ACS Central Science
Research Article
effectively pulled-down IPO5, as observed in a Western blot of
the elution fraction (Figure 3b). Addition of goyazensolide (10
μM) to the cell lysates prior to the addition of the beads
inhibited the pull-down of IPO5. Thus, the covalent
interaction between goyazensolide and IPO5 inhibits its
interaction with these two NLSs.
evidence of anticancer activity from mouse xenografts and
cellular assays, but the mode of action remained unknown. The
finding that goyazensolide selectively engages IPO5 covalently
and inhibits its interaction with cargo rationalizes its anticancer
activity and points to potential antiviral applications. IPO5 is
highly expressed in colorectal cancer31 and esophageal
cancer.36 As an antiviral target, there is clear evidence that
IPO5 is recruited by viruses and that its trafficking activity is
essential in the viral replication cycle.29,30
The fact that a small molecule can outcompete a high-
affinity interaction with larger molecular entities such as the
interaction of transportin with an NLS sequence of a protein
underpins the unique features of covalent inhibitors. Transport
of cargo between the cytoplasm and nucleus is a highly
regulated process with the nuclear pore complex acting as a
molecular sieve restricting diffusion. Translocation thus
requires active transport leveraged on the action of a
superfamily of karyopherin (importin and exportin).37 The
ability to modulate this traffic with small molecules is
empowering,38,39 but there are only few examples thus far.
The first example of a small molecule regulating a karyopherin
is leptomycin B, a polyketide bearing a Michael acceptor,
which selectively inhibits exportin-1 (XPO-1, also known as
chromosome region maintenance 1 (CRM1))40 by reacting
with its target.41 This discovery led to significant medicinal
chemistry efforts resulting in a molecule (selinexor) that is
FDA-approved for multiple myeloma.42
The second example, ivermectin, was identified from a high-
throughput screen for inhibitors of protein nuclear import.
Characterization of the nuclear transport inhibitory properties
of ivermectin demonstrated that it is a broad-spectrum
inhibitor of importin α/β nuclear import with potent antiviral
activity toward both HIV-1 and dengue virus, both of which
are strongly reliant on importins.43 Notably, this FDA-
approved drug also inhibits SARS-CoV-2 replication. Studies
on SARS-CoV proteins have revealed a potential role for
importin α/β1 heteroduplex in nucleocytoplasmic shuttling of
the SARS-CoV’s nucleocapsid protein.44 IPO5 has clearly been
shown to be implicated in the transport of influenza A’s RNA
polymerase,45 and mutations affecting binding to IPO5
severely attenuate viral growth,29 suggesting this activity
could be phenocopied with small molecule inhibitors of
IPO5. The crystal structure of IPO5 coupled to docking
studies of the NLS revealed critical interactions of influenza’s
PA−PB1 subcomplex with IPO5.46
Goyazensolide Inhibits the Cellular Function of IPO5.
IPO5 has been shown to be involved in the transport of RAS
protein activator-like 2 (RASAL2) to the nucleus, a negative
regulator of RasGAP. Thus, nuclear translocation of RASAL2
frees RasGAP to convert inactive Ras-GDP to active Ras-GTP
as shown in Figure 4a.31 The ratio of cytosolic RASAL2 and
nuclear RASAL2 was clearly shown to depend on IPO5 using
siRNA silencing of IPO5 (increased cytosolic concentration of
RASAL2) and IPO5 overexpression (decreased cytosolic
concentration of RASAL2).31 In order to understand if
goyazensolide could phenocopy the silencing activity of
siRNA targeting IPO5, an SW620 colon cancer cell line was
used to measure the RASAL2 ratio in the cytosol vs the
nucleus in the presence and the absence of goyazensolide
(Figure 4b). In the absence of treatment, the cytosolic/nuclear
ratio of RASAL2 is 1.2. Upon treatment with 1.0 or 2.5 μM
goyazensolide for 3 h, the ratio increased to 1.8 and 2.0,
respectively (Figure 4c). Thus, goyazensolide treatment led to
an increased cytosolic /nuclear ratio of RASAL2 in a dose
dependent fashion.
Since the inhibition of RASAL2 translocation to the nucleus
should have a downstream effect on the level of pAKT, we also
monitored the level of pAKT (Ser473) in the presence or
absence of drug. As shown in Figure 4d, treatment with 5 μM
goyazensolide led to a dramatic reduction of pAKT.
Collectively, these experiments demonstrate that IPO5 is the
target of goyazensolide and that covalent engagement of IPO5
inhibits its transport activity in cellulo.
Sesquiterpenes are a rich source of bioactive compounds,
and their syntheses continue to challenge synthetic chemists
(for recent examples, see refs 32−35). From a synthetic
chemistry perspective, goyazensolide stands out as a highly
oxidized member of the germacranolides with protean C3 and
C10 bridged furan ring featuring an anti-Bredt double bond.
The present work demonstrates that despite its complexity,
such natural products can now be accessed synthetically in less
than 13 steps. The strategy employed is compatible with a
build/couple/pair strategy facilitating the collective synthesis
of multiple natural products, including goyazensolide. This
required a cyclization to form a 10-membered ring, which is
notoriously difficult. The use of Barbier allylation to
simultaneously install the α-exo-methylene-γ-butyrolactone
and achieve this cyclization is unprecedented in this context,
and the work reported indicates that it is a robust strategy to
access the strained framework. The use of an alkyne in the ring
facilitates the pairing chemistry by mild and efficient
Sonogashira coupling, and the alkyne offers divergent
functionalization pathways to furanoheliangolide or related
heliangolides. The chemistry described enabled the collective
synthesis of 16 complex sesquiterpene lactones, many with
important biological activity (See Figure S1 for a complete set
of reference to biological activity).
CONCLUSION
■
In summary, we report the first total synthesis of goyazensolide
and 15 related natural products. Our studies revise the
stereochemical assignment of two natural products. Signifi-
cantly, this synthesis enabled the study of the covalent
interactome of goyazensolide, revealing IPO5 as its molecular
target. The covalent engagement inhibits the interaction of
IPO5 with cargos, provides a rationale for its cytotoxic activity
in several cancer cell lines, and points to potential antiviral
activity.
The resurging interest in covalent inhibitors brings renewed
interest to sesquiterpenes, particularly those that have already
been used in traditional medicine, affording a preliminary
assessment of toxicity. Covalent inhibition is particularly
attractive if target binding is in competition with strong
endogenous interactions such as in protein−protein inter-
actions or transcription factors interacting with DNA.
The use of some of these compounds in traditional medicine
gives them the benefit of a preliminary toxicology profile in
humans. However, the lack of information on the mode of
action often precludes a more insightful and targeted use of
such compounds. In the case of goyazensolide, there was clear
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ACS Cent. Sci. 2021, 7, 954−962
Barluenga, Sofia
