Highly cytotoxic and neurotoxic acetogenins of the Annonaceae: New putative biological targets of squamocin detected by activity-based protein profiling

Article abstract of DOI:10.1016/j.bmcl.2008.09.091

Acetogenins of the Annonaceae are strong inhibitors of mitochondrial complex I but discrepancies in the structure/activity relationships pled the search for other targets within the whole cell proteome. Combining hemisynthetic work, Cu-catalyzed Huisgen c

Full text of DOI:10.1016/j.bmcl.2008.09.091

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5741–5744
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Highly cytotoxic and neurotoxic acetogenins of the Annonaceae: New
putative biological targets of squamocin detected by activity-based protein
profiling
Séverine Derbré a, Sophie Gil b, Myriam Taverna c, Céline Boursier c, Valérie Nicolas c,
Emmanuelle Demey-Thomas d, Joëlle Vinh d, Santos A. Susin e, Reynald Hocquemiller a, Erwan Poupon a,
*
a Laboratoire de Pharmacognosie associé au CNRS, UMR 8076 (BioCIS), Faculté de Pharmacie, Université Paris-Sud 11, 5, rue Jean-Baptiste Clément,
92296 Châtenay-Malabry Cedex, France
b UPRES 2706 ‘‘Barrières et passage des médicaments”, Centre d’Études Pharmaceutiques, Université Paris-Sud 11, 5, rue Jean-Baptiste Clément,
92296 Châtenay-Malabry Cedex, France
c JE 2495 ‘‘Nanotechnologies et protéines” and ‘‘Plateformes Protéomique et Imagerie cellulaire”, IFR 141 Centre d’Études Pharmaceutiques, Université Paris-Sud 11, 5,
rue Jean-Baptiste Clément, 92296 Châtenay-Malabry Cedex, France
d Groupe ‘‘Spectrométrie de masse et neuroprotéome”, UMR 7637, ESPCI, 10, rue Vauquelin, 75231 Paris Cedex 5, France
e Laboratoire ‘‘Apoptose et système immunitaire”, Institut Pasteur, 28, rue du Dr Roux, 75015 Paris, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 July 2008
Revised 22 September 2008
Accepted 23 September 2008
Available online 30 September 2008
Acetogenins of the Annonaceae are strong inhibitors of mitochondrial complex I but discrepancies in the
structure/activity relationships pled the search for other targets within the whole cell proteome. Combin-
ing hemisynthetic work, Cu-catalyzed Huisgen cycloaddition and proteomic techniques we have identi-
fied new putative protein targets of squamocin ruling out the previously accepted ‘complex I dogma’.
These results give new insights into the mechanism of action of these potent neurotoxic molecules.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Acetogenin
Squamocin
Hemisynthesis
Proteomic
Microscopy
Acetogenins of the Annonaceae constitute a broad group of sec-
ondary metabolites with impressive biological activities and have
been considered as important leads for new anticancer drugs.1
But recently, acetogenins of the Annonaceae have been suspected
to be involved in neurodegenerative disorders such as fatal atypical
parkinsonisms in worldwide areas where annonaceous-derived
edible products or traditional medicines are comsummated.2 The
hyperactivity of the acetogenins of the Annonaceae in conjunction
with this new public health issue claim for urgent biological stud-
ies for a better understanding of their exact mechanisms of action.3
Therefore, we have embarked on synthetic investigations on
squamocin 1,4 an ubiquitous acetogenin of the Annonaceae
extracted, for example, from the seeds of Annona reticulata.
Annonaceous acetogenins are known to be strong inhibitors of
mitochondrial complex I (NADH-ubiquinone reductase)4b and
inducers of cell-apoptosis. One of the main facts resulting from
all pharmacomodulation studies conducted by us and many other
groups is the absence of any clear link between complex I inhibi-
tion and apoptosis and/or cytotoxicity. In this Communication, a
program directed towards the identification of acetogenin biologi-
cal targets is reported. It is inspired by Affinity-Based Protein
Profiling (ABPP) as part of chemical proteomic technologies.5
In fact, from a chemical reactivity point of view, the work of
Duval et al.4e suggested that the
a,b-unsaturated c-lactone could
constitute a possible electrophilic functional group that may cova-
lently bind to nucleophilic residues in specific protein targets.6
Among the chemical tools towards the design of chemical probes
for functional proteomics developed in the recent years we chose
to exploit the CuI-catalyzed version of the azide-alkyne Huisgen
cycloaddition7 (‘Click-chemistry-based ABPP’). To this aim, we
needed chemical probes derived from 1. The first target was mole-
cule 2, suitable for a ‘tag-free’ strategy. For this purpose we needed
a N3 group as a viable chemical reporter on an almost complete
Squamocin
-lactone and a central polar part consisting in two tetrahydrofu-
ran rings and three secondary alcohol functions.
1 (Fig. 1) possesses a terminal a,b-unsaturated
c
* Corresponding author. Tel.: +33 1 46 83 55 86; fax: +33 1 46 83 53 99.
E-mail address: erwan.poupon@u-psud.fr (E. Poupon).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.091

5742
S. Derbré et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5741–5744
mitochondrial membrane
recognition
OH
other targets ?
24
O
O
target protein
Nu
15
28
HO
HO
O
O
and/or
O
spacer
squamocin
1
(Annona reticulata seeds)
O
Nu
target protein
pharmacophore
complex I inhibition
O
O
Figure 1. Structure of squamocine 1, key RSA data and potential protein-reactive sites.
squamocin skeleton (keeping unchanged the lactone ring and the
bis-hydroxy/THF part) for detection of substrate-target covalent
adducts by tagging with a fluorophore in whole proteomes. Central
in our strategy was the search for a selective introduction of an
azido group at C-28. Interestingly, the direct introduction of the
azido group was successful in our case using Mitsunobu-type reac-
tion with diphenylphosphorazide8 leading to 2 as a probe for ‘tag-
free’ ABPP. Having reached this key-molecule, a new probe (3) was
synthesized in the classical conditions described for the catalyzed
Huisgen-type cycloaddition of azides with terminal alkynes.9 In
moving on to the next approach, a bifunctional derivative 4 bearing
(i) a reactive function for photoaffinity labeling in place of the ter-
minal lactone and (ii) the fluorescent group at position 28 was pre-
and 5 were predominantly, but not solely, addressed to the mito-
chondria (71% and 62%, respectively, LSM Image Browser
software).
Kinetic studies were also performed with 5 and shown a rapid
internalization into the cell (5–10 min) and a mitochondrial local-
ization after 15–25 min. This latter is completed after 40 min, that
is to say far before the observation of the mitochondrial transmem-
brane potential (MTP) disruption, an early stage feature of apopto-
sis (typically 3–6 h with annonaceous acetogenins.4c These
discrepancies, both in terms of localization and kinetic, prompted
us to further search for other targets than mitochondrial complex
I on which acetogenins such as 1 could covalently bind.
We first decided to study probe 3. Jurkat-T cells were incubated
9
pared.
with 3 (10 lM for 24 h). The optimal protocol for both 1D and 2D
Confocal microscopy with laser scanning allowed us to identify
more precisely the organelles targeted by 5 (with the fluorescein
moiety in place of the lactone ring).4a At first, intracellular localiza-
tion of probes 3 and 5 was studied (Fig. 2). In Hela cells, after 6 h, 3
(IEF/SDS–PAGE) electrophoresis was established in denaturating
conditions.
Affinity-isolated proteins were first separated and visualized by
1D (SDS–PAGE) and in-gel fluorescence scanning with Coomassie
Figure 2. Kinetic study of the penetration of probe 5 in Hela cells (A) and visualization of the colocalisation of 3 and 5 with mitochondria (B). (A) Hela mitochondria were
labeled with Mitotracker Red CMXRosÒ. At t = 0, 10
M of 5 were injected in the culture medium and acquisitions were realized. (i) Hela cells labeled with 5. (ii) Overlays of
l
CMXRos and 5 stainings. (B) Hela cells were treated with 3 or 5 (green) for 6 h and mitochondria were labeled with CMXRos (red). The overlay indicated that both probes were
predominantly addressed to the mitochondria (71% and 62%, respectively, LSM ImageÒ Browser software).

S. Derbré et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5741–5744
5743
Figure 3. 2D-electrophoresis of total proteins (A) and from Jurkat cells treated by probe 3 (B). Seven proteins (i–vii) are labeled by probe 3 in a reproducible manner (three
independent experiments): i, ii, vi and vii were identified (see text).
co-staining to directly visualize protein bands.9 Less than 10
protein bands (ranging from 20 to 80 kDa) were labeled in a
specific and reproducible manner. Interestingly, the electropho-
retic profile was identical when cells were treated with photoprobe
4 for 24 h and then photoactivated to establish a covalent link to its
nearby environmental proteins in vivo.10
analyzed by MALDI-TOF/TOF mass spectrometry. Spots (i), (ii),
(vi) and (vii) (Fig. 3) correspond, respectively, to the COP9 signalo-
some complex subunit 7b (MW 30 kDa, pI 5.83), an enzymatic
complex associated with the ubiquitinylation and phosphorylation
of transcription factors (p53 for example),11 the glutathione trans-
ferase omega 1 (MW 27 kDa, pI 6.23), an enzyme involved in
detoxification,12 the flavoprotein subunit of succinate dehydroge-
nase (MW 73 kDa, pI 7.06) also known as mitochondrial complex
The 2D gel showed seven proteins which were reproducibly
fluorescent. Protein spots were excised, digested by trypsin and
OH
O
DPPA, DEAD
PPh3
O
O
1
N3
O
HO
HO
2, probe for tag-free ABPP
OH
3, probe for ABPP
O
O
27
O
O
O
O
N
N
O
N
HO
O
4, photoprobe for ABPP
27
O
N
H
OH
O
N
O
N
HO
N
HO
O
O
5, probe for kinetic studies
OH
O
O
Scheme 1. Synthesis of squamocin-derived probes. See Supporting Information for experimental details.

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S. Derbré et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5741–5744
II13 and to the disulfide isomerase (MW 57 kDa, pI 4.76), a chaper-
one protein from endoplasmic reticulum.14 Proteins (iii)–(v) could
not be identified as the protein amount was not sufficient. How-
ever their molecular weights around 30 kDa as the ND1-subunit
of mitochondrial complex I is promising.3g
Org. Lett. 2008, 10, 717. and references cited therein; Interestingly, such natural
substances have been found recently in Ampelocissus sp. (Vitaceae): (e) Pettit,
G. R.; Mukku, V. J.; Cragg, G.; Herald, D. L.; Knight, J. C.; Herald, C. L.; Chapuis, J.-
C. J. Nat. Prod. 2008, 71, 130.
2. Eskobar-Khondiker, M.; Höllerhage, M.; Muriel, M.-P.; Champy, P.; Bach, A.;
Depienne, C.; Respondek, G.; Yamada, E. S.; Lannuzel, A.; Yagi, T.; Hirsch, E. C.;
Oertel, W. H.; Jacob, R.; Michel, P. P.; Ruberg, M.; Höglinger, G. U. J. Neurosci.
2007, 27, 7827. and references cited therein.
The ultimate challenge in our approach ideally entailed using
probe 2 for tag-free ABPP, that is, in vivo covalent interactions of
2 with its targets and subsequent labeling of the bio-orthogonal
azide chemical reporter group in vitro on whole proteomes. Unfor-
tunately, an unspecific labeling of proteins by propargylfluorescein
was systematically observed. Possible reasons can be put forward
for this failure and are inherent to acetogenin structures and phys-
icochemical properties. Previously reported successes of this latter
strategy15 dealt with the targeting/identification of soluble cyto-
solic proteins or expressed proteins. In our case, membrane associ-
ated targets and a strong affinity of acetogenins to lipids may
considerably complicate the task making the 28-azide totally
inaccessible for chemical modifications on whole proteome
(Scheme 1).
In conclusion, an acetogenin of the Annonaceae was converted
to activity-based probes for chemical proteomics. But mainly, we
were able to identify new putative targets including mitochondrial
(vi), but also cytosolic (i and ii) and reticulum associated (vii)
enzymes.16 This rules out the ‘complex I dogma’ and opens the
way to major new developments in biology for the comprehension
of both cytotoxicity and neurotoxicity of this class of secondary
metabolites which are found in edible products from the Annona-
ceae and constitute an important public health issue. These results
also validate the particular importance of natural products in the
exploration of proteome.17
3. For recent examples of studies directed towards the understanding of
annonaceaous acetogenins mechanisms of action, see inter alia: (a) Kojima,
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Acknowledgement
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9. See Supporting Information.
We gratefully thank Jean-Christophe Jullian for NMR assistance.
10. Considering that the association 3-proteins is resistant to protein denaturation,
a covalent link can be reasonably assumed between 3 and its targets.
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Supplementary data
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.09.091.
References and notes
16. The interaction with the known target of acetogenins, that is, mitochondrial
complex I, was not detected in the course of this study. In fact, this
interaction is known to be non-covalent: see among others, Tormo, J. R.;
González, M. C.; Cortes, D.; Estornell, E. Arch. Biochem. Biophys. 1999, 369,
119.
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