Synthesis of Fluorophores that Target Small Molecules to the Endoplasmic Reticulum of Living Mammalian Cells

Article abstract of DOI:10.1002/anie.201504156

The endoplasmic reticulum (ER) plays critical roles in the processing of secreted and transmembrane proteins. To deliver small molecules to this organelle, we synthesized fluorinated hydrophobic analogues of the fluorophore rhodol. These cell-permeable fluorophores are exceptionally bright, with quantum yields of around 0.8, and they were found to specifically accumulate in the ER of living HeLa cells, as imaged by confocal laser scanning microscopy. To target a biological pathway controlled by the ER, we linked a fluorinated hydrophobic rhodol to 5-nitrofuran-2-acrylaldehyde. In contrast to an untargeted nitrofuran warhead, delivery of this electrophilic nitrofuran to the ER by the rhodol resulted in cytotoxicity comparable to the ER-targeted cytotoxin eeyarestatin I, and specifically inhibited protein processing by the ubiquitin-proteasome system. Fluorinated hydrophobic rhodols are outstanding fluorophores that enable the delivery of small molecules for targeting ER-associated proteins and pathways.

Full text of DOI:10.1002/anie.201504156

.
Angewandte
Communications
International Edition: DOI: 10.1002/anie.201504156
German Edition: DOI: 10.1002/ange.201504156
Subcellular Targeting
Synthesis of Fluorophores that Target Small Molecules to the
Endoplasmic Reticulum of Living Mammalian Cells**
J. Matthew Meinig, Liqiang Fu, and Blake R. Peterson*
In memory of Lester A. Mitscher
Abstract: The endoplasmic reticulum (ER) plays critical roles
in the processing of secreted and transmembrane proteins. To
deliver small molecules to this organelle, we synthesized
fluorinated hydrophobic analogues of the fluorophore
rhodol. These cell-permeable fluorophores are exceptionally
bright, with quantum yields of around 0.8, and they were found
to specifically accumulate in the ER of living HeLa cells, as
imaged by confocal laser scanning microscopy. To target
a biological pathway controlled by the ER, we linked
a fluorinated hydrophobic rhodol to 5-nitrofuran-2-acrylalde-
hyde. In contrast to an untargeted nitrofuran warhead, delivery
of this electrophilic nitrofuran to the ER by the rhodol resulted
in cytotoxicity comparable to the ER-targeted cytotoxin
eeyarestatin I, and specifically inhibited protein processing by
the ubiquitin–proteasome system. Fluorinated hydrophobic
rhodols are outstanding fluorophores that enable the delivery
of small molecules for targeting ER-associated proteins and
pathways.
unfolded protein response (UPR) in the ER that initiates
complex signaling pathways. These pathways can promote
adaptive responses or, when the stress is excessive, cellular
[
3]
death. Pathologies associated with ER stress include neuro-
degenerative disease, stroke, heart disease, diabetes, and
[
4–7]
cancer.
Because of the importance of the ER in disease,
modulators of targets in this organelle are of substantial
interest as potential therapeutics and probes. A number of
[
2,4]
small molecules are known to induce ER stress,
eeyarestatin I (1, Figure 1).
including
This agent blocks the ubiq-
uitin–proteasome system (UPS) by accumulating in the ER
[8–14]
[8,9,12,13,15]
and inhibiting the protein p97.
This protein is
involved in the translocation of misfolded polypeptides
from the ER to the cytosol for eventual degradation by the
proteasome. Inhibition of p97 triggers a build-up of misfolded
proteins, which causes apoptosis in some cancer cell
[
10,16,17]
lines.
Previous structure–activity relationship (SAR)
[12]
studies
of eeyarestatin I (1) demonstrated that the 5-
T
he endoplasmic reticulum (ER), an organelle found in all
nitrofuran-2-acrylaldehyde hydrazone functions as an elec-
eukaryotes, generally contributes at least half of all of the
membranes of animal cells. Membranes of the ER spread
throughout the cytosol to define a network of tubes and sacs
that enclose a single internal space termed the lumen.
Numerous processes essential for cellular maintenance and
survival occur on and in the ER. The external surface of the
ER captures ribosomes involved in the translation of
membrane-bound and secreted proteins, whereas the lumen
stores intracellular calcium, regulates the folding and post-
translational processing of proteins in the secretory pathway,
[1]
and is a major site of cellular lipid biosynthesis. Stress
conditions can cause unfolded proteins to accumulate in the
ER. Stressors include hypoxia, oxidants or reductants,
glucose deprivation, altered calcium regulation, viral infec-
tion, and expression of aberrant proteins. This triggers an
[2]
[
+]
[+]
[
*] J. M. Meinig, Dr. L. Fu, Prof. Dr. B. R. Peterson
Department of Medicinal Chemistry
The University of Kansas, Lawrence, KS 66045 (USA)
E-mail: brpeters@ku.edu
+
[
] These authors contributed equally to this work.
[
**] B. Peterson acknowledges support from the NIH (RC1-GM091086,
R01-CA83831, and P20-GM103638) and the KU Cancer Center.
J.M.M. acknowledges support from the NIH Dynamic Aspects of
Chemical Biology Training Grant at the University of Kansas (T32-
GM08545).
Figure 1. Structures of the ER-targeted p97 inhibitor eeyarestatin I (1),
the fluorophore rhodol (2), a fluorinated hydrophobic analogue of
rhodol (3), and a related derivative (4) linked to an electrophilic 5-
nitrofuran warhead.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201504156.
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Chemie
[
18,19]
trophilic warhead
that interacts with the p97 protein
complex, whereas the aromatic urea moiety principally
localizes this warhead to ER membranes to gain selectivity
for ER-membrane-bound p97 over the cytosolic form of this
protein.
We report herein the synthesis of novel analogues of the
fluorophore rhodol (2, Figure 1) that uniquely accumulate in
the ER of mammalian cells. Analogues such as 3 (Figure 1)
incorporate fluorine atoms at the 2’- and 7’-positions to
enhance the photophysical properties, and substitute the
polar carboxylate of rhodol with a hydrophobic methyl group
to favor association with cellular membranes. Analogue 4
Figure 2. Photophysical properties and spectra of rhodol 3. Absorb-
ance (Abs., acquired at 10 mm) and normalized fluorescence emission
Em., acquired at 10 nm; Ex. at 500 nm) spectra are shown for
aqueous phosphate-buffered saline (PBS, pH 7.4), absolute ethanol
EtOH), and n-octanol (Oct.).
(
Figure 1) was designed to include a 5-nitrofuran-2-acrylal-
dehyde hydrazone, analogous to the electrophilic warhead
found in eeyarestatin I (1), to target the p97 protein of the
ER.
(
(
To synthesize these compounds, the fluorophore Pennsyl-
[
20,21]
vania Green (5, Scheme 1),
,6-dihydroxyxanthene-9-one,
prepared from 2,7-difluoro-
was converted into iodoar-
[22,23]
3
properties in ethanol (Abs. l = 512 nm; Em. lmax = 532 nm,
max
À1
À1
ene 6. This iodoarene was transformed into rhodols 3 and 8
e = 73,500m cm , F = 0.85) make it a potentially outstand-
ing probe for cellular studies.
Confocal laser scanning microscopy was used to examine
the subcellular distribution of rhodol 3 in the human cervical
carcinoma cell line HeLa. As shown in Figure 3, these
Scheme 1. Synthesis of the fluorinated rhodol 3, rhodol hydrazide 9,
rhodol nitrofuran 4, and the untargeted nitrofuran control 12.
Figure 3. Confocal laser scanning and differential interference contrast
through Buchwald-Hartwig cross-coupling with microwave
irradiation. This approach is similar to methods used to
(DIC) micrographs of living HeLa cells treated with ER-tracker Blue-
White DPX (0.1 mm, 0.5 h) and rhodol 3 (0.5 mm, 0.5 h). The fluores-
cence emission of ER-tracker Blue-White DPX can be observed in
green in the upper left panel and the fluorescence emission of the
spectrally orthogonal 3 can be observed in red in the upper right
panel. Colocalization of these two fluorophores is shown in yellow in
the lower left panel.
[24]
[25]
prepare other rhodols
and rhodamines
from triflates.
Rhodol 3 is a unique fluorinated hydrophobic (cLogP = 3.9,
calculated with ChemAxon MarvinView 6.2) analogue of
[24,26–30]
other previously reported
rhodol fluorophores. The
related rhodol 4 was derived through deprotection of 8,
preparation of the rhodol hydrazide 9, and condensation with
aldehyde 10 to install the warhead, whereas the analogous
nitrofuran derivative 12 was prepared as an untargeted
control compound.
experiments revealed the accumulation of 3 in distinct tubular
structures. These structures were identified as the ER by
colocalization with the spectrally orthogonal organelle
[32]
Rhodols are generally fluorescent in a wide variety of
marker ER-Tracker Blue-White (Figure 3). Although the
mechanism of the selectivity of fluorophores such as ER-
Tracker Blue-White for the ER is not completely understood,
[
24,26]
solvents.
To examine the photophysical properties of
rhodol 3, we obtained absorbance and fluorescence emission
spectra of this compound in PBS, ethanol, and octanol, and we
measured its extinction coefficient and quantum yield rela-
[33,34]
[35]
QSAR
and microscopy
studies of related probes
suggest that these types of hydrophobic amphipathic com-
pounds preferentially associate with cholesterol-poor ER
[
31]
tive
to rhodamine 6G (data shown in Figure S1 of the
[36]
supporting information). As shown in Figure 2, its spectral
membranes.
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Similar colocalization results were obtained in cells
treated with rhodol nitrofuran 4, thus indicating that the
ER-targeting ability of the rhodol was unaffected by attach-
ment of the warhead (see Figure S2 in the Supporting
Information). To examine the cytotoxicities of eeyarestatin I
(
1), the rhodol derivatives 4 and 9, and the untargeted
nitrofuran 12, HeLa cells were treated with these compounds
for 48 h and cellular viability was measured by flow cytom-
etry. The rhodol nitrofuran 4 (IC = 2.7 mm) and eeyarestatin
50
I (1, IC = 2.0 mm) were similar in potency, thus suggesting
5
0
that the ER-targeting motifs of both of these compounds are
effective at delivering the nitrofuran to this organelle
(
Figure 4). As controls, rhodol hydrazide 9, which lacks the
Figure 5. Confocal laser scanning and DIC micrographs of living HeLa
G76V
cells transiently transfected with the cyan fluorescent protein Ub
-
cerulean. The spectrally orthogonal rhodol nitrofuran 4 was observed
in the yellow channel. Cells were treated with the compounds for 12 h
prior to imaging.
Figure 4. Cytotoxicity of ER-targeted and untargeted compounds
towards HeLa cells after 48 h in culture. The half maximal inhibitory
concentration (IC ) values for 1, 4, and 12 are 2.0 mm, 2.7 mm, and
5
0
1
3 mm, respectively.
rapid degradation of this protein by the proteasome. How-
ever, when these cells were treated for 12 h with the p97
inhibitor eeyarestatin I (1, 5 mm), transfected cells but not the
adjacent non-transfected cells became highly cyan fluores-
cent. Similarly, when treated with rhodol nitrofuran 4 (5 mm),
cerulean was highly expressed by the transfected cells, thus
demonstrating that this compound is a specific inhibitor of the
UPS. By contrast, the nitrofuran control 12, either at 5 mm or
at an elevated concentration of 20 mm, did not activate the
reporter, thus demonstrating that targeting of this moiety to
the ER is critical for inhibition of this pathway.
cytotoxic warhead, showed no appreciable toxicity (IC >
50
5
0 mm), whereas the nitrofuran hydrazone warhead 12 alone
was less toxic (IC = 13 mm) than the ER-targeted com-
50
pounds.
Eeyarestatin I manifests toxicity by inhibiting p97 on ER
[
4]
membranes. This protein is required for maturation of the
transcription factor Nrf1, which activates genes that encode
[37]
subunits of the proteasome of the UPS. To characterize
inhibitors of the UPS, cells are often transfected with
fluorescent proteins fused to
signal. The fused ubiquitin of these reporters results in
rapid trafficking to and degradation by the proteasome,
whereas inhibitors of the UPS, such as eeyarestatin I,
prevent the degradation of these reporter proteins and
restore cellular fluorescence.
By targeting the nitrofuran to the ER, we hypothesized
that the rhodol nitrofuran 4 might selectively inhibit the UPS.
To test this hypothesis, we constructed a spectrally orthogonal
a
ubiquitin degradation
The results presented herein demonstrate that fluorinated
hydrophobic rhodols are outstanding fluorophores that can be
used to deliver small-molecule cargo to the ER. When this
cargo was an electrophilic nitrofuran warhead, delivery to the
ER resulted in inhibition of the UPS, with concomitant effects
on cellular proliferation. Given the widespread interest in
[
38,39]
[40]
[4]
[41]
[4,43]
targeting ER-associated proteins involved in cancer
[44]
[7]
neurodegeneration, and other diseases, these compounds
may provide useful tools for the imaging and modulation of
biological pathways associated with this critical organelle.
fluorescent reporter comprising an optimized G76V mutant
G76V [42]
of ubiquitin (Ub
)
fused to the cyan fluorescent protein
cerulean. This mutant of ubiquitin is efficiently degraded by
Keywords: chemical biology · cytotoxins ·
endoplasmic reticulum · fluorophores · proteasome
[42]
the proteasome when fused to fluorescent proteins.
As
shown in Figure 5, in the absence of inhibitors, HeLa cells
G76V
transfected with the Ub
-cerulean reporter showed no
How to cite: Angew. Chem. Int. Ed. 2015, 54, 9696–9699
Angew. Chem. 2015, 127, 9832–9835
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[
23] Z. R. Woydziak, L. Fu, B. R. Peterson, Synthesis 2014, 46, 158 –
164.
[
[
1] J. E. Vance, Traffic 2015, 16, 1 – 18.
2] N. Sovolyova, S. Healy, A. Samali, S. E. Logue, Biol. Chem. 2014,
[24] T. Peng, D. Yang, Org. Lett. 2010, 12, 496 – 499.
[25] J. B. Grimm, L. D. Lavis, Org. Lett. 2011, 13, 6354 – 6357.
[26] J. E. Whitaker, R. P. Haugland, D. Ryan, P. C. Hewitt, F. G.
Prendergast, Anal. Biochem. 1992, 207, 267 – 279.
[27] T. Peng, D. Yang, Org. Lett. 2010, 12, 4932 – 4935.
[28] J. Li, S. Q. Yao, Org. Lett. 2009, 11, 405 – 408.
[29] H. Komatsu, H. Harada, K. Tanabe, M. Hiraoka, S. Nishimoto,
MedChemComm 2010, 1, 50 – 53.
[30] S. C. Dodani, A. Firl, J. Chan, C. I. Nam, A. T. Aron, C. S. Onak,
K. M. Ramos-Torres, J. Paek, C. M. Webster, M. B. Feller, C. J.
Chang, Proc. Natl. Acad. Sci. USA 2014, 111, 16280 – 16285.
[31] A. T. Williams, S. A. Winfield, Analyst 1983, 108, 1067 – 1071.
[32] L. Cole, D. Davies, J. Hyde, A. E. Ashford, J. Microsc. 2000, 197,
239 – 249.
[33] J. Colston, R. Horobin, F. Rashid-Doubell, J. Pediani, K. K.
Johal, Biotech. Histochem. 2003, 78, 323 – 332.
[34] R. Horobin, J. Stockert, F. Rashid-Doubell, Histochem. Cell
Biol. 2013, 139, 623 – 637.
[35] M. Terasaki, J. Song, J. R. Wong, M. J. Weiss, L. B. Chen, Cell
1984, 38, 101 – 108.
[36] G. van Meer, D. R. Voelker, G. W. Feigenson, Nat. Rev. Mol. Cell
Biol. 2008, 9, 112 – 124.
3
95, 1 – 13.
[
[
[
3] I. Tabas, D. Ron, Nat. Cell Biol. 2011, 13, 184 – 190.
4] R. J. Deshaies, BMC Biol. 2014, 12, 94.
5] G. C. Shore, F. R. Papa, S. A. Oakes, Curr. Opin. Cell Biol. 2011,
2
3, 143 – 149.
6] I. Kim, W. Xu, J. C. Reed, Nat. Rev. Drug Discovery 2008, 7,
013 – 1030.
7] J. H. Lin, P. Walter, T. S. Yen, Annu. Rev. Pathol. Mech. Dis.
008, 3, 399 – 425.
[
[
[
[
1
2
8] E. Fiebiger, C. Hirsch, J. M. Vyas, E. Gordon, H. L. Ploegh, D.
Tortorella, Mol. Biol. Cell 2004, 15, 1635 – 1646.
9] Q. Wang, L. Li, Y. Ye, J. Biol. Chem. 2008, 283, 7445 – 7454.
[
[
[
[
10] Q. Wang, H. Mora-Jensen, M. A. Weniger, P. Perez-Galan, C.
Wolford, T. Hai, D. Ron, W. Chen, W. Trenkle, A. Wiestner, Y.
Ye, Proc. Natl. Acad. Sci. USA 2009, 106, 2200 – 2205.
11] B. C. Cross, C. McKibbin, A. C. Callan, P. Roboti, M. Piacenti, C.
Rabu, C. M. Wilson, R. Whitehead, S. L. Flitsch, M. R. Pool, S.
High, E. Swanton, J. Cell Sci. 2009, 122, 4393 – 4400.
12] Q. Wang, B. A. Shinkre, J.-g. Lee, M. A. Weniger, Y. Liu, W.
Chen, A. Wiestner, W. C. Trenkle, Y. Ye, PLoS ONE 2010, 5,
e15479.
13] M. O. Aletrari, C. McKibbin, H. Williams, V. Pawar, P. Pietroni,
J. M. Lord, S. L. Flitsch, R. Whitehead, E. Swanton, S. High,
R. A. Spooner, PLoS ONE 2011, 6, e22713.
[37] J. Steffen, M. Seeger, A. Koch, E. Krüger, Mol. Cell 2010, 40,
147 – 158.
[38] N. P. Dantuma, K. Lindsten, R. Glas, M. Jellne, M. G. Masucci,
Nat. Biotechnol. 2000, 18, 538 – 543.
[39] V. MenØndez-Benito, L. G. G. C. Verhoef, M. G. Masucci, N. P.
Dantuma, Hum. Mol. Genet. 2005, 14, 2787 – 2799.
[40] E. S. Johnson, P. C. M. Ma, I. M. Ota, A. Varshavsky, J. Biol.
Chem. 1995, 270, 17442 – 17456.
[41] T.-F. Chou, S. J. Brown, D. Minond, B. E. Nordin, K. Li, A. C.
Jones, P. Chase, P. R. Porubsky, B. M. Stoltz, F. J. Schoenen, M. P.
Patricelli, P. Hodder, H. Rosen, R. J. Deshaies, Proc. Natl. Acad.
Sci. USA 2011, 108, 4834 – 4839.
[42] N. P. Dantuma, K. Lindsten, R. Glas, M. Jellne, M. G. Masucci,
Nat. Biotechnol. 2000, 18, 538 – 543.
[43] J. Boelens, S. Lust, F. Offner, M. E. Bracke, B. W. Vanhoecke, In
Vivo 2007, 21, 215 – 226.
[44] M. Halliday, G. R. Mallucci, Neuropharmacology 2014, 76, 169 –
174.
[
14] G. J. Brem, I. Mylonas, A. Bruning, Gynecol. Oncol. 2013, 128,
3
83 – 390.
[
15] C. McKibbin, A. Mares, M. Piacenti, H. Williams, P. Roboti, M.
Puumalainen, A. C. Callan, K. Lesiak-Mieczkowska, S. Linder,
H. Harant, S. High, S. L. Flitsch, R. C. Whitehead, E. Swanton,
Biochem. J. 2012, 442, 639 – 648.
16] H. W. Auner, A. M. Moody, T. H. Ward, M. Kraus, E. Milan, P.
May, A. Chaidos, C. Driessen, S. Cenci, F. Dazzi, A. Rahemtulla,
J. F. Apperley, A. Karadimitris, N. Dillon, PLoS ONE 2013, 8,
e74415.
17] T.-F. Chou, K. Li, K. J. Frankowski, F. J. Schoenen, R. J.
Deshaies, ChemMedChem 2013, 8, 297 – 312.
18] E. Eder, C. Deininger, D. Muth, Mutagenesis 1991, 6, 261 – 269.
19] J. I. Borroto, A. Creus, R. Marcos, Mutat. Res. 2002, 519, 179 –
[
[
[
[
1
85.
[
[
[
20] L. F. Mottram, E. Maddox, M. Schwab, F. Beaufils, B. R.
Peterson, Org. Lett. 2007, 9, 3741 – 3744.
21] L. F. Mottram, S. Boonyarattanakalin, R. E. Kovel, B. R. Peter-
son, Org. Lett. 2006, 8, 581 – 584.
22] Z. R. Woydziak, L. Fu, B. R. Peterson, J. Org. Chem. 2012, 77,
Received: May 6, 2015
4
73 – 481.
Published online: June 26, 2015
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