAP) managed by the Australian Synchrotron,
part of ANSTO, funded by the Australian Government. We thank
Prof. Jeremy Baskin for allowing us to use their confocal fluores-
cence microscope, and Dr. Sierra Marker is thanked for her
assistance with some of these studies. We also thank Prof. Hening
Lin for allowing us to use their Bio-Rad ChemiDoc MP imaging
Fig. 3 (a) XFM elemental distribution maps of HeLa cervical cancer cells system for western blots.
treated with DMSO (0.06% v/v), 2 mM TRIP, or 3 mM I-TRIP. Correlation
analysis on the alignment of Re, I, and Zn distribution maps for (b) TRIP and
(c) I-TRIP. Scale bar = 20 mm.
Conflicts of interest
The authors declare no competing financial interests.
strong, as reflected by a Pearson’s correlation coefficient of
.684 (Fig. 3b, c and Table S2, ESI†). The colocalization of these
0
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