Chemoproteomics-enabled covalent ligand screen reveals a cysteine hotspot in reticulon 4 that impairs ER morphology and cancer pathogenicity

Article abstract of DOI:10.1039/c7cc01480e

Chemical genetics has arisen as a powerful approach for identifying novel anti-cancer agents. However, a major bottleneck of this approach is identifying the targets of lead compounds that arise from screens. Here, we coupled the synthesis and screening of fragment-based cysteine-reactive covalent ligands with activity-based protein profiling (ABPP) chemoproteomic approaches to identify compounds that impair colorectal cancer pathogenicity and map the druggable hotspots targeted by these hits. Through this coupled approach, we discovered a cysteine-reactive acrylamide DKM 3-30 that significantly impaired colorectal cancer cell pathogenicity through targeting C1101 on reticulon 4 (RTN4). While little is known about the role of RTN4 in colorectal cancer, this protein has been established as a critical mediator of endoplasmic reticulum tubular network formation. We show here that covalent modification of C1101 on RTN4 by DKM 3-30 or genetic knockdown of RTN4 impairs endoplasmic reticulum and nuclear envelope morphology as well as colorectal cancer pathogenicity. We thus put forth RTN4 as a potential novel colorectal cancer therapeutic target and reveal a unique druggable hotspot within RTN4 that can be targeted by covalent ligands to impair colorectal cancer pathogenicity. Our results underscore the utility of coupling the screening of fragment-based covalent ligands with isoTOP-ABPP platforms for mining the proteome for novel druggable nodes that can be targeted for cancer therapy.

Full text of DOI:10.1039/c7cc01480e

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F. Skibola, J. A. Olzmann and D. Nomura, Chem. Commun., 2017, DOI: 10.1039/C7CC01480E.
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Chemoproteomics-Enabled Covalent Ligand Screen Reveals a
Cysteine Hotspot in Reticulon 4 that Impairs ER Morphology and
Cancer Pathogenicity
Received 00th January 20xx,
Accepted 00th January 20xx
L.A. Bateman,a,b,† T.B. Nguyen,b,† A.M. Roberts,a,† D.K. Miyamoto,a W-M. Ku,a T.R. Huffman,a Y.
Petri,a M.J. Heslin,c C.M. Contreras,c C.F. Skibola,c J.A. Olzmann,b,* and D.K. Nomuraa,b, *
DOI: 10.1039/x0xx00000x
www.rsc.org/
Chemical genetics has arisen as a powerful approach for
major challenge of chemical genetics is identifying the targets of
leads that arise from screens. Oftentimes, lead compounds must be
derivatized to either bear bioorthogonal and/or photoaffinity
handles or conjugated to beads to facilitate chemoproteomic target
identifying novel anti-cancer agents. However, a major bottleneck
of this approach is identifying the targets of lead compounds that
arise from screens. Here, we coupled the synthesis and screening
of fragment-based cysteine-reactive covalent ligands with activity- identification.2 However, these approaches frequently require
based protein profiling (ABPP) chemoproteomic approaches to
identify compounds that impair colorectal cancer pathogenicity
and map the druggable hotspots targeted by these hits. Through
this coupled approach, we discovered a cysteine-reactive
acrylamide DKM 3-30 that significantly impaired colorectal cancer
cell pathogenicity through targeting C1101 on reticulon 4 (RTN4).
While little is known about the role of RTN4 in colorectal cancer,
this protein has been established as a critical mediator of
endoplasmic reticulum tubular network formation. We show here
that covalent modification of C1101 on RTN4 by DKM 3-30 or
genetic knockdown of RTN4 impairs endoplasmic reticulum and
nuclear envelope morphology as well as colorectal cancer
pathogenicity. We thus put forth RTN4 as a potential novel
additional synthetic efforts to make analogs of the lead molecule
thereby hindering or preventing target identification.
Here, we have generated a library of 75 fragment-based
cysteine-reactive covalent ligands and coupled the screening of this
library with an isotopic tandem orthogonal proteolysis-enabled
activity-based protein profiling (isoTOP-ABPP) platform to rapidly
identify covalent ligands that impair colorectal cancer pathogenicity
and to map the druggable hotspots targeted by these hits (Fig. 1A).
IsoTOP-ABPP uses reactivity-based chemical probes to map
proteome-wide reactive, functional, and ligandable hotspots. When
used in a competitive manner, covalent small-molecules can be
competed against the binding of their corresponding reactivity-
based probes to rapidly identify the targets of these molecules.3–5 In
colorectal cancer therapeutic target and reveal a unique druggable this study, upon identifying a cysteine-reactive lead fragment, the
hotspot within RTN4 that can be targeted by covalent ligands to lead molecules can compete with a broad cysteine-reactive probe
impair colorectal cancer pathogenicity. Our results underscore the to subsequently identify its targets and the specific sites of labelling.
utility of coupling the screening of fragment-based covalent
ligands with isoTOP-ABPP platforms for mining the proteome for
novel druggable nodes that can be targeted for cancer therapy.
There are several advantages to this combined approach. First,
our library already introduces specific covalent interactions through
the incorporation of cysteine-reactive acrylamide and
chloroacetamide warheads, thus avoiding the necessity for
introducing photoaffinity handles for target identification. Recent
studies by Backus et al. have shown that the reactivity of these
scaffolds can be tempered and made to confer substantial
selectivity through appending small-molecular weight fragments.4
Also, because these compounds are small molecular weight
fragment-based covalent ligands, they can sample more
macromolecular protein space and enable interrogation of more
druggable nodes, a notion explored by many pharmaceutical
companies with fragment-based ligand discovery.6 Second, the
advantage of this approach is that the lead molecule itself can be
directly competed against reactivity-based probes for target
identification without the need for additional synthetic efforts.
We screened our cysteine-reactive ligand library of acrylamides
and chloroacetamides to identify compounds that impair colorectal
cancer cell survival and proliferation in the highly metastatic and
tumorigenic SW620 colorectal cancer cell line (Fig. 1B, Table S1).
We identified a lead acrylamide DKM 3-30 as the top hit from this
screen which significantly impaired both serum-free cell survival
Traditional strategies for cancer target discovery often involve
searching for proteins or genes that are dysregulated or mutated in
tumors, which may miss promising therapeutic targets that may not
necessarily be changing in expression or activity. Screening chemical
libraries for anti-cancer small-molecules using chemical genetics
strategies has arisen as a powerful complementary approach to
traditional target discovery approaches for mining druggable nodes
that can be pharmacologically interrogated in cancer.1,2 However, a
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and proliferation in SW620 colorectal cancer cells (Fig. 1C, 1D). We
further showed that SW620 colorectal tumor xenograft growth was
significantly impaired upon in vivo treatment of mice with DKM 3-
protease digestion for subsequent quantitative proteomic analysis.
Through these studies, we identified the top hit for DKM 3-30 as
C1101 in reticulon 4 (RTN4, Uniprot ID Q9NQC3-1) with a light to
30, started 10 days after the initiation of the xenograft, without any heavy ratio of 3.0 (Fig. 2A; Table S2). We further validated this hit
changes in body weight or any signs of overt toxicity (Fig. 1E, Fig.
S1). Taken together, our data indicated that DKM 3-30 significantly
impaired SW620 colorectal cancer pathogenicity.
by competing DKM 3-30 against IAyne labeling of pure human RTN4
protein using gel-based ABPP methods (Fig. 2A).
Figure 2. DKM 3-30 Targets C1101 on RTN4. (A) IsoTOP-ABPP analysis of
DKM 3-30 in SW620 colorectal cancer cells. SW620 proteomes were pre-
treated with DMSO or DKM 3-30 (50 ꢀM) prior to labeling proteomes with
IAyne and isoTOP-ABPP analysis. Shown are mean light to heavy ratios for
those probe-modified peptides identified in at least 2 out of 3 biological
replicates. Also shown is gel-based ABPP validation of DKM 3-30 to RTN4.
Pure human RTN4 protein was preincubated with DMSO or DKM for 30 min
followed by IAyne labelling. Rhodamine-azide was conjugated by CuAAC and
probe-labeled RTN4 was visualized by SDS/PAGE and in-gel fluorescence. (B)
IsoTOP-ABPP analysis of cysteine-reactivity in pooled primary human
colorectal tumors. Nine primary human colorectal tumors were pooled
together and labeled with 100 or 10 ꢀM of IAyne followed by subsequent
isoTOP-ABPP analysis. Shown are ratios of heavy (100 ꢀM) to light (10 ꢀM)
peptides. (C, D) Serum-free cell survival and proliferation (48 h) and tumor
xenograft growth in SCID mice from transient siRNA or stable shRNA
knockdown of RTN4 in SW620 cells. Expression was determined by qPCR. All
data shown represents n=3-6/group. Data in (C,D) are presented as mean ±
sem. Significance is expressed as *p<0.05 compared to vehicle-treated or si
or shControls. Raw data for (A, B) can be found in Table S2.
Figure 1. Coupling Screening of Cysteine-Reactive Covalent Ligands with
isoTOP-ABPP to Identify Anti-Cancer Compounds and Druggable Hotspots
for Colorectal Cancer. (A) We screened a library of cysteine-reactive
fragment-based covalent ligands in colorectal cancer cells to identify
compounds that impair colorectal cancer pathogenicity and used isoTOP-
ABPP to identify the druggable hotspots targeted by hits. (B) We screened a
cysteine-reactive fragment library consisting of acrylamides and
chloroacetamides in SW620 colorectal cancer cells (50 ꢀM) to identify any
leads that significantly impaired SW620 serum-free cell survival. (C, D)
Shown is the structure of the lead covalent ligand DKM 3-30 (C) that
significantly (p<0.05) impaired SW620 cell survival and proliferation (D). (E)
SW620 tumor xenograft growth in immune-deficient SCID mice. Mice were
subcutaneously injected with SW620 cells to initiate the tumor xenograft
study and treatments of mice were initiated with vehicle or DKM 3-30 (50
mg/kg ip, once per day) ten days initiation of the xenograft study. Data in
(B, D, E) are presented as mean ± sem, n=3-8/group. Significance expressed
as *p<0.05 compared to vehicle-treated controls.
To determine the relevance of RTN4 in colorectal cancer, we
performed isoTOP-ABPP analysis to quantitatively map proteome-
wide reactivity of cysteines in pooled primary human colorectal
tumors through comparative ratiometric analysis of IAyne labeling
at 100 (heavy) versus 10 ꢀM (light) concentrations. Previous studies
by Weerapana et al. have shown that hyper-reactive cysteines,
which show saturated IAyne labeling at lower concentrations and
thus exhibit a lower (<3) heavy to light ratio, are highly enriched in
functional cysteines, compared to those sites that are not hyper-
reactive that show heavy to light ratios of ~10.8 We identify RTN4
labeling of C1101 in primary human colorectal tumors. RTN4 C1101
shows a ratio of 6.2 indicating that this cysteine is not hyper-
reactive (Fig. 2B). Our data therefore shows that RTN4 is present
We next performed isoTOP-ABPP studies to identify the direct
targets of these lead compounds. We competed either vehicle or
DKM 3-30 against labeling of SW620 proteomes with a broad
cysteine-reactive probe, iodoacetamide-alkyne (IAyne), followed by
appending probe-labeled proteins with a biotin-azide tag bearing a
TEV protease recognition site and an isotopically light (for vehicle-
treated) or heavy (for fragment-treated) tags via copper catalyzed
azide-alkyne cycloaddition (CuAAC).4,7 We then combined control
and treated proteomes in a 1:1 ratio, enriched probe-labeled
proteins with avidin, and digested proteomes with trypsin. Avidin-
enriched probe-modified tryptic peptides were released by TEV
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and that C1101 within RTN4 is accessible in primary human
colorectal tumors.
While little is known about the role of RTN4 in cancer
pathogenicity, RTN4 is known to be a critical mediator of
endoplasmic reticulum (ER) tubule formation.9–11 Interestingly,
Voeltz et al. found that tubular ER network formation in a
reconstituted in vitro system was disrupted by thiol modifying
agents and discovered that xenopus RTN4 was responsible for this
action.15 Intriguingly, one of these cysteines, C952 of xenopus
RTN4,10 corresponds to C1101 of human RTN4 identified in our
study (Fig. S3). C1101 is present in all human RTN4 isoforms, but is
absent in other reticulon family members (RTN1-3) (Fig. S4). This
cysteine is positioned within a cytosolically exposed linker between
two tandem hydrophobic regions (Fig. 3A), which allow RTN4 to
adopt a characteristic wedge-shaped hairpin conformation required
for generating highly curved membranes and tubular ER
structures.11 A solution NMR structure of a mouse RTN4 fragment
revealed that this linker region forms a compact helical bundle with
a portion associated with the membrane12 and a threaded
homology model of the human RTN4 linker region indicates that
C1101 is present in a cytosolically accessible helix (Fig. 3A).
Figure 3. DKM 3-30 disrupts the ER tubular network. (A) A schematic
illustration depicts the proposed topology of Rtn4 and the position of C1101
modified by DKM 3-30 (red arrow). A homology model of human Rtn4
illustrates the membrane-associated portion (blue), the cytosolically
accessible portion (green), and the position of C1101 (red). (B) U2OS cells
expressing GFP-tagged Sec61β an ER marker, were treated with DKM 3-30
(50 ꢀM) for 16 hr and the ER (green) and nucleus (blue) of fixed cells
visualized by fluorescence microscopy. Scale bar = 10 ꢀm. (C,D) U2OS cells
expressing GFP-tagged Sec61β were treated with vehicle (DMSO) (C) or DKM
3-30 (50 ꢀM) (D) and ER morphology visualized by time-lapse fluorescence
microscopy. Time (min) is indicated on each panel. Bottom panels indicate
boxed region. (E) U2OS cells were transiently transfected with control or
RTN4 siRNA and expression determined by qPCR. Data are presented as
mean ± sem, n=3. Significance is expressed as *p<0.05. (F) U2OS cells
expressing GFP-tagged Sec61β were transfected with siRNAs as in panel (D)
and the ER (green) and nucleus (blue) of fixed cells visualized by
Figure 4. DKM 3-30 disrupts nuclear envelope morphology during mitosis.
(A-C) U2OS cells expressing GFP-tagged Sec61β were treated with vehicle
(DMSO) or DKM 3-30 (50 ꢀM) and ER morphology of mitotic cells visualized
by time-lapse fluorescence microscopy. Time (min) is indicated on each
panel. Panels (A,B) provide examples of mitotic cells. Enlarged images
following mitosis show the nuclear envelope. White arrowheads indicate a
GFP-Sec61β structure bisecting the nucleus of a cell incubated with DKM 3-
30. (C) shows alterations in the nuclear envelope structure, followed by cell
death at the 800 min time point. Bottom panels indicate boxed region.
fluorescence microscopy. Scale bar = 10 µm.
We confirmed the relevance of RTN4 in colorectal cancer by
showing that transient or stable knockdown of RTN4 by RNA
interference phenocopies the impaired survival, proliferation, and
anti-tumorigenic effects observed with DKM 3-30 in SW620
colorectal cancer cells (Fig. 2C, 2D). To further confirm that the cell
viability impairments conferred by DKM 3-30 are due to RTN4, we
tested the effect of this compound in mouse embryonic fibroblasts
(MEF) with or without the expression of human RTN4. Mouse Rtn4
possesses a serine instead of cysteine at the analogous site to
human RTN4(C1101). We show that DKM 3-30 does not show
viability impairments in GFP-expressing MEF cells but induces
apoptosis in MEF cells expressing human RTN4-GFP (Fig. S2).
We postulated that covalent modification of RTN4(C1101) by
DKM 3-30 would impact the formation of ER tubular networks in
cells. We attempted to analyse the effects of DKM 3-30 in SW620
colorectal cancer cells, and while the images suggest alterations in
the ER morphology (Fig. S5), the reticular nature of the ER was
difficult to visualize in this cell type. Therefore, we utilized U2OS
osteosarcoma cells, which are a well-established cell line for the
analysis of ER morphology. As expected, control U2OS cells
expressing the ER marker GFP-Sec61β displayed a highly reticular
ER with clearly visible tubular ER in the cell periphery (Fig. 3B).
Treatment of U2OS cells with DKM 3-30 for 8 hr and 16 hr resulted
in a striking loss of nearly all peripheral ER tubules and an increase
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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.
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