Please do not adjust margins
ChemComm
Page 4 of 5
DOI: 10.1039/C7CC01480E
ARTICLE
Journal Name
in ER that exhibited sheet-like morphology (Fig. 3B). To more
precisely define the temporal dynamics of DKM 3-30 on ER
structure, we performed time-lapse imaging of GFP-Sec61β
expressing cells (Fig. 3C). In contrast to vehicle-treated control cells
(Fig. 3C and Video S1), treatment with DKM 3-30 resulted in the
loss of peripheral ER tubules and the accumulation of sheet-like ER
structures (Fig. 3D and Video S2). The alterations in the ER
morphology were evident as early as 0.5-1 hr and the ER
architecture became progressively more distorted, with some cells
exhibiting extremely aberrant, circular ER structures (Fig. S6).
Consistent with the importance of RTN4 in ER structure, siRNA-
mediated depletion of RTN4 resulted in the appearance of similarly
altered ER morphologies (Fig. 3E,F). Together, these results suggest
that DKM 3-30 acutely impairs RTN4 function in ER tubules
formation or maintenance.
envelope morphology and colorectal cancer pathogenicity. We also
show that DKM 3-30 impairs osteosarcoma cell survival as well,
suggesting that RTN4 may have broader impacts upon other types
of cancers. We recognize that DKM 3-30 may have additional off-
targets that may contribute its anti-cancer activity, but nonetheless
show compelling evidence that DKM 3-30 and its analogs
phenocopy what is observed with RTN4 knockdown and that DKM
3-30 confers sensitivity in MEF cells only when expressing human
RTN4. DKM 3-30 and AMR 1-125 may serve as initial starting points
for subsequent medicinal chemistry to develop a more potent and
selective RTN4 inhibitors for cancer therapy. Overall, we highlight
the utility of coupling the screening of covalent ligand libraries with
isoTOP-ABPP for mining the proteome for novel druggable nodes
that can be targeted for cancer therapy.
This work was supported by grants from the National Institutes of
Health (R01CA172667 for DKN, LAB, TRH, DKM, YP, R01GM112948
for JAO, TBN), American Cancer Society Research Scholar Award
(RSG14-242-01-TBE for DKN, LAB, TRH, DKM).
Notes and references
1
2
3
N. M. Gangadhar and B. R. Stockwell, Curr. Opin. Chem. Biol.,
2007, 11, 83–87.
I. Smukste and B. R. Stockwell, Annu. Rev. Genomics Hum.
Genet., 2005, 6, 261–286.
E. Weerapana, C. Wang, G. M. Simon, F. Richter, S. Khare, M. B.
D. Dillon, D. A. Bachovchin, K. Mowen, D. Baker and B. F.
Cravatt, Nature, 2010, 468, 790–795.
K. M. Backus, B. E. Correia, K. M. Lum, S. Forli, B. D. Horning, G.
E. González-Páez, S. Chatterjee, B. R. Lanning, J. R. Teijaro, A. J.
Olson, D. W. Wolan and B. F. Cravatt, Nature, 2016, 534, 570–
574.
Figure 5. DKM 3-30 and analogs. (A) Structures of DKM 3-30 and analogs.
(B) Gel-based ABPP analysis showing competition side-by-side competition
studies of DKM 3-30, YP 1-46, and AMR 1-125 against IA-rhodamine labelling
of pure human RTN4. Shown are the 50 % inhibitory concentration (IC50)
values for each compound. (C) Serum-free cell survival of U2OS (48 h) or
SW620 (24 h) cells treated with DMSO vehicle or each compound (50 ꢀM).
Data in (C) are presented as mean ± sem. Significance is expressed as
*p<0.001 compared to vehicle-treated controls.
4
Cell division requires elaborate rearrangements in the ER and
the nuclear envelope to ensure correct inheritance of DNA and
segregation of DNA within a single nucleus.13 During prophase the
nuclear envelope retracts into the ER and then reforms during
telophase. The reticulon family of proteins, and the transition
between ER tubules and sheets, have been implicated in nuclear
envelope assembly and disassembly during mitosis.14–16 Time-lapse
imaging of mitotic cells revealed that control cells divided rapidly
(~50-60 min) (Fig. 4A and Video S3). In contrast, DKM 3-30-treated
cells exhibited prolonged mitosis (~3-4 hr) (Fig. 4B and Video S4),
possibly reflecting complications in the division process. Indeed,
DKM 3-30-treated cells contained aberrant nuclei that were
bisected by GFP-Sec61β positive structures (Fig. 4B and Video S4).
Distortions in the nuclear envelope were also frequently observed
during interphase in DKM 3-30-treated cells, including multi-lobed,
cloverleaf-like nuclear envelope morphologies that often preceded
cell death (Fig. 4C and Video S5). Thus, disrupting RTN4-mediated
ER remodeling may impair colorectal cancer pathogenicity by
altering ER homeostasis and nuclear envelope assembly and
disassembly during mitosis.
We also synthesized analogs of DKM 3-30 and showed that YP
1-46 demonstrated less displacement of IAyne labelling of RTN4,
whereas AMR 1-125 exhibited ~7-fold improved potency compared
to DKM 3-30. We further showed that AMR 1-125, but not YP 1-46,
impaired cell survival in U2OS and SW620 cells and ER morphology
in U2OS cells (Fig. 5A-C and Fig. S7).
In summary, we identify RTN4 as a novel colorectal cancer
therapeutic target, and reveal a unique druggable hotspot within
this classically undruggable protein, which can be targeted by
cysteine-reactive ligands such as DKM 3-30 to impair ER and nuclear
5
6
7
8
9
C. Wang, E. Weerapana, M. M. Blewett and B. F. Cravatt, Nat.
Methods, 2014, 11, 79–85.
F. N. B. Edfeldt, R. H. A. Folmer and A. L. Breeze, Drug Discov.
Today, 2011, 16, 284–287.
V. V. Rostovtsev, L. G. Green, V. V. Fokin and K. B. Sharpless,
Angew. Chem. Int. Ed Engl., 2002, 41, 2596–2599.
E. Weerapana, G. M. Simon and B. F. Cravatt, Nat. Chem. Biol.,
2008, 4, 405–407.
L. Jozsef, K. Tashiro, A. Kuo, E. J. Park, A. Skoura, S. Albinsson, F.
Rivera-Molina, K. D. Harrison, Y. Iwakiri, D. Toomre and W. C.
Sessa, J. Biol. Chem., 2014, 289, 9380–9395.
10 G. K. Voeltz, W. A. Prinz, Y. Shibata, J. M. Rist and T. A. Rapoport,
Cell, 2006, 124, 573–586.
11 Y. Shibata, J. Hu, M. M. Kozlov and T. A. Rapoport, Annu. Rev.
Cell Dev. Biol., 2009, 25, 329–354.
12 S. V. Vasudevan, J. Schulz, C. Zhou and M. J. Cocco, Proc. Natl.
Acad. Sci. U. S. A., 2010, 107, 6847–6851.
13 S. Güttinger, E. Laurell and U. Kutay, Nat. Rev. Mol. Cell Biol.,
2009, 10, 178–191.
14 D. J. Anderson and M. W. Hetzer, J. Cell Biol., 2008, 182, 911–
924.
15 E. Kiseleva, K. N. Morozova, G. K. Voeltz, T. D. Allen and M. W.
Goldberg, J. Struct. Biol., 2007, 160, 224–235.
16 A. Audhya, A. Desai and K. Oegema, J. Cell Biol., 2007, 178, 43–
56.
17 R. Yan, Q. Shi, X. Hu and X. Zhou, Cell. Mol. Life Sci. CMLS, 2006,
63, 877–889.
18 T. Karnezis, W. Mandemakers, J. L. McQualter, B. Zheng, P. P.
Ho, K. A. Jordan, B. M. Murray, B. Barres, M. Tessier-Lavigne and
C. C. A. Bernard, Nat. Neurosci., 2004, 7, 736–744.
4 | Chem. Commun., 2017, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins