Ipomoeassin F Binds Sec61α to Inhibit Protein Translocation

Article abstract of DOI:10.1021/jacs.8b13506

Ipomoeassin F is a potent natural cytotoxin that inhibits growth of many tumor cell lines with single-digit nanomolar potency. However, its biological and pharmacological properties have remained largely unexplored. Building upon our earlier achievements

Full text of DOI:10.1021/jacs.8b13506

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Article
Ipomoeassin F Binds Sec61# to Inhibit Protein Translocation
Guanghui Zong, Zhijian Hu, Sarah O’Keefe, Dale Tranter, Michael J. Iannotti, Ludivine Baron,
Belinda Hall, Katherine Corfield, Anja Paatero, Mark Henderson, Peristera Roboti, Jianhong Zhou,
Xianwei Sun, Mugunthan Govindarajan, Jason Rohde, Nicolas Blanchard, Rachel Simmonds, James
Inglese, Yuchun Du, Caroline Demangel, Stephen High, Ville Oskari Paavilainen, and Wei Q. Shi
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b13506 • Publication Date (Web): 06 May 2019
Downloaded from http://pubs.acs.org on May 6, 2019
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Ipomoeassin F Binds Sec61α to Inhibit Protein
Translocation
Guanghui Zong,a,b,† Zhijian Hu, Sarah O’Keefe, Dale Tranter,
a,†
c,#
d,e,#
Michael J. Iannotti, Ludivine
f,#
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Baron,g,h,# Belinda Hall, Katherine Corfield, Anja O. Paatero,d,e,# Mark J. Henderson, Peristera
i,#
i, #
f,#
c,#
j
a,k
a,l
f
Roboti, Jianhong Zhou, Xianwei Sun, Mugunthan Govindarajan, Jason M. Rohde, Nicolas
m
i,
f,
j,
g,h,
Blanchard, Rachel Simmonds, * James Inglese, * Yuchun Du, * Caroline Demangel, * Stephen
c,
d,e,
a,n,
High, * Ville O. Paavilainen, * and Wei Q. Shi *
a
j
Department of Chemistry and Biochemistry, and Department of Biological Sciences, University of
Arkansas, Fayetteville, Arkansas 72701, USA
b
Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742,
United States
c
School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester,
Manchester, M13 9PT, United Kingdom.
d
e
University of Helsinki, HiLIFE, and Institute of Biotechnology, Helsinki, Finland
f
National Center for Advancing Translational Sciences, National Institutes of Health, Rockville,
Maryland 20850, USA.
g
h
Institut Pasteur, Immunobiology of Infection Unit, 75015 and INSERM, U1221, 75005 Paris, France
Department of Microbial Sciences, School of Biosciences and Medicine, University of Surrey, Guildford,
i
Surrey GU2 7XH, United Kingdom
k
Department of Radiology, Baylor College of Medicine, Houston, Texas 77030, USA
l
Emory Institute for Drug Development, Emory University, 954 Gatewood Road, Atlanta, Georgia 30329,
USA
m
Université de Haute-Alsace, Université de Strasbourg, CNRS, LIMA, UMR 7042, 68000 Mulhouse,
France
n
Department of Chemistry, Ball State University, Muncie, Indiana 47306, USA
†
#
*
Equal contribution as first-author
Equal contribution as second-author
Corresponding authors
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ABSTRACT
Ipomoeassin F is a potent natural cytotoxin that inhibits growth of many tumor cell lines with single-digit
nanomolar potency. However, its biological and pharmacological properties have remained largely
unexplored. Building upon our earlier achievements in total synthesis and medicinal chemistry, we used
chemical proteomics to identify Sec61α (protein transport protein Sec61 subunit alpha isoform 1), the pore-
forming subunit of the Sec61 protein translocon, as a direct binding partner of ipomoeassin F in living cells.
The interaction is specific and strong enough to survive lysis conditions, enabling a biotin-analogue of
ipomoeassin F to pull down Sec61α from live cells, yet it is also reversible as judged by several experiments
including fluorescent streptavidin staining, delayed competition in affinity pulldown, and inhibition of
TNF-biogenesis after washout. Sec61α forms the central subunit of the ER protein translocation complex,
and the binding of ipomoeassin F results in a substantial, yet selective, inhibition of protein translocation
in vitro and a broad ranging inhibition of protein secretion in live cells. Lastly, the unique resistance profile
demonstrated by specific single nucleotide point mutations in Sec61α provides compelling evidence that
Sec61α is the primary molecular target of ipomoeassin F and strongly suggests that the binding of this
natural product to Sec61α is distinctive. Therefore, ipomoeassin F represents the first plant-derived,
carbohydrate-based member of a novel structural class that offers new opportunities to explore Sec61α
function and to further investigate its potential as a therapeutic target for drug discovery.
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INTRODUCTION
Historically, natural products have significantly contributed to the development of drugs for human
1
2
disorders, most notably as anticancer chemotherapeutics. Structurally and functionally unique natural
products also provide a spectrum of valuable chemical tools for examining biological systems in
translational biomedical research.3-4 To continue our battle against various unsolved problems of human
health, including those arising from drug resistance, it is crucial to systematically investigate underexplored
areas of existing chemical space such as those offered by bioactive natural products with unique structures
and/or mechanisms.5
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Resin glycosides, also called glycoresins, are a large collection of amphiphilic glycolipids isolated from
the morning glory family of plants,6 and these compounds are considered active ingredients of many
morning glory-based traditional medicines that are used worldwide. Resin glycosides consist of a
differently-acylated oligosaccharide glycosylated with a mono- or dihydroxy C14 or C16 fatty acid, with
more than 300 family members discovered to date. Through an ester bond, the fatty acid chain is usually
folded back to form a macrolactone ring of various sizes spanning one or more carbohydrate units. Because
of their unique macrocyclic architecture with embedded carbohydrates, and their broad spectrum of
biological activities exhibited in phenotypic screens, resin glycosides have attracted considerable attention
from the synthetic chemistry community, but not much beyond.7-9
In 2005, a new family of glycoresins, ipomoeassins A–E, was isolated from the leaves of Ipomoea
squamosa found in the Suriname rainforest and shown to inhibit the proliferation of A2780 human ovarian
cancer cells.10 Among these compounds, ipomoeassin D (Figure 1) displayed the greatest potency with an
IC value of 35 nM. Two years later, a new member of the family, ipomoeassin F (Figure 1), was isolated
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and showed a cytotoxicity comparable to ipomoeassin D.11 Because of their promising antiproliferative
activity and unique molecular skeleton with carbohydrates being part of the macrocycle, these newly
discovered natural glycoconjugates quickly inspired synthetic chemists, including ourselves, to tackle their
total syntheses.12-16 Subsequently, ipomoeassin F was confirmed to be the most cytotoxic resin glycoside
discovered to date with single-digit nanomolar IC50 values against several cancer-derived cell lines.13
Intriguingly, ipomoeassins A and F (see Figure 1) have distinct cytotoxicity profiles, as revealed in the NCI
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0-cell lines screen, suggesting that the ipomoeassins may possess an unusual mode of action.16-17 On this
basis, ipomoeassin F is a very promising candidate for molecular probe and even chemotherapeutic
development; however, the absence of knowledge about the cellular targets of ipomoeassin F has
significantly impeded such efforts. To overcome this challenge, after improving our understanding of the
structure–activity relationship (SAR) of ipomoeassin F through medicinal chemistry studies,18-20 we
employed a chemical proteomics approach to identify its binding partner(s) in human cells. Here we
describe the evolution of our chemoproteomic studies that enabled the discovery of Sec61α (protein
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transport protein Sec61 subunit alpha isoform 1) as a primary molecular target of ipomoeassin F, and our
subsequent mechanistic investigations which validate this component as a key target mediating cytotoxicity.
To this end, we compared the activity of ipomoeassin F to mycolactone, a recently discovered broad-
spectrum inhibitor of Sec61-dependent translocation.21-22 This polyketide lactone is the pathogenic exotoxin
of Buruli ulcer disease (Mycobacterium ulcerans infection) that has well-established immunosuppressive
and cytotoxic activities.23-24 We define the inhibitory effects of ipomoeassin F on Sec61-mediated protein
translocation into and across the membrane of the endoplasmic reticulum (ER), corroborating our
contemporaneous observations of the potent inhibition of cellular protein secretion by ipomoeassin F.
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R
O
O
cinnamate
n
O
O
O
O
O
O
O
O
O
OH
HO
OAc
D-glucose D-fucose
tiglate
Ipomoeassin A: R = H, n = 1
Ipomoeassin D: R = OAc, n = 1
Ipomoeassin F: R = H, n = 3
Figure 1. Structures of ipomoeassins A, D, and F.
RESULTS AND DISCUSSION
Design, Synthesis and Proteomic Evaluation of Chemical Probes derived from Ipomoeassin F.
Michael acceptor systems are present in many electrophilic natural products, allowing the formation of
covalent adducts with bio-macromolecules, which are often responsible for their biological activities.25
Because of significant activity loss (up to 160-fold) after reducing one of the two double bonds in the α,β-
18
unsaturated esters to a single bond, we hypothesized that the cinnamate and/or the tiglate (Figure 1) may
enable irreversible binding between ipomoeassin F and its biological target(s). Therefore, we initially
designed and synthesized a potential activity-based probe26-28 3 (Scheme 1) by introducing a small
propargyloxy group to the para-position of the benzene ring.
Probe 3 was synthesized in two steps from key intermediate 1 obtained during the total synthesis of
ipomoeassin F.15 The alkyne-functionalized cinnamic acid 2 was synthesized as previously described.29
DCC-mediated Steglich esterification was applied to couple 1 with 2 in the presence of a catalytic amount
of DMAP to give the TBS-protected intermediate in 90% yield. Subsequently, exposure of the intermediate
to TBAF led to the removal of the TBS silyl protecting groups and delivered the final product 3 in good
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yield. As expected, the small bioorthogonal alkyne tag did not affect cytotoxicity and 3 was equally potent
as compared to the original natural product (Table 1).
Scheme 1. Synthesis of Ipomoeassin F Probes 3, 5 and 7
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O
a, b
4 or 6
OH
O
O
O
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O
+
O
O
O
HO
O
O
O
O
O
O
c
O
O
O
O
O
O
O
O
O
O
O
O
OTBS TBSO
OH
HO
OH
HO
OAc
OAc
O
OAc
R
O
N
N
N
2
1
3
5 or 7
O
O
S
NO2
O
HN
H
NH
H
HN
H
NH
H
O
O
O
O
O
N3
H
H
H
N
O
N3
N
O
N
O
S
O
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OMe
O
4
O
S
6
NO2
O
HN
H
NH
H
HN
H
NH
H
O
O
O
O
O
R =
H
R =
H
N
H
N
O
O
N
O
O
S
O
O
O
O
O
O
OMe
O
5 (94%)
7 (72%)
a
o
Reagents and conditions: a) 4-propargyloxycinnamic acid (2), DCC, DMAP, CH Cl , 0 C→RT,
2
2
overnight, 90%; b) TBAF, THF, –10 °C, 6 h, 80%; c) CuSO , sodium ascorbate, CH Cl /t-BuOH 3/2.
4
2
2
a
Table 1. Cytotoxicity (IC , nM) of Ipomoeassin F, Cotransin (CT8) and Probes 3, 5 and 7–15
5
0
Cell Line:
Ipomoeassin F
MDA-MB-231b
6.3
MCF7
36.4
‒
22.2
187.4
19.8
> 25,000
> 25,000
172.2
21.1
MCF-10A
HCT116 b,c
5.3
‒
9.6
21.7
5.1
‒
8
243
‒
‒
‒
2832
‒
‒
‒
‒
d
CT8
‒
6.3
29.5
3
5
7
8
9
7.0
> 25,000
> 25,000
8.9
‒
1
0
11.3
2.0
133.8
‒
11
12
2.8
253.4
172.2
1
8
18
1
1
4
5
980
429
2560
477
1874
480
18
18
‒
a
The data were obtained from at least two independent experiments, and the standard errors are within
b
c
d
2
0%; See Figure S1 for IC curves; Assayed separately; “‒” indicates “not determined”.
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With probe 3 in hand, we conducted activity-based protein profiling experiments for target
identification both in the soluble fraction of cell lysates made from MDA-MB-231 breast cancer cells and
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live MDA-MB-231 cells. Unfortunately, after in situ click chemistry with a fluorescent rhodamine azide
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S27 (see Supporting Information), we detected no differences in labeled proteins between negative control
lane 1), probe 3 (lane 2), and probe 3 plus ipomoeassin F (competition; lane 3) (Figure S2A), while an
(
unrelated probe used as a positive control for pulldown gave clear signals (Figure S2A, lanes 4 and 5). We
considered two possible explanations for this negative result: firstly, ipomoeassin F is not a covalent protein
modifier; secondly, target proteins of ipomoeassin F are in low abundance. Due to the dramatic loss in
activity with alkene-reduced compounds, we favored the second possibility.
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In order to enrich for relatively low abundance cellular proteins, we next synthesized a biotin-labeled
3
1
analogue 5 of ipomoeassin F by reacting the alkyne probe 3 with biotin azide 4 under chemo-selective
copper-catalyzed alkyne-azide coupling (CuAAC) conditions (Scheme 1).32 The synthesis was successful
in producing biotin probe 5 that remained cytotoxic (Table 1), showing only ~5-fold decrease. After
incubating 5 with either detergent solubilized cell lysate or intact cells, we used streptavidin beads to
recover the biotin probe 5-bound proteins followed by electrophoresis and silver staining of the captured
material. However, no proteins were selectively enriched by probe 5 (Figures S3A and S3C, lanes 1 to 3),
nor was any enrichment apparent when these samples were probed using a fluorophore-labeled streptavidin
(Figure S3B, lanes 1 to 3).
We hypothesized that if the target protein(s) of ipomoeassin F is of relatively low abundance, then
endogenously biotinylated proteins could outcompete, and thereby mask, any positive signal. To minimize
interference from endogenously biotinylated proteins, we synthesized a new biotin probe 7 (Scheme 1)
33
containing a photo-cleavable nitrobenzene moiety by coupling the alkyne analogue 3 with the biotin azide
fragment 6 containing an o-nitrobenzyl photolabile linker, the latter synthesized by modifying previously
reported procedures31-33 (see Supporting Information). Notably, the capacity of 7 to inhibit cancer cell
proliferation was close to that of parental ipomoeassin F (Table 1), despite the large size of the appendage
at the para-position of the cinnamate benzene ring. We then used probe 7 to label target proteins in live
MDA-MB-231 cells, performed a streptavidin pulldown on the resulting cell lysate and selectively released
bound proteins by UV photocleavage. When the resulting products were analyzed by SDS-PAGE, probe 7
strongly enriched a ~40 kDa protein and, to a lesser extent, a second protein of ~100 kDa (Figure 2A, lane
2, red arrows), both of which were absent from the negative control (Figure 2A, lane 1). In support of their
bona fide interaction with 7, these two proteins were lost when a 100-fold excess of unmodified
ipomoeassin F was included during the incubation (Figure 2A, lane 3). Because no proteins were selectively
recovered with probe 7 when it was incubated with detergent-solubilized (1% NP40 and 0.25% sodium
deoxycholate) extracts of MDA-MB-231 cells (Figure S3C, lanes 1, 4 and 5), we believe that the interaction
between ipomoeassin F and its target protein(s) may require a particular subcellular environment that is
detergent sensitive.
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A.
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B.
C.
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3
7
(M)
0.2 0.2
20
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3
0.2 0.2
7
(M)
Ipom-F (M)
0.2
0.2
10
10
7
(M)
Ipom-F (M)
hv
+
+
+
Ipom-F (M)
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100 kDa
Sec61
1
00 kDa
Input
AP
Actin
4
0 kDa
4
0 kDa
Sec61
Figure 2. SDS-PAGE images (silver staining) for affinity pulldown using probe 7 with (A) or without
(B) photo cleavage. Red arrows indicate specifically pulled down protein species; (C) Target validation
by western blot with a Sec61α antibody. Ipom-F, ipomoeassin F; AP, affinity pulldown.
We next investigated whether photocleavage is essential for the selective recovery of the two putative
target protein bands using probe 7. Strikingly, the ~40 kDa protein was also recovered without
photocleavage. It was substantially enriched by 7 when total streptavidin-bound products were analyzed,
and was lost in the presence of excess ipomoeassin F (Figure 2B, lanes 1–3). In contrast, an enrichment of
the ~100 kDa component under these conditions was not apparent (Figures 2A and 2B). Since
photocleavage was not essential for probe 7 to selectively recover the ~40 kDa protein, it is very likely that
the increased linker length between the biotin moiety and the ipomoeassin F region, but not its capacity for
selective cleavage, significantly improved the performance of probe 7 over probe 5. Our data also suggest
that, once formed, the complex between probe 7 and the ~40 kDa protein is tight and can survive detergent-
containing cell lysis. This is further supported by pulldown of the ~40 kDa target protein with low
concentrations of probe 7 (10 nM) that was out-competed by a 5-fold excess of ipomoeassin F (50 nM)
(Figure S4A, lanes 1–3). Fluorescent streptavidin staining suggested that a stable covalent adduct between
the ~40 kDa protein and probe 7 was not formed under our experimental conditions (Figure S3B, lanes 4
and 5). We next submitted both the ~40 kDa protein(s) (Figure 2B, lane 2) and the corresponding regions
of the gel for the negative control (Figure 2B, lane 1) and the ipomoeassin F competition sample (Figure
2
B, lane 3) for mass spectrometry analysis. When spectral counts were compared, Sec61α (protein transport
protein Sec61 subunit alpha isoform 1) stood out as the only protein that was both substantially enriched in
the pulldown with probe 7 and showed a corresponding reduction when excess ipomoeassin F was present
(Table S1). The selective recovery of Sec61α by probe 7 was further confirmed by Western blotting (Figure
2C, cf. signals between input and AP).
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To further support Sec61α as a target protein responsible for the biological activity of ipomoeassin F,
we synthesized two inactive reference compounds 8 and 9 (Figure 3 and Table 1) by removing both double
bonds in cinnamate and tiglate (see Supporting Information). As predicted, pulldown experiments with
inactive biotin probe 9 revealed a loss of the ~40 kDa protein, even with 500 nM of 9 (Figure S4B, lanes 1
and 4). Additionally, inactive analogue 8 was ineffective at competing with probe 7 binding, even when 8
was present in 50-fold excess (Figure S4B, cf. lanes 1 to 3). Taken together, these findings further support
the specificity of Sec61α as a primary cellular target of ipomoeassin F.
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R =
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Figure 3. Structures of two inactive derivatives of ipomoeassin F.
Cell Imaging Studies. The Sec61 protein translocon plays an essential role in translocating newly
synthesized membrane and secretory polypeptides into and across the membrane of the ER.34-35 Sec61α
forms the membrane conduit that nascent secretory polypeptides pass through. To examine the subcellular
localization of ipomoeassin F, we performed live cell imaging studies using three fluorescent analogues
10–12 (Figure 4) prepared from alkyne probe 3 using CuAAC (see Supporting Information). Both
coumarin-coupled probe 10 and NBD-coupled probe 11 retained substantial cytotoxicity with 11 being even
more potent than ipomoeassin F (see Table 1). Unfortunately, 11 underwent photobleaching very easily
and could not be used for imaging studies. In contrast, 10 gave a rather weak fluorescent signal, presumably
due to an internal photo-induced electron transfer effect combined with its shorter excitation maximum
(365 nm). To overcome these technical limitations, rhodamine analogue 12 was synthesized from the
rhodamine azide precursor S27. Although 12 is between 5- and 40-fold less potent than ipomoeassin F
depending on the cell type analyzed, it retains substantial cytotoxicity when compared to inactive analogues
8
and 9 (see Table 1), and we therefore used it for our imaging studies.
R =
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Figure 4. Structures of fluorescent derivatives of ipomoeassin F.
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Subsequently, we confirmed that 12 could penetrate the cell membrane in a concentration- and time-
dependent manner and gave a strong fluorescent signal. Hence, 12 stained cells within 30 minutes when
present at 2 μM, but took >3 h to give clear images when used at 20 nM. When cells labelled with 12 were
co-stained with markers for either the ER or the nucleus, it was apparent that 12 strongly labeled the ER
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(Figure 5), but not the nucleus (Figure S5A). To confirm the specificity of ER labelling by 12, competition
experiments were performed using ipomoeassin F or inactive compound 8. The fluorescence signal of 12
was almost completely abolished when the cells were preincubated for 30 minutes with a 100-fold excess
of free ipomoeassin F (Figure S5B), yet preincubation with compound 8 had little effect (Figure S5C). The
apparent ER localization of the cellular interacting partner(s) of ipomoeassin F strongly supports the notion
that its target is Sec61α.
12, 0.2 μM
ER Tracker
Merge
Figure 5. Cell imaging studies with fluorescent probe 12 in MDA-MB-231 cells. Rhodamine-conjugated
ipomoeassin F analogue 12 (0.2 μM) was added to cells and after 1 h, cells were imaged to analyze
localization of 12 relative to ER staining.
Pulldown from ER Microsomes. As evidenced by the negative results from our pulldown experiments
using cell lysates prepared with detergent, we propose that the membrane environment of the ER may be
required to maintain Sec61α in a conformation that can bind to ipomoeassin F effectively. In this regard,
it would be unlikely that purified Sec61α alone could recapitulate a direct interaction with the natural
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product. However, since the biological function of Sec61α is maintained in isolated ER microsomes, and
once pre-bound to probe 7 in intact cells the resulting complex appears stable to detergent treatment (Figures
2
A and 2B), we attempted to pulldown Sec61α from purified ER-derived microsomes. After incubation
with probe 7 and detergent solubilization (1% n-dodecyl-ß-D-maltoside) to release biotin-bound proteins
from the phospholipid bilayer, only a single protein was visible after pulldown (Figure S6A). The identity
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of this product was confirmed as Sec61α by western blotting (Figure S6B). This finding strongly supports
our previous conclusion made with live cells, namely that Sec61α directly interacts with ipomoeassin F.
Competition of Ipomoeassin F with Cotransin for Binding to Sec61α. To test whether ipomoeassin
F competes for binding to Sec61α with known Sec61 ligands, such as cotransin and mycolactone,36-38 we
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exploited CT7, a cotransin photoaffinity probe that can covalently label Sec61α. As reported earlier,
mycolactone efficiently competes with CT7 and prevents its photo-crosslinking to Sec61α (Figure 6). A
similarly potent competition was observed with ipomoeassin F, suggesting it binds directly to Sec61α,
potentially at the same or a partially overlapping interaction site as that used by cotransin. Ipomoeassin F
competes with CT7 photo-crosslinking with similar potency as mycolactone, consistent with its ability to
inhibit protein ER translocation with high potency.
Figure 6. Sheep rough microsomes were preincubated with ipomoeassin F (Ipom-F) or mycolactone (Myco)
at 10 μM. Subsequently 100 nM photocotransin CT7 was added and samples photolyzed. The covalent
CT7/Sec61α adduct was detected using click chemistry to install a rhodamine-azide reporter and in-gel
fluorescent scanning.
In vitro Inhibition of Protein Translocation by Ipomoeassin F. In order to understand the
consequences of ipomoeassin F binding for Sec61 function, we used a well-established in vitro system to
study the Sec61-dependent translocation of radiolabeled precursor proteins into ER microsomes derived
from canine pancreas.22-23 In the presence of 1 µM ipomoeassin F, the translocation of two model secretory
proteins, bovine preprolactin and yeast prepro-alpha-factor, was almost completely prevented. The
blockade of protein translocation by ipomoeassin F was comparable to that achieved using 1 µM
mycolactone, and resulted in a loss of signal sequence cleavage and protein N-glycosylation (see Figures
7
A and 7B; cf. lanes 1, 3 and 4), both events specific to the ER lumen. The unusual ability of tail-anchored
membrane proteins to insert into the ER membrane independently of the Sec61 complex provides a useful
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control for validating the specificity of ER translocation inhibitors. The in vitro integration of two model
tail-anchored proteins bearing C-terminal N-glycosylation reporters was unaffected by ipomoeassin F
treatment (Figures 7C and 7D, cf. lanes 1 and 3), mirroring the specificity of the Sec61-selective inhibitor
mycolactone21-22 (see also Figures 7C and 7D, lane 4). Furthermore, despite the presence of carbohydrate
moieties in ipomoeassin F (see Figure 1), it has no effect on the N-glycosylation of our model tail-anchored
proteins, and we conclude that protein N-glycosylation of model substrates provides a robust reporter for
the ipomoeassin F-mediated inhibition of Sec61-dependent protein translocation.
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Figure 7. Ipomoeassin F inhibits Sec61-mediated protein translocation into and across the ER in a substrate
selective manner. Phosphorimages of membrane associated in vitro products resolved by SDS-PAGE
together with outline structures of the protein substrates generated in the presence or absence of
ipomoeassin F (Ipom-F) or mycolactone (Myco): secretory proteins (A) bovine preprolactin, PPL and (B)
yeast prepro-alpha-factor ppF; tail-anchored proteins (C) C-terminally tagged Sec61 subunit,
Sec61OPG2 and (D) C-terminally tagged cytochrome b5, Cytb5OPG2; type I single pass transmembrane
protein (TMP) (E) vascular cell adhesion molecule 1, VCAM1; type II single pass TMP (F) isoform 2
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(residues 17 to 232) of the short form of HLA class II histocompatibility antigen gamma chain, Ii; type III
single pass TMP (G) glycophorin C, GypC; short secretory protein (H) C-terminally tagged Hyalophora
cecropia preprocecropin A, ppcecAOPG2. Samples were treated with endoglycosidase H (Endo H) where
indicated to distinguish N-glycosylated (XGly) from non-glycosylated (0Gly) products. nc, signal sequence
not cleaved; sc, signal sequence cleaved; TM1, transmembrane domain 1. Proteins are of human origin
unless otherwise stated.
Known small molecule inhibitors of Sec61-mediated protein translocation show varying degrees of
substrate selectivity,23,41 and we therefore studied the effects of ipomoeassin F on the Sec61-mediated
translocation of four other classes of precursor proteins (see Figure S7) into and across the ER membrane
using the in vitro system outlined above.22,42 We find that ipomoeassin F potently inhibits the membrane
insertion of representative type I and type II integral membrane proteins that have a single transmembrane
spanning domain (Figures 7E and 7F). In contrast, the compound has no effect on the membrane insertion
of the single-spanning type III integral membrane protein glycophorin C and only a marginal impact on the
translocation of the short secretory protein prepro-cecropin A (Figures 7G and 7H). Interestingly, it has
recently been shown that type III membrane proteins have the unusual ability to exploit an alternative,
Sec61-independent, mechanism for ER insertion, which relies on the ER membrane protein complex acting
4
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as an integrase. On this basis, we conclude that the integration of glycophorin C is completely refractive
to ipomoeassin F (this study) and mycolactone (See Ref. 42) because it can insert into the ER via a Sec61-
independent mechanism. Based on the model membrane and secretory proteins that we have studied here,
the substrate selectivity for the ipomoeassin F-mediated inhibition of ER protein translocation appears
remarkably similar to that of mycolactone.42
To further investigate ipomoeassin F specificity, we used the ER insertion of the type II membrane
protein Ii (HLA class II histocompatibility antigen gamma chain; cf. Figure 7F) to compare the
effectiveness of a subset of structural variants. The differences we observe in this in vitro assay (see Figure
S8) are strikingly similar to the variations in cytotoxicity that the same compounds display in cell-based
assays (Table 1). Hence, ipomoeassin F and the open-chain analogue 1318 (Figure 8) strongly inhibit
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membrane insertion, whilst 14 (Figure 8) is a far less effective inhibitor and analogue 8 (Figure 3) has no
measurable inhibitory activity in vitro (see Figures S8A and S8B). Ii membrane insertion was also used to
estimate IC values for ipomoeassin F (~50 nM) and 13 (~120 nM) (see Figures S8C and S8D). The fact
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that we observe IC50 values in the nM range using in vitro translocation inhibition and cell-based
cytotoxicity assays, combined with the similarity in the structure-activity relationship observed in both
systems, leads us to conclude that the inhibitory effect of ipomoeassin F on Sec61-mediated protein
translocation is most likely the principal molecular basis for its cytotoxic activity. Furthermore, since the
ER acts as the entry point into the eukaryotic secretory pathway, our in vitro findings predict that if cells
are exposed to ipomoeassin F their capacity to produce and deploy membrane and/or secretory proteins is
likely compromised.
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HO
OH
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OAc
OAc
OAc
13
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Figure 8. Structures of ipomoeassin F analogues 13–15.
Inhibition of Protein Secretion by Ipomoeassin F in Live Cells. Through a partnership with the
National Center for Advancing Translational Sciences (NCATS), ipomoeassin F was included within a
collection of natural products for screening against a panel of assays to interrogate a variety of biological
pathways and assess cell type-dependent toxicity.44 Ipomoeassin F was identified as an inhibitor of protein
secretion in two distinct quantitative high-throughput screening (qHTS) assays using different cell lines and
secretory reporters.45 In U2-OS human osteosarcoma cells, a protein reporter consisting of secreted
NanoLuc (secNLuc) fused to the Z mutant of alpha-1 antitrypsin (secNLuc-ATZ) was designed to monitor
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the extracellular accumulation of this protein. Ipomoeassin F demonstrated acute, highly potent secretion
inhibition (EC = ~5 nM) in U2-OS cells prior to the onset of cytotoxicity as assessed by decreasing cellular
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ATP after 24 h (Figure 9A). A second reporter of protein secretion was also examined, using SH-SY5Y
human neuroblastoma cells expressing a secreted Gaussia luciferase (GLuc). Ipomoeassin F inhibited
secretion of the reporter protein with a lower potency (EC = ~120 nM) than in U2-OS cells (Figure 9B)
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reflecting a potential cell type-dependent functional effect. The secretion phenotype in cells treated with
ipomoeassin F is strongly consistent with Sec61α as its inhibitory protein target, since most secretory
proteins must pass through the Sec61 translocon into the ER prior to traversing the secretory pathway for
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extracellular secretion. The difference in potency between different cell lines is, again, in line with previous
observations with mycolactone.47-48
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Figure 9. Differential effect of acute ipomoeassin F treatment on protein secretion and cell viability.
Bioluminescence outputs from secretion sensor proteins ()and cellular viability based on total well ATP
content ( ). (A) secNLuc-ATZ secretion and cell viability measured in U2-OS cells after 24 h. (B) GLuc

secretion and cell viability measured in SH-SY5Y cells after 48 h. Error bars represent the SEM for n=2
or 3 replicates.
Selective Prevention of Biogenesis of Secreted Proteins by Ipomoeassin F. To assess ipomoeassin
F effects on cellular protein production, we employed a metabolic labeling strategy. Cells were pretreated
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with ipomoeassin F or its analogue 8 (Figure 3), 14 or 15 (Figure 8), for 30 minutes before addition of S-
labelled methionine/cysteine for further 90 minutes. The lack of inhibition of nascent protein biogenesis
observed in total cellular lysates and cytosolic fractions (Figures 10A and 10B) demonstrates that
ipomoeassin F does not acutely influence global cellular processes such as transcription or translation, and
is in line with previous observations using mycolactone.2 In contrast, strong inhibition was observed for
the production of secreted proteins for cells treated with 100 nM ipomoeassin F (Figure 10C). Importantly,
ipomoeassin F inhibits the production of most newly synthesized secretory proteins, suggesting its
mechanism of action leads to an inhibition of the biogenesis of a broad range of Sec61 substrate proteins
that is comparable to known Sec61 inhibitors such as mycolactone21,36 and apratoxin A38,50. Ipomoeassin F
analogues 8, 14, and 15 all inhibit the production of secreted proteins less potently when tested at 1 µM
concentration. Furthermore, the rank order of potencies with which ipomoeassin F and its analogues inhibit
protein secretion (Ipom-F > 15 > 14 > 8) correlates with their observed cytotoxicity against HCT-116 cells
1,49
(cf. Table 1 and Figure S1C), suggesting a link between cytotoxicity and inhibition of cellular protein
secretion, as previously suggested for other Sec61 inhibitors.36,38,51-52
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Figure 10. HCT-116 cells were washed twice with PBS and incubated in the presence of indicated
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concentrations of compounds under media lacking methionine and cysteine for 30 minutes. S-labelled
methionine and cysteine were then introduced to the media, and cells incubated for a further 90 minutes.
Cells were harvested by scraping into ice cold PBS, homogenized and analyzed by SDS-PAGE and
autoradiography. (A). The collected cells were subsequently homogenized and analyzed by SDS-PAGE
and autoradiography; (B) As for (A) but the samples are the cytosolic contents of harvested cells following
partial permeabilization with 0.15% Digitonin; (C) As for (A) but the samples are from TCA-precipitated
culture medium from the same experiment. Ipom-F denotes ipomoeassin F. AprA denotes control samples
treated with Apratoxin A to block protein translocation into the ER. CHX denotes samples treated with
cycloheximide and chloramphenicol to inhibit total cellular protein synthesis. Cotransin CT8 is a substrate-
selective inhibitor of protein translocation into the ER.
HepG2 cells secrete a range of endogenous proteins, but these products are almost completely absent
following treatment with the ER translocation inhibitor eeyarestatin I or the vesicular transport inhibitor
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brefeldin A. To extend our studies with HCT116 cells (Figure 10), we therefore treated HepG2 cells with
ipomoeassin F or mycolactone overnight and then followed the fate of newly synthesized proteins by pulse
chase analysis. When the cell media was analyzed for secretory proteins, a substantial reduction was
observed for cells treated with ipomoeassin F or mycolactone when compared to control cells treated with
DMSO or compound 8 (see Figure S9, media, lanes 1 to 4). Whilst the effect of these Sec61 inhibitors was
akin to that of brefeldin A, treatment with the proteasome inhibitor bortezomib had no effect (see Figure
S9, media, lanes 3 to 6). When the levels of total radiolabeled proteins recovered from the same cells were
analyzed, they were directly comparable across all treatments confirming that ipomoeassin F did not inhibit
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global protein synthesis during the treatment used (Figure S9, whole cell extract, cf. lanes 1 to 4). In fact,
overnight exposure to ipomoeassin F increases at least one prominent cellular component (Figure S9, whole
cell extract, lane 3, 80 kDa), perhaps reflecting the early stages of a cellular stress response.51,53
Immunomodulatory Effects of Ipomoeassin F. While ultimately cytotoxic in most cell types,
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mycolactone has immediate immunosuppressive effects. Recent studies have indicated that these effects
are a direct consequence of mycolactone’s inhibitory activity on Sec6121,36,53-54, and we find the effects of
ipomoeassin F and mycolactone are equivalent in cell-free assays of Sec61-dependent protein translocation
(Figure 7). By blocking Sec61, mycolactone prevents the production of both secreted proteins and
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membrane receptors that are key to the generation of immune responses , as is clearly illustrated by the
biogenesis of CD62L, a cell surface receptor mediating naive lymphocyte homing to secondary lymphoid
organs, which is highly susceptible to mycolactone.36 In the human Jurkat T cell line, we find that
ipomoeassin F and mycolactone downregulated CD62L expression with comparable potency (Figure 11A).
Likewise, they were also equivalent in capacity to prevent Interleukin (IL)-2 production by activated T cells
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Figure 11B). Together, these data suggest that, like mycolactone, ipomoeassin F may display systemic
immunosuppressive effects in vivo.
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Figure 11. Compared effects of Ipomoeassin F and synthetic mycolactone (A/B form) in assays of CD62L
expression (A) and activation-induced production of IL-2 (B) by Jurkat T cells. Data are mean +/- SEM of
biological duplicates and are representative of two independent experiments.
Resistance of Sec61α Mutants to Ipomoeassin F Cytotoxicity. Having established that binding of
ipomoeassin F to Sec61α potently impairs protein translocation at the ER both in vitro and in vivo, we
sought further evidence that this interaction also underlies the cytotoxic effects of ipomoeassin F (cf. Table
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Chemogenetic screening has identified mutations in the SEC61A1 gene that confer resistance to known
inhibitors of Sec61α-dependent protein translocation,56 and established that these mutations can confer
resistance when engineered into a naïve cell background.37-38 We therefore compared the effects of
ipomoeassin F and mycolactone on the growth of HEK293 and HCT116 cells expressing either wild type
Sec61α, or a previously characterized point mutant of Sec61α that is resistant to mycolactone.36,41 As
previously observed using mycolactone in HEK293 cells engineered to express mutant alleles of
Sec61α,36,41 point mutations R66I and S82P confer significant and dominant desensitization to ipomoeassin
F in a viability assay of HEK293 cells, yet T86M confers resistance to mycolactone but not to ipomoeassin
F (see Figures 12A and 12B). Furthermore, whilst isolated drug-resistant HCT116 cells carrying mutations
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to Sec61α of R66K and D60G were strongly resistant to mycolactone (Figure 12D) as previously reported ,
any decrease in sensitivity to ipomoeassin F was comparatively modest (Figure 12C). Taken together, these
results strongly suggest that ipomoeassin F and mycolactone bind to a similar region as other known
inhibitors of Sec61, but indicate that the interaction of ipomoeassin F may be partially dependent upon the
basic side chain of residue R66 but does not require conservation of D60 or T86.
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Figure 12. Human HEK293 cells or cells engineered to express Sec61α alleles bearing indicated point
mutations were treated with increasing concentrations of ipomoeassin F (A) or mycolactone (B), and cell
viability assayed at 72h with the Alamar Blue assay. (Mean ± SD; n = 4 technical quadruplicates). Parental
or HCT116 cells containing mutations in Sec61A1 were seeded at 3 x 104 /well in 96-well plates in triplicate.
ipomoeassin F (C) or mycolactone (D) were added at various concentrations with the carrier DMSO used
as a control. Cell viability was measured at 96h with the Alamar Blue assay (Mean ± SEM of n = 3
independent experiments performed in triplicate).
Strong yet Reversible Binding of Ipomoeassin F to Sec61α. Although our SAR studies suggested
that a covalent adduct may be formed between ipomoeassin F and Sec61α, no such adduct could be detected
by fluorescent streptavidin staining of a Western blot. In order to further address the nature of the interaction
between ipomoeassin F and Sec61α, we designed a delayed competition experiment by adding 100-fold
excess of ipomoeassin F only after a preincubation with biotin probe 7 (lane 4 in Figures 13A and 13B).
Our cytotoxicity data show that probe 7 closely resembles ipomoeassin F, and we reasoned that if a stable
covalent complex was formed between 7 and Sec61α, the later addition of ipomoeassin F would not
significantly decrease the signal of Sec61α in an affinity pulldown. When this experiment was carried out,
we observed a time dependent decrease in the probe 7-dependent recovery of Sec61α in the presence of
excess ipomoeassin F, such that after 60 minutes, the signal was substantially reduced (Figures 13A and
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3B, cf. lanes 2 and 4; see also Supporting information, Figure S10, for complete gel images).
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Independently, the reversibility of ipomoeassin F activity was also investigated using a cellular assay.
This measured Sec61-dependent production of TNF by LPS-stimulated macrophage-like RAW264.7 cells.
Mycolactone’s ability to completely block TNF production directly after a 1 h pre-incubation (Figure 13C,
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white bars) was previously shown not to be restored following a 24 h washout and this was confirmed
here (Figure 13C, purple bars). In contrast, while ipomoeassin F similarly blocked TNF production in the
direct assay, after extensive washing and 24 h recovery, LPS-dependent TNF production was restored,
albeit at a reduced level. Therefore, in contrast to mycolactone, the effects of ipomoeassin F on cells are, at
least partially, reversible under these conditions.
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On the basis of these results, we conclude that the binding of ipomoeassin F to Sec61α is strong yet
apparently reversible, suggesting that it does not involve a stable covalent interaction.
Figure 13. SDS-PAGE images (silver staining) for affinity pulldown using probe 7 with 0.5 h (A) or 1 h
(B) competition in both regular (lane 3) and reverse (lane 4) order. Green arrows indicate the protein band
for Sec61α; (C) TNF abundance in RAW264.7 cell supernatants 4 h after stimulation with 100 ng/ml LPS
in the presence of medium alone (ctrl), DMSO, 125 ng/ml mycolactone (Myco) or 250 nM ipomoeassin F
(Ipom-F). Cells were either pre-incubated for 1 h prior to addition of LPS (‘direct’) or incubated for 1 h,
washed and allowed to recover for 24 h in complete medium before addition of LPS (‘24 h washout’). TNF
was quantified by ELISA in triplicate, and the dashed line shows detection limit of the assay. Controls for
each condition without LPS stimulation did not reach this detectability threshold. For each replicate, data
were normalized to control levels and the mean of n=2 independent cellular assays are presented.
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CONCLUSIONS
Here, we report comprehensive chemical biology studies of ipomoeassin F to understand the molecular
basis for its potent cytotoxicity. By designing and evolving our chemical probes on an empirical basis,
Sec61α was ultimately identified and validated as a direct and physiologically relevant target of
ipomoeassin F. The results from fluorescent streptavidin staining, delayed competition in affinity pulldown,
and TNF-production washout experiments indicate that ipomoeassin F is unlikely to form a stable covalent
complex with Sec61α, despite the presence of two Michael acceptor systems in its structure (cinnamate and
tiglate, Figure 1). We therefore conclude that the pulldown of Sec61α with probe 7 represents a successful
biotin affinity enrichment of a reversible ligand-membrane protein complex formed in live cells, most likely
reflecting a slow disassociation rate of ipomoeassin F from Sec61α. The binding of ipomoeassin F to
cellular Sec61α is supported by the ER staining seen with a fluorescent version of the compound, the highly
selective pulldown of Sec61α from ER-derived microsomes and the ability of ipomoeassin F to compete
with cotransin for Sec61α binding.
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The impact of ipomoeassin F on Sec61 function was first investigated in vitro, revealing a strong
inhibition of protein translocation into the ER that affected secretory, type I and type II membrane proteins.
The in vitro effect of ipomoeassin F translated into potent secretion inhibition in two cellular models
utilizing independent luciferase secretory reporters. Likewise, ipomoeassin F treatment downregulated the
release of a broad range of endogenous secretory proteins from cultured cells, acting at a stage after their
translation. At this point in our studies, it was striking that, although structurally quite distinct, the molecular
mechanisms of ipomoeassin F, and the functional consequences of its application, closely resemble those
seen with the immunosuppressive macrolide mycolactone that is released by the human pathogen
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Mycobacterium ulcerans. We therefore studied the effects of ipomoeassin F on immune cells and found
that its inhibition of membrane receptor expression and cytokine production is comparable to that achieved
with mycolactone, Previous studies have illustrated the translational potential of mycolactone against
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inflammatory disorders , but further exploitation of mycolactone-inspired molecules has been limited by
their intrinsic structural complexity that required extensive synthetic efforts.57-62 Given that ipomoeassin
variants are easier to generate by synthetic chemistry and equally inhibitory in cellular assays of
inflammation, future studies of their pharmacokinetics and therapeutic efficacy against inflammatory
diseases and inflammatory pain are an obvious area to pursue in future.
Lastly, we could establish an excellent correlation between the functional Sec61 inhibition and the
cytotoxic potency of ipomoeassin F using cell lines expressing inhibitor-resistant mutants of Sec61α mutant.
The loss of cytotoxicity we observed in HEK293 cells expressing R66I and S82P mutants is consistent with
the notion that ipomoeassin F binds to a location that overlaps with the interaction site for other small
molecule inhibitors of Sec61α, most likely at or near its so-called lateral gate.41 However, differences
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between ipomoeassin F and mycolactone in response to other mutant cell lines strongly suggest that the
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mode of ipomoeassin F binding to Sec61α is distinctive from other known Sec61α inhibitors . The
sustained sensitivity of the mycolactone resistant T86M (in HEK293) and D60G (in HCT116) mutant cell
lines to ipomoeassin F that we report is particularly intriguing, highlighting that ER protein translocation
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can be complex and our current understanding is far from complete.
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In short, we provide compelling evidence that the core α subunit of the Sec61 translocation complex is
the major target for ipomoeassin F in a cellular context, thereby opening up the way for the exploitation of
ipomoeassin F for target-based drug discovery. More broadly, given its comparatively advantageous
synthesis, we anticipate that ipomoeassin F and its analogues/probes will provide accessible new molecular
tools that will help our efforts to fully define the molecular basis for Sec61-mediated protein translocation
at the ER membrane.
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ASSOCIATED CONTENT
Supporting Information. Experimental procedures and analytical data for all the new compounds and
biological assays. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
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Corresponding Author:
*
E-mail: rachel.simmonds@surrey.ac.uk; jinglese@mail.nih.gov;
ydu@uark.edu; demangel@pasteur.fr; stephen.high@manchester.ac.uk; ville.paavilainen@helsinki.fi;
weishi@uark.edu, shiwei43@gmail.com.
Author Contributions: G.Z. and J.Z. contributed equally as first-author. S.O’K, D.T., M.J.I., L.B., B.H.,
K.C., A.O.P., M.J.H. and P.R. contributed equally as second-author.
Notes: The authors report no conflicts of interest.
ACKNOWLEDGMENTS
We thank the Arkansas Nano & Bio Materials Characterization Facility at the Institute for Nano Sciences
&
Engineering for our imaging studies, and Prof Yoshito Kishi (Harvard University) for the kind gift of
synthetic mycolactone A/B used by S.H. and R.S. W.S. is supported by Grant Number R15GM116032 from
the National Institute of General Medical Sciences of the National Institutes of Health (NIH) and startup
funds from the University of Arkansas. This work was also supported in part by Grant Number P30
GM103450 from the National Institute of General Medical Sciences of the National Institutes of Health
(NIH) and by seed money from the Arkansas Biosciences Institute (ABI). S.O’K. is the recipient of a
Biotechnology and Biological Sciences Research Council (BBSRC) Doctoral Training Programme Award
BB/J014478/1 and S.H. holds a Welcome Trust Investigator Award in Science 204957/Z/16/Z. The alpha-1
antitrypsin work was supported by the Alpha-1 Foundation (J.I. and M.J.I.). J.I. and M.J.H were supported
by the intramural program of NCATS, National Institutes of Health, projects 1ZIATR000048-03 (JI) and
ZIATR000063-04 (MJH). RS holds Welcome Trust Investigator Award in Science 202843/Z/16/Z. CD
received funding from the Institut Pasteur, the Institut National de la Santé et de la Recherche Médicale and
the Fondation Raoul Follereau. NB’s synthesis and chemical biology studies of mycolactone were
supported by CNRS, Université de Strasbourg, Fondations Potier et Follereau and the Investissement
d’Avenir (Idex Unistra). V.O.P is supported by the Academy of Finland (grants 289737 and 314672), and
the Sigrid Juselius Foundation.
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