Identification of a Strigoterpenoid with Dual Nrf2 and Nf-?°B Modulatory Activity

Article abstract of DOI:10.1021/acsmedchemlett.8b00604

The sesquiterpene-coumarin ether samarcandone provided a suitable framework to replace the apocarotenoid A-C ring system of strigol (1), replicating, after linking to a butenolide moiety, the activity of the natural phytohormone on Nrf2 and also showing potent NF-kB inhibitory activity, overall modulating two critical pathways of inflammation and cancer.

Full text of DOI:10.1021/acsmedchemlett.8b00604

Letter
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Cite This: ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
Identification of a Strigoterpenoid with Dual Nrf2 and Nf-κB
Modulatory Activity
¶,⊥,#,‡
Federica Rogati,†,‡ Estrella Millan,
Giovanni Appendino,† Alejandro Correa,¶,⊥,#
́
Diego Caprioglio,† Alberto Minassi,*,† and Eduardo Munoz*
,¶,⊥,#
̃
†
̀
Dipartimento di Scienze del Farmaco, Universita del Piemonte Orientale, Via Bovio 6, Novara 28100, Italy
¶
́
Maimonides Biomedical Research Institute of Cordoba, Avda Menendez Pidal s/n, 14004 Cordoba, Spain
⊥Department of Cellular Biology, Physiology and Immunology, University of Cordoba, Avda Menendez Pidal s/n, 14004 Cordoba,
Spain
#University Hospital Reina Sofía, Avda Menendez Pidal s/n, 14004 Cordoba, Spain
S
* Supporting Information
ABSTRACT: The sesquiterpene−coumarin ether samarcan-
done provided a suitable framework to replace the
apocarotenoid A−C ring system of strigol (1), replicating,
after linking to a butenolide moiety, the activity of the natural
phytohormone on Nrf2 and also showing potent NF-kB
inhibitory activity, overall modulating two critical pathways of
inflammation and cancer.
KEYWORDS: Strigolactones, terpenes, Nrf2, NF-κB
mall-molecule endogenous hormones modulate basic plant
Sfunctions like growth, differentiation, and reproduction as
well as their response to abiotic- and biotic stress. Ethylene was
the first member of this structurally heterogeneous class to be
identified, and the inventory has then significantly expanded to
include as major members indoleacetic and abscisic acids,
brassinosteroids, cytokinins, gibberellins, jasmonoids, salicylic
acid, and strigolactones (SLs). In addition to their role in plant
physiology, certain plant hormones can also bind mammalian
targets, or even be produced by mammalian cells, as
exemplified by abscisic acid.1 Furthermore, plant hormones
can also serve as a scaffold for drug discovery, as shown by
aspirin and, more recently, by the cyclin-dependent kinase
inhibitors olomoucine and roscovitine, whose structure was
inspired by cytokinins, a class of N6-substituted adenine
derivatives.2 Despite these interesting clues, the potential of
plant hormones to serve a lead structure for drug discovery has
not yet been systematically evaluated. This gap has provided a
rationale to explore the potential of strigolactones (SLs), the
most recent addition to the plant hormone inventory, to
modulate mammalian targets of medical relevance.
responsible for the plant hormone activity of SLs, which is
mediated by covalent binding to a reactive cysteine residue in
their macromolecular targets.3 Synthetic and natural SLs have
been extensively investigated as germination stimulants of
parasitic weed seeds as well as biopesticides for crop
protection,4 but only limited knowledge exist on their
involvement in animal cell function and their potential cross-
kingdom activity, despite promising results in the realm of
anticancer drug discovery.5 Within the possible mammalian
targets of SLs, the transcription factor Nrf2 (nuclear factor
(erythroid-derived 2)-like 2) seemed of particular relevance
because of its sensitivity to nucleophilic trapping and its role in
the regulation of many cytoprotective enzymes involved in the
adaptive oxidative stress response.6 Nrf2 is the target of
dimethyl fumarate, a compound used in the management of
multiple sclerosis,7 and of bardoxolone methyl, currently under
phase III clinical study for the management of pulmonary
hypertension.8
In the event, strigolactone (1) turned out to be a potent
activator of the Nrf2 pathway (EC50 = 1.2
0.9 μM),
providing a rationale for investigating the structure−activity
relationship of this chemotype. Strigol and SLs in general have
a very limited availability, and we therefore attempted to
Strigolactones (SLs) are C15 apocarotenoid dilactones
involved in shoot- and root architecture and in plant responses
to environmental stress. Strigol (1), the first member of this
family, was isolated in 1966, and the inventory of
strigolactonoids now includes more than 15 additional
analogues. SLs are characterized by a tricyclic scaffold linked
to a butenolide D ring by an enolic oxymethine. There is
convincing evidence that the reactive CD ring system is
Special Issue: Highlighting Medicinal Chemistry in Italy
Received: November 30, 2018
Accepted: January 8, 2019
Published: January 8, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.8b00604
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ACS Medicinal Chemistry Letters
Letter
Table 1. Synthesis of Homoterpeno-strigoids (A) and Terpenostrigoids (B)
B
DOI: 10.1021/acsmedchemlett.8b00604
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ACS Medicinal Chemistry Letters
Letter
Table 1. continued
a
b
Strigol (1) EC50: NRF2 (EC50) 1.2 0.9; NF-κB (IC50) > 50.
identify a surrogate of the apocarotenoid A−C ring system of
the natural hormone within more easily available isoprenoids.
To this purpose, analogues where ring D is implanted in
various isoprenoids scaffolds were designed. Two series of
analogues were prepared, differing for the way ring D and the
isoprenoid core are linked (oxymethine- or oxygen tether), and
all compounds were then comparatively evaluated with strigol
(1) for their capacity to modulate the activity of Nrf2. All
compounds were also investigated for their capacity to inhibit
NF-κB, another transcription factor sensitive to electrophilic
modulation. Although strigol was inactive in this assay, dual
modulators of Nrf2 and Nf-κB hold great pharmacological
potential,7,8 and we hope to discover compounds with this
bioactivity profile.
Compounds with an oxymethine tethered were built by
Claisen formylation of an isoprenoid ketone and then coupling
with the bromofuranone 2 (Table 1). The reaction was
stereoselective regarding the configuration of the oxymethine
C
DOI: 10.1021/acsmedchemlett.8b00604
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ACS Medicinal Chemistry Letters
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In all cases, an almost equimolecular mixture of isomers at the
furanone C-5 carbon was, however, obtained. The diastereo-
meric mixture was difficult to separate, and all compounds
were assayed as such.
The strigoids obtained from pentacyclic triterpene ketones
(3−9) were totally devoid of activity, suggesting that the
isoprenoid scaffold was too large to access the site hosting the
reactive thiol group of Nrf2, and similar results were observed
for the inhibition of NF-κB. However, bicyclic and monocyclic
isoprenoid ketones afforded active strigoids, some of which
showed potency similar to strigol, with EC50 in the one-digit
micromolar range (Table 1). Within p-menthane derivatives,
the cross-conjugated dienones 11 and 12, obtained from,
respectively, carvone and pulegone, were significantly more
potent than the enone 10, derived from menthone (EC50 = 1.6
0.3 μM and 5 0.2 μM vs 17.8 1,7 μM, respectively),
suggesting that electronic factors are important for activity.
Also 13, derived from α-thujone, was significantly active (EC50
= 1.9 0.6 μM), and one-digit micromolar activity was also
retained in the bicyclic strigoids 14 (EC50 = 1.2 0.03 μM),
and 15 (EC50 = 2.5 0.2 μM), derived, respectively, from the
sesquiterpene coumarin ether samarcandone and the triterpe-
noid mirranone B.
The second series of terpenostrigoids was obtained by
condensing the butenolide lactol 22 with a series of isoprenoid
alcohols according to the Feringa protocol (heating at 120 °C
in the absence of solvent)9 (Table 1). Also in this case, the
reaction gave a mixture of diastereomeric furanones (16−21)
that were assayed as such. Overall, the oxygen-tethered
terpenostrigoids were one order of magnitude less potent
than the oxymethine-linked homoterpeno-strigoids.
Figure 1. Compound 14 activates Nrf2 without inducing ROS. (A)
ROS production in HaCaT cells. Images were obtained after 3 h of
treatment. (B) Nrf2 transcription activity was analyzed in HaCaT-
ARE-Luc. Cells were treated with 14 in absence or presence of NAC
(15 mM) at the doses indicated during 6 h.
Strigol (1) could activate Nrf2, but had no effect on TNFα-
induced NF-κB activation, another upstream regulatory
process sensitive to thiol trapping that critically relies on the
presence of cysteine as an on/off switch. This profile was
replicated by all active compounds we identified with the
exception of the bicyclic drimane coumarin ether 14, that
could, surprisingly, also inhibit NF-κB activity at low μM
concentrations (IC50 = 7.9 μM). This dual profile of activity is
interesting, and was further investigated. We first clarified the
Nrf2 activation mechanism, which can be electrophilic (direct
thiol trapping) or oxidative (oxidation of the cysteine sulfur
atom) and mediated by reactive oxygen species (ROS). To this
purpose, we investigated the relationship between the
induction of Nrf2 activity and the increase of cellular ROS.
Figure 1A shows that, in contrast to tert-butyl hydroperoxide
(TBHP), 14 was unable to affect the intracellular levels of
ROS. Interestingly, pretreatment with N-acetyl cysteine
(NAC) inhibited the activity of 14 on Nrf2 activation (Figure
1B). NAC is a scavenger of oxygen free radicals and a
precursor of L-cysteine. Since 14 was not able to induce ROS,
NAC might react with its coumarin moiety, which has
Michael-acceptor properties,10 to generate an inactive adduct.
Next, we investigated the effect of 14 on the canonical
pathway of NF-κB activation by analyzing the steady-state
levels of phosphorylated IκBα and p65 (a subunit of the more
common form of NF-κB heterodimers). Both IκBα and p65
proteins are phosphorylated by the IκB kinase β (IKKβ), which
is activated by TNFα through the so-called canonical pathway.
The drimane strigoid 7 clearly inhibited the phosphorylation of
both IκBα and p65 induced by TNFα in NIH-3T3-KBF-Luc
cells. Phosphorylation of IκBα is required for its degradation,
Figure 2. Effects of 14 on NF-κB activation. (A) Levels of NF-κB
proteins expression and phosphorylation by immunoblot. (B) IKKβ-
induced NF-κB activation is inhibited by 14.
linker, with the predictable exclusive formation of the E-
isomer, evident from the downfield shift of the oxymethine (δ
ca. 7.40), diagnostic of a syn-relationship with the carbonyl.
Thus, the nucleophilic displacement reaction occurred with
formal inversion of configuration of the enol double bond,
which was in the intramolecularly hydrogen-bonded Z-
configuration in the starting enol (compounds B, Table 1).
D
DOI: 10.1021/acsmedchemlett.8b00604
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ACS Medicinal Chemistry Letters
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(6) Cuadrado, A.; Manda, G.; Hassan, A.; Alcaraz, M. J.; Barbas, C.;
and we found that 14 could also prevent TNFα-induced IκBα
degradation (Figure 2A).
Furthermore, 14 could also inhibit specifically the NF-κB
activation induced by overexpression of IKKβ (Figure 2B).
Taken together, these observations suggest that 14 could
directly interact with the Cys-179 of this kinase and inhibit its
activity.
In conclusion, we have identified a sesquiterpene-coumarin
strigoid (14) that not only replicates the activity of natural
strigol (1) on the activation of Nrf2, but also targets the NF-kB
pro-inflammatory pathway. Within terpeno-strigoids, the
activation of Nrf2 was sensitive to the size of the isoprenoid
moiety, tolerating mono- and bicyclic systems but not more
complex polycyclic constructs, while the inhibition of NF-κB
was specific of 14. The cross-talk between inflammation and
the oxidative response plays an important role in cancer, and
compounds capable to modulate both pathways are interesting
leads to prevent and treat malignancies, qualifying 14 for
further studies.11
́
́
Daiber, A.; Ghezzi, P.; Leon, R.; Lopez, M. G.; Oliva, B.; Pajares, M.;
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Rojo, A. I.; Robledinos-Anton, N.; Valverde, A. M.; Guney, E.;
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Dimethyl fumarate treatment in multiple sclerosis: Recent advances in
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(8) Cheng, Y.; Gong, Y.; Qian, S.; Mou, Y.; Li, H.; Chen, X.; Kong,
H.; Xie, W.; Wang, H.; Zhang, Y.; Huang, Z. Identification of a Novel
Hybridization from Isosorbide 5-Mononitrate and Bardoxolone
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ASSOCIATED CONTENT
* Supporting Information
■
S
(11) Khurana, N.; Sikka, S. C. Targeting Crosstalk between Nrf-2,
NF-κB and Androgen Receptor Signaling in Prostate Cancer. Cancers
2018, 10, 352.
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.8b00604.
Materials and methods; original spectroscopic data for
the novel compounds (PDF)
AUTHOR INFORMATION
Corresponding Authors
*Phone: +39 0321375843. E-mail: alberto.minassi@uniupo.it
(A.M.).
■
*Phone: +34 957213766. E-mail: fi1muble@uco.es (E.M.).
ORCID
Alberto Minassi: 0000-0002-3640-2912
Author Contributions
‡These authors contributed equally. The manuscript was
written through contributions of all authors. All authors have
given approval to the final version of the manuscript.
Notes
The authors declare no competing financial interest.
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DOI: 10.1021/acsmedchemlett.8b00604
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX