Iriomoteolides: Novel chemical tools to study actin dynamics

Article abstract of DOI:10.1039/c7sc04286h

Despite its promising biological profile, the cellular targets of iriomoteolide-3a, a novel 15-membered macrolide isolated from Amphidinium sp., have remained unknown. A small library of non-natural iriomoteolide-3a analogues is presented here as a result of a novel, highly convergent, catalysis-based scaffold-diversification campaign, which revealed the suitable sites for chemical editing in the original core. We provide compelling experimental evidence for actin as one of iriomoteolides' primary cellular targets, establishing the ability of these secondary metabolites to inhibit cell migration, induce severe morphological changes in cells and cause a reversible cytoplasmic retraction and reduction of F-actin fibers in a time and dose dependent manner. These results are interpreted in light of the ability of iriomoteolides to stabilize F-actin filaments. Molecular dynamics simulations provide evidence for iriomoteolide-3a binding to the barbed end of G-actin. These results showcase iriomoteolides as novel and easily tunable chemical probes for the in vitro study of actin dynamics in the context of cell motility processes including cell invasion and division.

Full text of DOI:10.1039/c7sc04286h

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Iriomoteolides: novel chemical tools to study actin
dynamics†
Cite this: DOI: 10.1039/c7sc04286h
a
a
a
b
a
b
*
A. Unzue,‡ R. Cribiu,§ M. M. Hoffman, T. Knehans,‡ K. Lafleur,{ A. Caflisch
´
a
*
and C. Nevado
Despite its promising biological profile, the cellular targets of iriomoteolide-3a, a novel 15-membered
macrolide isolated from Amphidinium sp., have remained unknown. A small library of non-natural
iriomoteolide-3a analogues is presented here as a result of a novel, highly convergent, catalysis-based
scaffold-diversification campaign, which revealed the suitable sites for chemical editing in the original
core. We provide compelling experimental evidence for actin as one of iriomoteolides' primary cellular
targets, establishing the ability of these secondary metabolites to inhibit cell migration, induce severe
morphological changes in cells and cause a reversible cytoplasmic retraction and reduction of F-actin
fibers in a time and dose dependent manner. These results are interpreted in light of the ability of
iriomoteolides to stabilize F-actin filaments. Molecular dynamics simulations provide evidence for
iriomoteolide-3a binding to the barbed end of G-actin. These results showcase iriomoteolides as novel
and easily tunable chemical probes for the in vitro study of actin dynamics in the context of cell motility
processes including cell invasion and division.
Received 2nd October 2017
Accepted 10th March 2018
DOI: 10.1039/c7sc04286h
rsc.li/chemical-science
cells was recognized early on.2 These results are in line with
those reported for amphidinolides, polyketides also isolated
Introduction
from Amphidinium species, most of which exhibit respectable
levels of cytotoxicity against standard murine and/or human
tumor cell lines in vitro, in some cases with potencies that rival
those of renowned anticancer agents. As it is typical for
secondary metabolites with high level of molecular complexity,
additional biological evaluation is hampered by the low yield in
which they can be isolated from the natural source (0.015% of
the wet weight of the dinoagelate in the case of 1). Thus, in
depth studies to unravel both the mode of action and primary
cellular targets of these molecules must rely on chemical
synthetic efforts. Both the challenging structural features and
the potential of these natural products as biological probes have
brought amphidinolides3–10 and iriomoteolides11–16 to the fore-
front of synthetic efforts in recent years. Our group reported the
rst total synthesis of iriomoteolide-3a (1), which enabled the
conrmation of its proposed structure.17 However, even if
access to the desired compound was granted, our initial strategy
fell short to provide signicant amounts of this secondary
metabolite and derivatives thereof for further evaluation
(Scheme 1A).
Screening of free-swimming dinoagellates Amphidinium sp. via
genetic and metabolomic analyses enabled the identication of
the HYA024 strain as a source of unknown bioactive metabo-
lites.1 Three new 20-membered ring cytotoxic lactones,
iriomoteolides-1a-c, could be isolated together with a novel
vinyl epoxide containing 15-membered ring macrolide named
iriomoteolide-3a (1) and its 7,8-isopropylidene analogue (2).2
The carbon chain length as well as the C1- and oxygen distri-
bution observed in 1 differ from that of most macrolides which
possess an even-numbered lactone ring and a C1–Me/C2–O
pattern as a result of the polyketide synthase machinery. The
unique pattern found in 1 thus hints towards a non-successive
incorporation of acetates or propionates in the assembly of the
polyketide chain (Scheme 1, top le).
The ability of iriomoteolides to impair cell growth on human
B lymphocyte DG-75 and Epstein–Barr virus positive (EBV+) Raji
a
¨
Department of Chemistry, University of Zurich, Winterthurerstrasse 190, CH-8057,
¨
Zurich, Switzerland. E-mail: cristina.nevado@chem.uzh.ch
b
¨
Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057,
Herein we present a new synthetic approach towards
iriomoteolide-3a which has enabled a scaffold-diversication
campaign and solved the supply problem (Scheme 1B). The
suitable sites for chemical edition of the original core have been
identied and the impact of conformation in the bioactivity of
these molecules has been proved.18 In addition, based on
a varied array of cell-based and in vitro studies, we demonstrate
¨
Zurich, Switzerland. E-mail: caisch@bioc.uzh.ch
† Electronic supplementary information (ESI) available: Details for all biological
assays as well as experimental and computational procedures and spectroscopic
data. See DOI: 10.1039/c7sc04286h
‡ Present Addresses: Discovery and Development Technologies, Merck KGaA,
Frankfurter Str. 250, 64293 Darmstadt, Germany.
§ Present Address: Biosynth AG, Sankt Gallen, Switzerland.
{ Present Address: Novartis, Fabrikstrasse 2, 4056 Basel, Switzerland.
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Scheme 1 Iriomoteolide-3a: key fragments (top) and different strategies for final assembly (bottom). (A) Original route via esterification/CM-
RCM: (a) EDC$HCl, 4-pyrrolidinopyridine, CH2Cl2, 25 ꢀC, 72%; (b) 5d (5 eq.), Grubbs II 11 (5 mol%), toluene, 25 to 50 ꢀC; (c) TBSOTf, 2,6-lutidine,
THF, 0 ꢀC, 80%; (d) Grubbs II 11 (12 mol%), toluene, 25 ꢀC, 76%. (B) Optimized route via CM/esterification/RCM: (e) Grubbs II 11 (5 mol%), toluene,
50 ꢀC, 82%; (f) 2,6-lutidine, TBSOTf, CH2Cl2 followed by EtOAc/MeOH/water, Na2CO3, 78%; (g) 3, 4-pyrrolidinopyridine, EDC$HCl, CH2Cl2, 84%;
(h) Grubbs II 11 (5 mol%), toluene, 25 ꢀC, 81%; (i) NH4F, MeOH, 58%; (j) DMP, CH2Cl2, then 6, K[N(SiMe3)2], THF, 0 ꢀC, 93 : 7 E/Z, 76%; (k) TBAF, THF,
86%. (l) 2,3-Dimethoxypropane, PPTS, CH2Cl2, 25 ꢀC, 20%. EDC ¼ 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. DMP ¼ Dess–Martin
periodinane.
that actin is one of the primary cellular receptors for sequence involving an asymmetric dihydroxylation, and in situ
iriomoteolide-3a and derivatives thereof. This study provides lactonization followed by functional group interconversion to
a comprehensive characterization of the mode of action of these produce fragment 3. The southern portion was produced in 6
metabolites as actin reversible stabilizers. The iriomoteolides steps from 3-(benzyloxy)propanal 823 via catalytic asymmetric
therefore constitute a new class of chemical probes towards the cyclocondensation with acetyl bromide24,25 followed by treatment
reversible modulation of actin dynamics beyond their potential with dimethyl cuprate to install the key C3–Me stereocenter in
relevance as cytotoxic targets.
fragment 4. L-Tartaric acid was used as precursor for the 1,3-
dienes 5a–d following previously reported procedures.26,27
Finally, sulfone 6 was prepared from (E)-5-bromo-2-pentene 928
in only two steps via halogen displacement and oxidation.29 The
original approach to assemble the macrocycle (Scheme 1A)
started with an intermolecular esterication between the
secondary alcohol in 3 and acid 4 using EDC as activating agent
in the presence of 4-pyrrolidinopyridine to produce ester 10 in
72% yield. To complete the macrolide we were inspired by the
C2-symmetric nature of the C5,C10-portion of the molecule. A
“capping” strategy via a highly E,E-stereoselective cross metath-
esis (CM)/-ring closing metathesis (RCM) sequence between 10
Results and discussion
Strategic considerations and new synthetic approach
Iriomoteolide-3a (1) was divided into four key fragments of
comparable complexity (3–6),17 which were prepared from
readily available starting materials (Scheme 1, top right).19 The
north skeleton of the molecule, containing the sensitive vinyl
epoxide moiety and the three additional stereocenters, was
assembled from Evans oxazolidinone20 and (E)-tert-butyl((5-
iodopent-3-en-1-yl)oxy)diphenylsilane (7)21,22 in
a 14 steps
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and C2-symmetric fragment 5 using Grubbs 2nd generation
catalyst 1130 was sought.31 While the high reactivity of diol 5a and
acetonide 5b resulted in complex reaction mixtures, bis-silylated
olen 5c delivered a contracted 11-membered ring lactone
together with self-immolative CM products as a result of the
steric hindrance imposed by the two bulky silyl groups anking
the double bonds in 5c (data not shown).27,32 We envisioned that
the TBS group in 5d could prevent the coordination of the Ru-
catalyst (11) and thus the CM reaction on its neighbouring
double bond. As expected, the cross-metathesis between ester 10
and mono-TBS protected diol (5d) afforded a mixture of
regioisomers in a ca. 1 : 1 ratio but, unfortunately, the RCM of
this mixture of regioisomers did not afford the desired products
but rather truncated lactones and dimeric 5d signalling the lack
of reactivity of the olen vicinal to the silyl group even in the case
of a RCM event.33–35 Fortunately, aer per-silylation of the
mixture, a productive RCM was observed to give lactone 12 as
a single isomer in 76% overall yield. The usefulness of Grubbs-
type catalysts for the assembly of molecular complexity in chal-
lenging settings is once again portrayed here.36–40
Preparation of a focused library of iriomoteolide analogues
Next, a structural diversication campaign18,43,44 was designed
to map the macrocyclic framework of the natural lead by
straightforward modications of the 3–6 subunits (Scheme 2).
Our rst goal was to explore the conformational landscape of
the macrolide, as conformation is known to play a key role in
the biological activity of drugs entitling medium or large ring
sizes.19,45–48 The southern portion of the molecule, in particular
the substitution pattern at C3 was studied rst. This goal
seemed easy to attain as it would only require the replacement
of a single building block (i.e. 4) in the entire sequence. Inter-
estingly, we noticed that the 2JH2a/H2b and 3JH2a/H3 determined
in our synthetic sample of 1 differed in ca. 4 and 2 Hz from the
ones reported for the isolated sample, respectively.2 Since
a sample of the natural material could not be made available to
us, we focused on the preparation of the C3-epimer of 1 rst.
Applying the strategy described in Scheme 1B,19 persilylated
derivative 16 could be prepared in multi-mg scale. To our
surprise, and in striking contrast to the smooth removal of the
silicon protecting groups observed en route to compound 1, the
reaction of 16 in the presence of TBAF delivered two products. A
careful purication of the reaction mixture revealed the
formation of the desired irio-C3epimer 17 together with a ring-
enlarged analogue 18 formed via transesterication between
the C15–OH and the lactone moiety in 63 and 15% yields
respectively (Scheme 2A). Both compounds displayed
While this approach secured access to the desired compound
in four additional steps (namely, deprotection of the primary
OTBDPS,41,42 oxidation of the primary alcohol with Dess–Martin
periodinane followed by a Julia–Kocienski olenation with
sulfone 6 and nal TBAF deprotection of the remaining silyl
groups), further chemical edition of the macrolide as well as
access to a sufficient supply of the natural product itself for
subsequent biological evaluation were hampered by the lack of
scalability of this strategy. As a result, we embarked in the design
of an alternative synthetic route to access key intermediate 12 in
substantial amounts. The new approach encoded the previously
described fragments (3–6) but aimed at a more productive
assembly strategy both in terms of scalability and synthetic
efficiency. In short, the southern and western part of the mole-
cule exemplied by fragments 4 and 5, respectively, would be
coupled rst via cross metathesis reaction prior to the inter-
molecular esterication with northern fragment 3. Ring-closing
metathesis would then construct the 15-membered lactone
converging with our previously reported strategy. tert-Butyl ester
13 and 1,5-diene 5d were coupled via cross metathesis reaction
in the presence of Grubb's 2nd generation catalyst 11 in excellent
yield with complete E-stereocontrol (Scheme 1B). Silylation of
the secondary alcohol under standard conditions followed by
hydrolysis of the tert-butyl ester moiety produced fragment 14 in
78% yield. Intermolecular esterication with fragment 3 under
the conditions previously reported for fragment 4 produced ester
15 in 84% yield. The strategic potential of RCM was again
illustrated here with the use of 5 mol% of 11 to efficiently close
the 15-membered lactone with complete E-stereoselectivity
converging with our original synthesis. This approach afforded
iriomoteolide-3a 1 in only 7 steps from the corresponding
1
a completely different array of spectroscopic signals in H and
13C NMR compared to parent compound 1, qualitatively
reecting the population of a different conformational space
elicited by the change in conguration at a single stereocenter
(see ESI†). Although ring enlargements are not uncommon in
macrolides under basic conditions,49–51 the result summarized
in Scheme 2A was completely unexpected given the clean desi-
lylation observed to produce 1 in both the original as well as the
more recently developed total syntheses campaigns summa-
rized in Scheme 1.
Molecular dynamics simulations with the MMFF94 force
eld in the CHARMM program52,53 with implicit solvent were
performed on 1 and 17 to nd a qualitative explanation for this
highly distinct behaviour (see molecular dynamics simulations
section in the ESI† for further details).19 Two sets of clusters
were found using a 10 kcal molꢁ1 maximum energy difference
with the corresponding global minima as capping criteria. This
restriction afforded 62 clusters for the natural product (1) and
31 for the C3-epimer (17).
For the two molecules investigated, the lower energy clusters
(clusters 1–5 with a max. DE ¼ 2 kcal molꢁ1 and comprising
more than 1/3 of the total number of conformers) present
a rather distinct conformational blueprint as depicted in
Scheme 2B (irio-3a 1: Scheme 2B.1 and C3-epimer analogue 17:
Scheme 2B.2). Especially remarkable is the inuence that the
methyl group at position C3 exerts on the dihedral angle O1–
building blocks.19 7,8-O-Isopropylidene derivative
2
was
prepared by treatment of 1 with 2,2-dimethoxypropane in the
presence of pyridinium p-toluene sulfonate. Multi-mg quantities
of these two molecules could be prepared with the new route,
thus demonstrating the advantage of this strategy compared to
the originally reported one.
C
14–C15–OH, which is shied from ꢁ63ꢀ in the natural
compound to +62ꢀ in 17. In turn, the p* C]O orbital in 17 is
favorably aligned with the OH group explaining the observed
transesterication reaction. The relative disposition of the
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Scheme 2 (A) Final deprotection towards C3-irio-3a epimer (17) and C7,C8-irio-3a diastereoisomer (20) in the presence of TBAF. (B) Blueprint of
the most stable conformers for iriomoteolide-3a (1, B.1) and its C3-epimer (17, B.2). 1: O1–C14–C15–OH ¼ ꢁ63ꢀ. 17: O1–C14–C15–OH ¼ +62ꢀ. (C)
Synthetic iriomoteolide derivatives using the general strategy reported in Scheme 1B and preliminary evaluation of their anti-proliferative activity.
Values in brackets represent the % of growth inhibition of Daudi cells at 10 mM concentration of the compound. N.d.: not determined.
lateral chain compared to the macrocycle is also strongly obtained from D-tartaric acid following the route detailed in
affected, with the dihedral C1–O1–C14–C15 going from ꢁ5ꢀ in 1 Scheme 1B. As in the case of the C3-epimer, formation of a one-
to ꢁ44ꢀ in the synthetic analogue 17. This effect also translates carbon atom expanded lactone 21 could also be observed,
into a macrocycle “folding” in 17 compared to the relatively at together with the desired C7,C8-irio-3a diastereoisomer 20 in the
structure observed in the parent compound 1. Interestingly, nal desilylation step (Scheme 2A). Aiming to further investi-
these clusters present structural features that are in congruence gate the effect of C3 in the conformation and biological activity
with the recorded NMR data of these compounds in different of these molecules, a derivative of 1 bearing two methyl groups
organic solvents. Thus, the resonance for H9 appears at higher at C3 (22) and a second one devoid of any substituent at this
eld for compound 17 than for the natural molecule, which position (23) were also prepared following the general strategy
reects a shielding effect in the former case as this hydrogen is outlined in Scheme 1B. An additional criterion in our design
pointing towards the interior of the macrocyclic cavity.7 To involved the modication of the hydrogen bond network of the
further explore the conformational landscape of the macro- molecule. We investigated whether the hydroxy group at C15
cycle, the (7R,8R)-diastereoisomeric precursor 19 was easily plays a key role in the activity of these macrolides, and thus the
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methyl ether derivative at this position (24) was also prepared. thus not surprising that the discovery of secondary metabolites
Along the same lines, a tetrahydrofuran derivative 25 was that interact with actin by mimicking the effect of the above-
included as a result of the cyclization of a vinyl ester truncated mentioned proteins has attracted growing attention,58–62 to such
analogue. The vinyl epoxide was conserved throughout the an extent that some actin binding macrolides have undergone
analogues as it has been recognized as a critical motif for similar molecular editing exercises and computational studies
covalent binding to protein targets in related macrolides.54 as the one presented in this paper.63–65 These compounds,
Finally, it is important to note that the abovementioned mostly of marine origin, have been broadly classied as
chemical edition could not have been probed by standard “destabilizers” (if they destabilize actin laments or inhibit
derivatization of the natural product (Scheme 2C).
their assembly) and “stabilizers”. Cytochalasins66 and latrun-
As a way to compare the performance of the synthetic culins,67,68 among others, destabilize F-actin by capping one of
derivatives with that of the parent compound in an already the lament ends and by inhibiting their assembly through
established setting, a preliminary evaluation of cell viability on monomer sequestration, respectively. Swinholides,69 bis-
two different human lymphoma cell lines (Daudi and Akata) tramides70–72 and related molecules73 destroy the actin cyto-
was carried out. Iriomoteolide-3a 1, C7,C8-acetonide 2 and skeleton primarily by severing and/or sequestering actin. On the
derivatives 17, 20, 22–24 were tested at two different concen- other hand, jasplakinolide74,75 promotes polymerization by
trations 10 and 2.5 mM (% of growth inhibition at 10 mM on triggering nucleation whereas molecules such as phalloidin,76,77
Daudi are shown in Scheme 2C. For complete data set, see amphidinolide H54 or cucurbitacin E78 are able to stabilize
Fig. S1 in the ESI†). All compounds showed anti-proliferative F-actin, in the case of amphidinolide H and cucurbitacin E, by
activity at 10 mM except for C3-epimer 17, which was formation of a covalent adduct. Osada and co-workers demon-
completely inactive. These results conrm the striking inu- strated that amphidinolide H, an exceptionally cytotoxic
ence that a single-point mutation in the stereochemical metabolite against KB human epidermoid carcinoma cells
composition of a molecule can have in its conformational (IC50: 0.52 ng mLꢁ1
)
79 forms a covalent bond with Tyr200 in the
landscape, and subsequently, in its biological activity.19,45–48 subdomain IV of actin via opening of its epoxide unit.54 In
´
Along the same lines, both the addition and the elimination of contrast, a recent report by Dıaz and co-workers showed that
a methyl group at C3 (analogues 22 and 23) resulted in a reduced amphidinolides J and X, lacking the vinyl epoxide moiety in
anti-proliferative activity. Although the methylation of the their scaffold, behave as actin-assembly inhibitors.80 These
hydroxy group at C15 (24) was tolerated, the cytotoxicity of this studies prompted us to study the effects of iriomoteolide-3a
derivative substantially decreased compared to the parent and our small library of synthetic analogues on the actin
compound, indicating that the presence of a hydrogen bond cytoskeleton. For this purpose, a well-established assay involves
donor at this position is key for the activity.
the incubation of broblasts with the compound of choice
followed by staining of actin with uorescence marked
phalloidin. Morphological changes can be easily monitored by
uorescence microscopy providing a straightforward read-out
Characterization of cellular targets
Study of actin interaction properties. Actin and tubulin are of the impact of such molecules on the actin network. The
the two major constituents of the cytoskeleton in eukaryotic results of the inuence of irio-3a 1, acetonide 2 and synthetic
cells. Tubulin is a recognized target for anti-cancer therapies analogue 20 on the actin cytoskeleton in NIH/3T3 cells, Swiss
and microtubule-stabilizers such as paclitaxcel or epothilones mouse embryo broblasts, are shown in Fig. 1 (for complete
are now in clinical use.55 The key role of actin in numerous data set, see ESI†). At concentrations as low as 250 nM, 1 and 2
diseases is starting to be unravelled. The actin cytoskeleton is of elicit profound morphological changes in cells starting shortly
utmost importance to a wide variety of cellular processes aer toxin application. As shown in Fig. 1, irio-3a 1 caused
ranging from cell shape and locomotion to cell division, cell rounding of NIH/3T3 cells and loss of cell contacts aer
adhesion and cell transport (endo- and exo-cytosis).56 These only 2 hours. Well spread cells lose their smooth contour and
functions demand an exquisite control of the dynamics inter- a diminution of the microlament bundles (stress bers),
change between monomeric G-actin and polymeric F-actin together with cell shrinkage, is also observed (Fig. 1B). Inter-
laments. The regulation of this precise machinery is carried estingly, and in sharp contrast with the covalent binding of
out by a large number of endogenous proteins with four main amphilidinolide H to actin,54 the effect of iriomoteolide on the
modes of action.57 They can “sequester” monomeric G-actin, actin cytoskeleton is reversible. Aer 8 hours (Fig. 1C) cells have
inhibiting its addition to the growing lament (e.g. prolin). completely recovered and show their original morphology with
Alternatively, laments of actin can be either “capped” by a well spread actin network. Complete recovery of the microla-
inhibition of both monomer addition and dissociation (e.g. ment organization is also observed aer 2 hours of drug incu-
CapZ), or “severed” via active lament breaking (e.g. colin) or bation followed by a 22 hour period in new media (see Fig. S2 in
both (e.g. gelsolin). Finally, some proteins can also “promote” the ESI†). At higher concentrations of the drug (1 and 4 mM),
the polymerization of G-actin by inducing nucleation from depletion of F-actin from the central region and formation of
a pool of actin monomers (e.g. formins). However, despite the actin bundles at the cell margins can be observed (Fig. 1D, E).
extensive use of genetic approaches to study actin regulation, Similar phenotypes were also observed upon treatment with
substantial part of our knowledge on its biological roles has synthetic analogue 20 at 10 mM (Fig. 1F). Remarkably, many of
been acquired through its interaction with small molecules. It is the cells became binuclear (see Fig. 1B, D and E) suggesting that
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Fig. 1 (A–F) Effect of iriomoteolides-3a on mouse fibroblast: fluorescence micrographs (40ꢂ) of the actin cytoskeleton of NIH/3T3 cells. Actin
cytoskeleton is stained with FITC-phalloidin (green) and nuclei with 2-(4-amidinophenyl)-6-indolecarbamidine hydrochloride (DAPI) (blue). Cell
viability >80% according to a fluorimetric assay with resazurin. (A) Control cells. (B) Cells incubated with irio-3a (1) at 250 nM for 2 hours and (C)
after 8 hours showing complete recovery of their normal morphology and microfilament organization. (D and E) Cells incubated with 1 at 1 and
4 mM for 2 hours. (F) Cells incubated with synthetic analogue 20 (10 mM) for 8 hours. (G and H) Determination of the effects of irio-3a (1) and
synthetic analogues 2 and 20 on cell migration by means of the scratch-wound assay on NIH/3T3 cells. (G) Percentage of surface area filled 18
hours after treatment with 1, 2 and 20 at two different concentrations (4 and 1 mM). The results are presented as mean ꢃ SEM of three inde-
pendent experiments. (H) Scratch wound carried out by a pipette tip and recolonized after 18 h under the effect of compound 20 (4 mM). (I and J)
Effect of iriomoteolide 3a (1) and acetonide (2) on actin polymerization in vitro. (I) Effect of 1 and 2 (500 nM) on the copolymerization of actin and
pyrenyl labeled G-actin. Actin/pyrenyl-labeled actin (4 mM) was incubated with 1 (red), 2 (blue) or without either (black). Polymerization was
started by the addition of inducing salts (50 mM KCl, 2 mM MgCl2, 1 mM ATP) and drugs solved in DMSO at time ¼ 0 (less than 5% DMSO). (J)
Effects of 1 (1 mM, red) and 2 (1 mM, blue) on the rate of pyrenyl F-actin (2.3 mM, final concentration) depolymerization. Pyrenyl-labeled actin
F-actin (23 mM) was diluted to 2.3 mM and the drugs were added. The samples were mixed to give a 1 mM final concentration of drug.
iriomoteolides might most likely inhibit cytokinesis rather than the scratch on a monolayer of subconuent cells, broblasts
affect mitosis. The phenotype described here aer treatment with migrate into the wound reaching complete coverage aer 18
1 (cell shrinkage, reduction of F-actin bers, accumulation of hours. Upon treatment with synthetic analogue 20 (4 mM), only
F-actin in the perinuclear region and cells binucleation) ca. 20% of the area was covered aer 18 hours (Fig. 1H.
could also be observed at the same range of concentrations for Complete set of pictures and dose dependent experiments can
acetonide 2 (see Fig. S3 in the ESI†), and is in full accord with be found in Fig. S5 of the ESI†).
earlier studies on actin-stabilizing compounds. In all
Effects of iriomoteolides on actin polymerization. To eluci-
studied cases, cell viability was indirectly evaluated in parallel by date the mechanism by which iriomoteolides interfere with the
a uorimetric assay with resazurin as uorescent dye showing cell cytoskeleton, we investigated the ability of these compounds
that, at the used concentrations/times more than 80% of the to affect actin polymerization and depolymerization in vitro by
cells were able to reduce resazurin at the mitochondria. a standard uorescence assay (copolymerization of actin and
Importantly, no major effect on the tubulin cytoskeleton could be pyrenyl-labeled actin). We found that at submicromolar concen-
observed in a parallel assay (see Fig. S4 in the ESI†).
trations, irio-3a 1 and acetonide 2 increased the degree of poly-
Evaluation on cell motility. Cell movement through tissue merization of globular monomeric actin (G-actin) (Fig. 1I). At
requires a series of distinct but concerted biological events in lower concentrations of actin, lag times were observed for both
which actin plays a crucial role.81,82 Along these lines, several the control and the drug treated samples, indicating that irio-
natural products have been described to be cell migration moteolides are not inducing actin nucleation (e.g. as jasplaki-
inhibitors.82–86 Thus, we decided to examine the effect of irio- nolide). In line with this result, no actin polymerization was
moteolides on cell migration and invasion by a “scratch-wound observed in non-polymerizing conditions (data not shown). In
healing” assay on NIH/3T3 cells (muscle NIH/3T3 (myc-) cells addition, and in correlation with the data shown in Fig. 1I, both 1
were obtained as a donation from the UZH Cancer Institute). and 2 slowed down depolymerization of F-actin at 1 mM (Fig. 1J).
The expansion of the cell population was quantied by calcu-
These two sets of data indicate that iriomoteolides are likely
lating the percentage of recolonization of the wound surface to stabilize F-actin laments, enhancing, as a consequence
over time. As shown in Fig. 1G, natural irio-3a (1), and synthetic actin polymerization (Fig. 1I) and inhibiting F-actin depoly-
analogues 2 and 20 inhibited cell migration in a dose depen- merization (Fig. 1J). Interestingly, amphidinolide H, which is
dent manner. Acetonide 2 showed the most pronounced effect isolated from the Amphidinium species as iriomoteolide-3a (1),
with no cell-coverage in relation to the total wound area at 4 mM has also been classied as an actin stabilizer whereas its effect is
concentration. In the control experiment, aer generation of due to covalent binding to actin through its epoxide moiety.54,79
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In contrast, no covalent adduct could be detected by MALDI- interactions and clustering results, see Fig. S7–10 in the ESI†).
TOF upon incubation of 1 with G-actin, indicating that irio- The two binding modes are similar in their relative arrange-
moteolides, despite their epoxide moiety, do not form covalent ment of the hydrophobic tail of iriomoteolide-3a (1). The tail is
adducts with actin, in line with the reversible effects seen in inserted into the barbed end interacting with mainly hydro-
Fig. 1C (see Fig. S6 in the ESI†).
phobic residues (Fig. 2A, D). The conformation of the tail
MD simulations study of the complex formation of iriomo- section for the concatenated trajectories is similar for both
teolide 3a (1) and monomeric G-actin. Explicit solvent molec- binding modes (Fig. 2C, F red line). In contrast, the macrocycle
ular dynamics simulations were performed to elucidate the is either extended from the tail in an orientation more similar to
interactions between iriomoteolide-3a (1) and G-actin (Fig. 2). other natural products like reidispongiolide A,73 kabiramide C87
Thirty independent simulations were conducted starting from and trisoxasoles88 (cluster 1, Fig. 2A, B) or is located on top of
random positions and orientations of iriomoteolide-3a in the the tail section (cluster 2, Fig. 2D, E). The time series of RMSD
simulation box which showed preferential binding of the from the cluster representatives conrm the structural hetero-
natural product to the “barbed end” of G-actin with persistent geneity for the macrolide ring in both putative binding modes
contacts with aminoacids Tyr143, Leu346, Phe352, and Phe375 (Fig. 2C, F black) while the tail moiety remains structurally more
residues. Secondary hot spots, albeit weaker than barbed end homogeneous (Fig. 2C, F red).
residues, were observed at Met227 and Met283 which are
Additionally, although the macrolide conformation for basin 2
located in subdomains 4 and 3 of actin, respectively. All has been sampled in more simulations than basin 1, once basin 1
trajectories for which association of iriomoteolide-3a (1) with was reached it was structurally more stable than basin 2 (Fig. 2C).
the barbed end was observed (cumulative sampling time of The high sampling rate of the iriomoteolide-3a tail moiety in
3.6 ms) were clustered, revealing two free-energy basins sepa- close contact with barbed end residues within the combined
rated by a main barrier of 5.5 kcal molꢁ1
.
trajectories would be consistent with previous reports that sug-
Representatives of these two clusters are shown in Fig. 2 (for gested that interactions of actin binding macrolide tails at the
complete set of simulations, heat map analysis of contact barbed end of actin affect polymerization.61,73,89 The different
Fig. 2 Representatives of the two most populated clusters sampled upon binding of iriomoteolide-3a (1) at the barbed end of monomeric G-
actin (A, B cluster 1; D, E cluster 2). Binding modes are shown with residues within 4.5 A˚ highlighted (A, D magenta sticks) and overlayed with
crystal structure coordinates from reidispongiolide A (B, E blue lines, PDB: 2asm). For the representative of cluster 1 (green sticks), a binding mode
similar to natural products like reidispongiolide A is observed (B). The hydrophobic tail is inserted into the barbed and the macrocycle is located
along the hydrophobic patch (A, B). The representative of cluster 2 (cyan sticks) shows a similar arrangement of the hydrophobic tail while,
contrary to cluster 1, the macrolide resides on top of the tail section and not along the hydrophobic patch of the barbed end periphery (D, E).
Time series of RMSD from the representative structures of clusters 1 (C) and 2 (F) for ring (black) or tail (red) moiety. Individual trajectories are
concatenated in the time series, and starting points are marked (black vertical lines). Images rendered in Pymol http://www.pymol.org.
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binding sites of iriomoteolide-3a (within the barbed end) and
amphidinolide H, which covalently binds to actin within sub-
domain IV,54 could explain the non-covalent interaction between
iriomoteolide-3a and actin (see Fig. S6 in the ESI†). The simula-
tion results thus indicate that iriomoteolide-3a (1) likely binds to
the barbed end of monomeric actin although the macrocycle
might be able to adopt several conformations.
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Conclusions
Iriomoteolides are a novel family of cytotoxic macrolides, whose
highly-convergent, catalysis-based synthesis has been accom-
plished attesting the usefulness of alkene cross- and ring-
closing metathesis reactions.
8 D. R. Williams and K. G. Meyer, J. Am. Chem. Soc., 2001, 123,
765–766.
9 A. K. Ghosh and C. Liu, J. Am. Chem. Soc., 2003, 125, 2374–
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In this study, conformation has been unraveled as a key 10 A. Furstner, Isr. J. Chem., 2011, 51, 329–345.
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to at least equal the activity of the natural product, thus high-
3127.
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for in depth studies of complex biologically active molecules. 13 J. Xie, Y. Ma and D. A. Horne, Chem. Commun., 2010, 46,
Small molecules that target the actin cytoskeleton have long
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profound morphological changes in the actin network in cells.
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Iriomoteolides appear to stabilize F-actin laments by 17 R. Cribiu, C. Jager and C. Nevado, Angew. Chem., Int. Ed.
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Multiple molecular dynamics simulations with explicit solvent 19 E. Moulin, V. Zoete, S. Barluenga, M. Karplus and
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dynamics of the actin cytoskeleton in order to understand cell
motility processes including cell invasion and division.
2140.
22 K. Ohtsuki, K. Matsuo, T. Yoshikawa, C. Moriya, K. Tomita-
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23 P. Perlmutter, W. Selajerern and F. Vounatsos, Org. Biomol.
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Conflicts of interest
There are no conicts to declare.
24 S. G. Nelson, T. J. Peelen and Z. H. Wan, J. Am. Chem. Soc.,
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25 S. G. Nelson, Z. H. Wan and M. A. Stan, J. Org. Chem., 2002,
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Acknowledgements
We thank Dr Navid Nadal for a generous loan of lymphoma cell
lines and Dr Sabrina Sonda for helpful discussions and tech- 26 M. A. Robbins, P. N. Devine and T. Oh, Org. Synth., 1999, 76,
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