Marine-derived 2-aminoimidazolone alkaloids. Leucettamine B-related polyandrocarpamines inhibit mammalian and protozoan DYRK & CLK kinases

Article abstract of DOI:10.3390/md15100316

A large diversity of 2-aminoimidazolone alkaloids is produced by various marine invertebrates, especially by the marine Calcareous sponges Leucetta and Clathrina. The phylogeny of these sponges and the wide scope of 2-aminoimidazolone alkaloids they produce are reviewed in this article. The origin (invertebrate cells, associated microorganisms, or filtered plankton), physiological functions, and natural molecular targets of these alkaloids are largely unknown. Following the identification of leucettamine B as an inhibitor of selected protein kinases, we synthesized a family of analogues, collectively named leucettines, as potent inhibitors of DYRKs (dual-specificity, tyrosine phosphorylation regulated kinases) and CLKs (cdc2-like kinases) and potential pharmacological leads for the treatment of several diseases, including Alzheimer’s disease and Down syndrome. We assembled a small library of marine sponge- and ascidian-derived 2-aminoimidazolone alkaloids, along with several synthetic analogues, and tested them on a panel of mammalian and protozoan kinases. Polyandrocarpamines A and B were found to be potent and selective inhibitors of DYRKs and CLKs. They inhibited cyclin D1 phosphorylation on a DYRK1A phosphosite in cultured cells. 2-Aminoimidazolones thus represent a promising chemical scaffold for the design of potential therapeutic drug candidates acting as specific inhibitors of disease-relevant kinases, and possibly other disease-relevant targets.

Full text of DOI:10.3390/md15100316

marine drugs
Article
Marine-Derived 2-Aminoimidazolone Alkaloids.
Leucettamine B-Related Polyandrocarpamines Inhibit
Mammalian and Protozoan DYRK & CLK Kinases
Nadège Loaëc 1 , Eletta Attanasio , Benoît Villiers , Emilie Durieu , Tania Tahtouh ,
,2
1
1
1
1
1
3
ID
4
5
Morgane Cam , Rohan A. Davis
, Aline Alencar , Mélanie Roué ,
5
6
7
7
Marie-Lise Bourguet-Kondracki , Peter Proksch , Emmanuelle Limanton , Solène Guiheneuf ,
François Carreaux *, Jean-Pierre Bazureau *, Michelle Klautau * and Laurent Meijer *
7
,
7,
4,
1,
ID
1
ManRos Therapeutics, Perharidy Research Center, 29680 Roscoff, Bretagne, France;
nadege.loaec@univ-brest.fr (N.L.); attanasio@manros-therapeutics.com (E.A.); bvilliers@yahoo.fr (B.V.);
durieu@manros-therapeutics.com (E.D.); tania.tahtouh@gmail.com (T.T.); cam.morgane@live.fr (M.C.)
Station Biologique de Roscoff, CNRS, ‘Protein Phosphorylation and Human Disease’ Group, Place G.
Teissier, 29680 Roscoff, Bretagne, France
Griffith Institute for Drug Discovery, Griffith University, Brisbane, QLD 4111, Australia;
r.davis@griffith.edu.au
Universidade Federal do Rio de Janeiro, Instituto de Biologia—Departamento de Zoologia, Av. Carlos
Chagas Filho 373-CCS-Bloco A-Sala A0-100, Ilha do Fundão, 21941-902 Rio de Janeiro, Brazil;
aalencar@biologia.ufrj.br
Molécules de Communication et Adaptation des Micro-Organismes, UMR 7245 CNRS, Muséum National d’
Histoire Naturelle, 57 rue Cuvier (C.P. 54), 75005 Paris, France; mroue@mnhn.fr (M.R.);
bourguet@mnhn.fr (M.-L.B.-K.)
2
3
4
5
6
7
Institut für Pharmazeutische Biologie und Biotechnologie, Universitätsstr. 1, 40225 Düsseldorf, Germany;
proksch@duesseldorf-uni.de
Université de Rennes 1, Institut des Sciences Chimiques de Rennes, ISCR UMR CNRS 6226, Groupe Chimie
Organique et Interfaces (CORINT), Bât. 10A, Campus de Beaulieu, Avenue du Général Leclerc, CS 74205,
3
5042 Rennes CEDEX, Bretagne, France; emmanuelle.limanton@univ-rennes1.fr (E.L.);
solene.guiheneuf@univ-rennes1.fr (S.G.)
*
Correspondence: francois.carreaux@univ-rennes1.fr (F.C.); jean-pierre.bazureau@univ-rennes1.fr (J.-P.B.);
mklautau@biologia.ufrj.br (M.K.); meijer@manros-therapeutics.com (L.M.); Tel.: +33-223-235-734 (F.C.);
+
33-223-236-603 (J.-P.B.); +55-21-3938-6551 (M.K.); +33-608-605-834 (L.M.)
Received: 30 August 2017; Accepted: 12 October 2017; Published: 17 October 2017
Abstract: A large diversity of 2-aminoimidazolone alkaloids is produced by various marine
invertebrates, especially by the marine Calcareous sponges Leucetta and Clathrina. The phylogeny of
these sponges and the wide scope of 2-aminoimidazolone alkaloids they produce are reviewed in this
article. The origin (invertebrate cells, associated microorganisms, or filtered plankton), physiological
functions, and natural molecular targets of these alkaloids are largely unknown. Following the
identification of leucettamine B as an inhibitor of selected protein kinases, we synthesized a family of
analogues, collectively named leucettines, as potent inhibitors of DYRKs (dual-specificity, tyrosine
phosphorylation regulated kinases) and CLKs (cdc2-like kinases) and potential pharmacological
leads for the treatment of several diseases, including Alzheimer’s disease and Down syndrome.
We assembled a small library of marine sponge- and ascidian-derived 2-aminoimidazolone alkaloids,
along with several synthetic analogues, and tested them on a panel of mammalian and protozoan
kinases. Polyandrocarpamines A and B were found to be potent and selective inhibitors of DYRKs
and CLKs. They inhibited cyclin D1 phosphorylation on a DYRK1A phosphosite in cultured cells.
2-Aminoimidazolones thus represent a promising chemical scaffold for the design of potential
therapeutic drug candidates acting as specific inhibitors of disease-relevant kinases, and possibly
other disease-relevant targets.
Mar. Drugs 2017, 15, 316; doi:10.3390/md15100316
www.mdpi.com/journal/marinedrugs

Mar. Drugs 2017, 15, 316
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Keywords: marine sponge; Porifera; Calcarea; ascidian; Polyandrocarpa; 2-aminoimidazolone
alkaloids; leucettamine B; leucettine; polyandrocarpamines; protein kinases; DYRK; CLK; kinase
inhibitor; Alzheimer’s disease; Down syndrome
1
. Introduction
Protein kinases catalyze the phosphorylation of proteins on serine, threonine and tyrosine residues [
They represent a large family of intracellular regulators which, after their initial discovery by Edwin
Krebs and Edmund Fisher in the 1950s [ ], has seen massive research investment on their functions and
1].
2
regulations. The human kinome comprises 518 kinases, many of which display some abnormal activity or
deregulation in human disease. This prompted both academic and pharmaceutical laboratories to search
for, optimize, characterize and develop pharmacological inhibitors of selected disease-relevant protein
kinases. As a matter of fact, kinases have become the leading therapeutic target in the pharmaceutical
industry, before G-protein coupled receptors, and over 30 inhibitors have already reached the clinical
market [1,3,4]. Although many inhibitors were initially derived from natural products, all drug candidates
are chemically synthesized at some stage of their development towards a therapeutic drug.
During a general screen for inhibitors of cell signaling and cell cycle control kinases, we identified
the marine sponge product leucettamine B as a weak inhibitor of various protein kinases [5,6].
Following this initial discovery, a large number of analogues and derivatives were synthesized
and optimized as kinase inhibitors, especially as inhibitors of the DYRKs (dual-specificity, tyrosine
regulated kinases) and CLKs (cdc2-like kinases) kinase families. Leucettamine B-derived inhibitors
are collectively named leucettines [6,7]. These inhibitors have a potential as drug candidates for the
treatment of cognitive disorders associated with Down syndrome (DS) and Alzheimer’s disease (AD),
but also for some cancers, inflammation, diabetes, and so forth.
Leucettamine B was initially identified in the marine, calcareous sponge Leucetta microraphis [8].
The physiological function of this marine alkaloid is unknown, as is the biosynthetic pathway
leading its production. Furthermore, whether leucettamine B is produced by the sponge itself or
by some associated microorganisms remains to be determined. Interestingly, leucettamine B shows a
central 2-aminoimidazolone scaffold shared by several marine products produced by various marine
sponges, most of which belonging to the class Calcarea, and by other marine invertebrates, such as
ascidians and nudibranchs (Figure 1). Several extensive reviews on these marine alkaloids [9–11]
and 2-aminoimidazolones in general [12] have been recently published. We thought it might be
interesting to obtain some of these leucettamine B-related natural products to evaluate them as
potential kinase inhibitors. This was also an opportunity to review the sponge species producing such
molecules, with particular emphasis on Leucetta and Clathrina species, as well as the wide scope of
2
-aminoimidazolones produced by marine organisms.
OH
OR
O
OH
O
H2N
HN
N
H2N
Me
N
H2N
HN
N
N
O
O
O
Polyandrocarpamine A (R= Me)
Polyandrocarpamine B (R= H)
Leucettamine B
Phorbatopsin A
Br
O
Br
H
N
R1HN
R2
2
1
N
Me
NH
HN
3
N
5
HN
N
4
H2N
HN
N
N
O
Me
2-aminoimidazolin-4-one
scaffold
O
O
Aplysinopsine
Dispacamide A
R2
O
NH
R1
R
N
O
H2N
HN
N
O
NH
Me
N
N
NH
Me
O
N
O
Hymenialdisine (R1= H; R2= Br)
Spongiacidin A (R1= Br; R2= Br)
Spongiacidin B (R1= Br; R2= H)
Clathridine (R= O)
Clathridimine (R= NH)
Figure 1. Structures of a selection of marine natural products sharing the 2-aminoimidazolin-4-one scaffold.

Mar. Drugs 2017, 15, 316
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2
. Results
2
.1. Phylogeny of Calcareous Sponges with Special Emphasis on Leucetta and Clathrina
Since most of the compounds described here are produced by marine calcareous sponges, we first
present a phylogeny of these 2-aminoimidazolones-producing sponges, with specific focus on the
Leucetta and Clathrina species. Sponges (phylum Porifera) are sessile, filter-feeding animals that utilize
flagellate cells (choanocytes) to pump water through their bodies. They are morphologically very
simple, as they do not have, for example, organs, sensorial cells or a nervous system. They are
considered the oldest present metazoan (600 Ma) [13]. Four classes of extant sponges are currently
accepted: Demospongiae, Hexactinellida, Homoscleromorpha, and Calcarea.
The class Calcarea Bowerbank, 1864, consists of a monophyletic group of marine sponges whose
skeleton is formed by Mg-calcite spicules, while the others have spicules made of silica [14]. This class
is composed of two subclasses, both monophyletic—Calcinea Bidder, 1898, and Calcaronea Bidder,
1898—which differ by cytological, embryological, ontogenetic and skeleton characteristics. In the
Calcinea subclass, spicules are mainly regular (equiangular and equiradiate) and the first spicule
to be synthesized during ontogeny is the triactine, while in Calcaronea, spicules are irregular and
diactines are the first spicules to be produced. Moreover, the larvae in Calcinea is the calciblastula and
the nucleus of the choanocytes is basal, while in Calcaronea the larvae is the amphiblastula and the
nucleus of the choanocytes is apical. Calcareous sponges are considered viviparous and their larvae
are lecytotrophic. Calcinea has two orders, Clathrinida Hartman, 1958, and Murrayonida Vacelet,
1981, the former being the most diverse. The genera Leucetta Haeckel, 1872, and Clathrina Gray, 1867,
are part of the Clathrinida order (see Supplementary Data and Supplementary Figures S1–S4, and
Supplementary Table S1 for detailed description and phylogeny of Leucetta and Clathrina species).
2
.2. Polyandrocarpamines Are Potent Inhibitors of the DYRK and CLK Kinases
Leucettamine B [ ] shares a 2-aminoimidazolone scaffold with various marine natural
products such as polyandrocarpamines [15 16], dispacamide [17 19], aplysinopsine [20 21],
clathridine/clathridimine [ 22], hymenialdisine/spongiacidin B [23 30] and phorbatopsin [31 32
Figure 1). A summary of all marine aminoimidazolones described so far, and their natural sources
and structures, is presented in Supplementary Table S2 and Supplementary Figure S5.
8
,
–
,
8,
–
, ]
(
We tested a selection of these 2-aminoimidazolones (Figure 1) on a battery of 27 purified kinases
14 mammalian and 13 unicellular parasites (Supplementary Table S3)). Dose-response curves run
(
with the active compounds provided IC50 values that are reported in Table 1. As expected [29,30],
hymenialdisine and the closely related spongiacidin B were very potent inhibitors of most kinases (IC50
values in the 1–10 nM range). Yet they were not very selective, inhibiting essentially all tested kinases.
With the exception of PfGSK-3, the Plasmodium falciparum orthologue of glycogen synthase kinase 3
(GSK-3) [33], and LmCK1, the Leishmania major orthologue of casein kinase 1 (CK1) [34], unicellular
parasite kinases tended to be less sensitive than their mammalian counterparts.
Aplysinopsine, dispacamide, clathridine and clathridimine were essentially inactive in the
kinase panel. As expected [6], leucettamine B was modestly active (sub-micromolar IC50 values),
and leucettine L41 was potent on members of the DYRKs and CLKs families. Again, little inhibitory
activity was detected with kinases from unicellular parasites. Finally, both polyandrocarpamines A
and B were found to display significantly selective inhibitory effects on DYRKs and somewhat less
on CLKs (Table 1). To investigate the selectivity of polyandrocarpamines, polyandrocarpamine A
was screened on the large-scale DiscoveRx KinomeScan panel (Figure 2, Supplementary Table S4).
This interaction assay provides a semi-quantitative scoring view of the affinity of a compound for
any of 442 human kinases [35]. This comprehensive screening approach confirmed the rather good
selectivity of polyandrocarpamine A for DYRKs, and detected additional affinity for CSNK2A1/A2,
CLK1/4 (Table 2).

Mar. Drugs 2017, 15, 316
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Figure 2. TREEspot
™ kinase interaction map of polyandrocarpamine A with 442 human kinases
®
(DiscoveRx KinomeScan , San Diego, CA, USA). Polyandrocarpamine A was tested at a 1 µM final
concentration in the kinase interaction panel. A semi-quantitative scoring of this primary screen was
estimated. This score relates to a probability of a hit rather than strict affinity. Scores >10, between
1–10 and <1 indicate that the probability of being a false positive is <20%, <10%, <5%, respectively.
Results are presented in Table 2 and here as a TREEspot kinase interaction maps. Circles indicate the
™
major hits and their size is proportional to the scores. All 442 values are provided in the Supplementary
Table S4.

Mar. Drugs 2017, 15, 316
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Table 1. Protein kinase selectivity of a selection of 10 marine natural products sharing the 2-aminoimidazolone scaffold: leucettamine B and its synthetic leucettine L41
derivative; polyandrocarpamines A & B, aplysinopsine, dispacamide, hymenialdisine, spongiacidin B, clathridine, and clathridimine. All compounds were tested at
various concentrations against 27 purified kinases (14 mammalian and 13 unicellular parasites). IC50 values (concentrations inducing 50% inhibition of maximal,
non-inhibited kinase activity), calculated from the dose-response curves, are reported in µM. IC50 values below 1 µM are shown in bold.
Kinases
Leucettamine B
Leucettine L41
Polyandrocarpamine A
Polyandrocarpamine B
Aplysinopsine Dispacamide
Hymenialdisine
Spongiacidin B
Clathridine
Clathridimine
CDK1/cyclin B
CDK2/cyclin A
CDK5/p25
CDK9/cyclin T
CK1δ/ε
CLK1
CLK2
CLK3
CLK4
DYRK1A
DYRK1B
DYRK2
>10
>10
>10
>10
>10
0.1
0.91
9.7
0.12
0.42
1.8
0.49
0.6
7.3
>10
0.79
>10
>10
4.2
>10
>10
>10
>10
4.2
0.039
0.21
2.4
0.031
0.032
0.05
0.088
0.42
1.1
>10
>10
>10
0.93
3.3
1.0
1.2
2.6
0.32
0.27
0.43
0.42
0.17
>10
>10
0.65
>10
>10
5.9
>10
>10
>10
5.2
>10
2.7
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.005
0.011
0.0061
0.011
0.0037
0.003
0.0041
0.0053
0.0029
0.0033
0.0032
0.0031
0.0049
0.0032
0.0041
0.011
0.0025
0.4
0.031
0.068
0.042
0.2
0.031
0.022
0.0073
0.049
0.0036
0.78
0.0058
0.021
0.044
0.044
0.038
0.085
0.025
5.2
9.0
0.5
0.3
>10
0.71
1.1
0.13
0.12
>10
>10
>10
>10
>10
6.9
6.0
>10
3.9
>10
>10
>10
>10
>10
7.2
6.1
>10
3.5
5.2
5.6
6.2
6.1
>10
>10
>10
>10
>10
>10
>10
7.9
>10
>10
>10
>10
>10
>10
4.9
8.6
0.82
0.47
1.2
0.76
0.88
>10
>10
3.1
>10
>10
>10
>10
>10
>10
>10
>10
0.2
7.8
>10
7.1
DYRK3
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
GSK-3α/β
PfGSK-3
PfCLK1
LmCK1
LmCLK
LmDYRK2
LdDYRK1B
LdDYRK3
LdDYRK4
TbCLK1
TcCLK1
CpLAMMER
GlCLK
8.5
0.42
>10
>10
2.9
0.82
>10
>10
>10
>10
0.1
0.38
6.9
1.1
0.042
0.021
>10
0.53
0.51
0.016
0.012
0.041
>10
>10
>10
>10
1.8
>10
>10
>10
>10
0.17
0.95
>10
3.1
4.1
>10
>10
2.2
>10
TgCLK
0.48

Mar. Drugs 2017, 15, 316
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Table 2. Protein kinase selectivity of polyandrocarpamine A in a kinase interaction assay (DiscoveRx
®
KinomeScan ). Polyandrocarpamine A was tested at 1
µM on a 442 kinases interaction panel.
A semi-quantitative scoring of this primary screen was obtained. This score relates to a probability of a
hit rather than strict affinity. Scores >10, between 1–10 and <1 indicate that the probability of being
a false positive is <20%, <10%, <5%, respectively. The 11 best scores are presented. Full results are
available in Supplementary Table S4.
Kinases
Abbreviation
Score
Dual specificity tyrosine-phosphorylation-regulated kinase 1A
Casein kinase 2 α
DYRK1A
CSNK2A1
HIPK3
3
12
19
21
22
24
25
26
28
30
32
Homeodomain interacting protein kinase 3
Proviral Integrations of Moloney virus 2
Ribosomal protein S6 kinase alpha-4 (Kin.Dom.2-C-terminal)
Dual specificity tyrosine-phosphorylation-regulated kinase 2
Dual specificity tyrosine-phosphorylation-regulated kinase 1B
Casein kinase 2 α’
PIM2
RPS6KA4
DYRK2
DYRK1B
CSNK2A2
IRAK1
Interleukin-1 receptor-associated kinase 1
Cdc2-like kinase 4
CLK4
CLK1
Cdc2-like kinase 1
To confirm the effects of the active products on native DYRK1A in a cellular context, we made use
of a SH-SY5Y neuroblastoma cell line expressing human DYRK1A [36] and antibodies directed against
cyclin D1 phosphorylated at Threonine 286, a reported DYRK1A phosphorylation site [36
SH-SY5Y-DYRK1A cells were exposed for 24 h to each product (30 M, except for hymenialdisine
and leucettine L41, which were tested at 3 M), cells were harvested and their proteins resolved by
,37] (Figure 3).
µ
µ
SDS-PAGE, followed by Western blotting with antibodies against P-Thr286-cyclin D1, total cyclin D1
and GAPDH (loading control) (Figure 3). Phosphorylation of cyclin D1 at Thr286 was partially inhibited
by polyandrocarpamine A (better than polyandrocarpamine B, leucettine L41 and hymenialdisine,
confirming the potent effects on DYRK1A in a cellular context.
Figure 3. Inhibition of cyclin D1 Thr286 phosphorylation. SH-SY5Y cells expressing human
DYRK1A were treated for 24 h with dimethylsulfoxide (DMSO) (
−
) or 30 µM of each compound
(
+) (3 M for leucettine L41 and hymenialdisine). Proteins were resolved by SDS-PAGE and
µ
analyzed by Western blotting with antibodies directed against P-T286-cyclin D1, total cyclin D1
or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (used as loading control). Numbers in
parentheses indicate the level of cyclin D1 phosphorylation relative to that of control, DMSO-treated
cells (100%).
To extend these results we synthesized both polyandrocarpamines, as well as a series of
derivatives [38] (Figure 4), and tested them on a panel of 16 purified kinases. Synthesized (Table 3,
PAC 1 and 2) and natural (Table 3, PAC 12 and 13) polyandrocarpamines showed very similar effects
on the target kinases. In contrast, none of the synthetic analogues displayed significant efficacy in this
kinase panel.

Mar. Drugs 2017, 15, 316
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Figure 4. Structure of synthetic analogues and derivatives of polyandrocarpamines (PAC1-PAC11)
used in this study.
Table 3. Protein kinase selectivity of synthetic products derived from polyandrocarpamines A and B.
All compounds were tested at various concentrations on 16 purified kinases (14 mammalian and two
unicellular parasites). IC50 values, calculated from the dose-response curves, are reported in
µM. IC50
values below 1 M are shown in bold. PAC1 and PAC2, PAC12 and PAC13 are synthetic and natural
µ
polyandrocarpamines A and B, respectively.
Polyandrocarpamines (PAC)
Kinases
1
2
3
4
5
6
7
8
9
10
11
12
13
CDK1/cyclin B
CDK2/cyclin A
CDK5/p25
CDK9/cyclin T
CK1δ/ε
>10
>10
>10
1.1
5.2
0.43
1.2
2.3
0.4
0.23
0.4
0.14
0.28
>10
>10
>10
>10
>10
>10
2
>10
1
2.5
9.2
0.95
0.75
1.1
0.48
0.9
>10
>10
>10
>10
≥10
>10
>10
≥10
2.2
6.3
>10
2.3
2.1
8.2
2.2
>10
>10
>10
>10
>10
≥10
>10
>10
≥10
≥10
>10
>10
≥10
4
>10
4.5
5.1
>10
>10
>10
>10
>10
>10
≥10
>10
2.2
7.5
>10
2.4
1.3
6.1
0.64
5.7
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.51
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
3.6
≥10
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.63
>10
>10
>10
>10
>10
>10
>10
>10
>10
3
>10
3.4
>10
>10
>10
>10
>10
>10
>10
>10
>10
4.2
≥10
>10
3.2
2.1
6.2
1.3
2.4
>10
>10
>10
0.9
>10
>10
>10
1.8
>10
0.59
2.42
10
0.91
0.59
0.96
0.38
0.68
>10
>10
>10
5
CLK1
CLK2
CLK3
CLK4
DYRK1A
DYRK1B
DYRK2
0.47
0.64
2.7
0.47
0.3
0.47
0.24
0.34
>10
>10
>10
DYRK3
GSK-3α/β
PfGSK-3
LmCK1
>10
>10
>10
2
>10
>10
3
. Discussion
Calcareous sponges of the Leucetta and Clathrina genera and a few other marine invertebrates
produce a wealth of related yet diverse 2-aminoimidazolone alkaloids (Supplementary Table S2 and
Supplementary Figure S5). The existence of these alkaloids in several Calcareous sponges raises several
questions regarding their production. What is/are the biosynthetic pathway/s involved (see reference
19 for first hypothesis)? Are these molecules actually produced by the sponges or by associated
microorganisms? In addition, these molecules may derive from a plankton source taken up (and
possibly metabolized) by these filter-feeders. The fact that polyandrocarpamines were identified in
ascidians (Polyandrocarpa sp.) [15] and that most 2-aminoimidazolones were discovered in calcareous
sponges of the Leucetta and Clathrina genera (Supplementary Table S2) supports the hypothesis
of microorganism-derived metabolites. However, cellular localization of clathridimine revealed
its presence in sponge cells rather than in associated bacteria [22]. Several 2-aminoimidazolones

Mar. Drugs 2017, 15, 316
8 of 15
were found in Notodoris nudibranchs and in the calcareous sponges they crawl and feed on [39–41].
Altogether these scattered results suggest possible transfers of 2-aminoimidazolones, their precursors
and/or metabolites from one organism to another.
Many questions remain open regarding the physiological/ecological functions of these natural
products. Are they used as repellant for predators or to control the expansion of associated
microorganisms? Do they have specific functions for the sponge/ascidian/nudibranch metabolism
itself? What are their natural molecular and cellular targets? Are they synthesized as kinase inhibitors
or do they target other enzymes or structural proteins? Some of these may have orthologues in humans.
The biological source, functions and natural targets of marine 2-aminoimidazolone alkaloids should be
further investigated.
Only a few of these 2-aminoimidazolones have been chemically synthesized (Supplementary
Table S2, right column) and are thus relatively easy to access for biological studies of their
pharmacological properties. Nevertheless, they have already proved to be promising scaffolds
for the design of potential therapeutic drug candidates. The identification of leucettamine B [6]
and polyandrocarpamines A and B [this article] as potent and relatively selective inhibitors
of disease-relevant kinases opens the way to the development of drug candidates for various
applications [
various kinases which have been solved [
leucettines as DYRK1A inhibitory drug candidates [6,7,42,43] to address cognitive deficits associated
7,
42]. This could be guided by the crystal structures of leucettines in complex with
7]. At ManRos Therapeutics we are currently developing
with DS [Nguyen et al., in preparation] and AD [44
diabetes treatment should be investigated, too.
,45], but applications in cancer, inflammation and
Besides kinase inhibition, polyandrocarpamine derivatives have been recently identified
as inhibitors of cystathionine -synthase, another potential therapeutic target for DS [46].
-Aminoimidazolones have been tested for evaluation of their antibacterial activity, anti-biofilm,
β
2
antifungal, anticancer [32,47], anti-tuberculosis, anti-protozoan, antiviral, anti-inflammatory,
adrenergic, anti-histaminic, anti-cholinergic, anti-serotoninergic, cardiac, immunosuppressive,
leukotriene B receptor antagonistic [ 48] and AD drug activities [12]. It would be of great interest to
8,
4
run more marine derived 2-aminoimidazolone alkaloids on wider enzyme/cell screening panels to
identify novel targets and potentially novel therapeutic applications. Hopefully these molecules will
add to the growing list of marine drugs and drug candidates developed in recent years [49,50].
4
. Experimental Section
4
.1. DNA Sequencing, Alignment and Phylogenetic Analyses
The methods used for molecular phylogenetic analyses of Leucetta and Clathrina species are
detailed in the Supplementary Data.
4
.2. Chemistry: Purification or Synthesis of Polyandrocarpamines A and B, and Analogues
For the purification of clathridine and clathridimine, specimens of Clathrina clathrus were collected
by scuba diving in the northwestern Mediterranean Sea (Marseille, France) between 10 and 15 m depth.
Lyophilized samples (4.2 g) were extracted with 1:1 CH Cl /MeOH (3 20 mL, sonication for 15 min)
at room temperature. The 1:1 CH Cl /MeOH extract was concentrated under reduced pressure to
×
2
2
2
2
yield a dark brown viscous oil (208.1 mg), which was chromatographed on a C18 SPE column in
a vacuum chamber (H O, H O/MeOH 1:3, MeOH, CH Cl , 100 mL of each). The fraction eluted
2
2
2
2
with H O/MeOH, 1:3 (47.9 mg) was subjected to semi-preparative reversed-phase HPLC (Gemini
2
C -phenyl 10
×
250 mm) with increasing amounts of CH CN/0.1% formic acid in H O/0.1% formic
3 2
6
acid as eluent (flow rate: 5 mL/min, wavelength: 254 and 280 nm) and afforded clathridimine (4.6 mg,
0
.11% of sponge dry weight) and clathridine (2.8 mg, 0.07% of sponge dry weight).
PAC1 to 11 were supplied by Rohan A. Davis The synthesis, purification and spectroscopic
characterization of this focused polyandrocarpamine library has been described elsewhere [38].

Mar. Drugs 2017, 15, 316
9 of 15
Aplysinopsine and leucettamine B were synthesized according to a previously described
protocol [51,52] respectively. Dispacamide was synthesized as described in [53].
The synthesis of polyandrocarpamines A & B (PAC1 and PAC2) was performed
from corresponding benzaldehyde derivatives, n-propylamine and 2-aminoimidazolin-4-one,
the preparation of which was previously described in literature [53] (Figure 5). Guanidine (2.95 mmol,
◦
1
eq) and HClaq 6N (9 mL) were heated at 120 C for 22 h. Water was removed under vacuum and the
crude product was solubilized in hot ethanol (9 mL). Diethylether (6 mL) was added, then the mixture
◦
was cooled to 0 C. After 6 h, filtration was performed and the 2-aminoimidazolin-4-one was isolated
1
13
as a white powder with a 49% yield. RMN H (D O):
C-1); 174.9 (C=O); [M
then produced by mixing the corresponding benzaldehyde (vanillin or catechaldehyde 1.48 mmol,
δ
4.18 (s, 2H, H-1); RMN C (D O):
δ
48.5
2
2
+
◦
(
−
HCl] = 99.0427 (99.0433 C H N O); F = 200–202 C. PAC1 and PAC2 were
3
5
3
1
eq) with n-propylamine (2.95 mmol, 2 eq). The imine formation was performed under microwave
◦
irradiation at 60 C for 30 min (maximized authorized power 60 W). Acetonitrile was added to the
crude imine with 2-aminoimidazolin-4-one (1.48 mmol, 1 eq), and the mixture was stirred at reflux for
6
3 h. The solvent was removed under reduced pressure.
PAC1 was isolated as a beige powder with a 71% yield. RMN H (DMSO-d ):
1
δ
3.79 (s, 3H, OCH3);
6
6
9
.23 (s, 1H, C=CH); 6.76 (d, 1H, J = 8.0 Hz, H-5); 7.10 (sl, 2H, NH ); 7.35 (sl, 1H, H-6); 7.56 (sl, 1H, H-2);
2
.24 (sl, 1H, OH); 10.38 (sl, 1H, NH); RMN 13C (DMSO-d ):
δ
56.2 (OCH3); 114.6 (C-2); 116.0 (C-5);
6
+
◦
1
24.0 (C=CH); 127.6 (C-6); 147.1 (C-3); 147.9 (C-4); [M] = 233.0797 (233.0800 C H N O3); F > 260 C.
11
11
3
1
PAC2 was isolated as a beige powder with a 80% yield. RMN H (DMSO-d ):
δ
6.14 (s, 1H, C=CH);
6
6
.70 (d, 1H, J = 8.1 Hz, H-5); 6.92 (s, 2H, NH ); 7.27 (d, 1H, J = 7.5 Hz, H-6); 7.53 (s, 1H, H-2); 8.81 (s,
2
13
1H, NH); 9.19 (s, 1H, OH); 10.45 (s, 1H, OH); RMN C (DMSO-d ):
δ
112.5 (C=CH); 116.6 (C-5); 118.2
6
◦
(C-2); 123.1 (C-6); 128.2 (C-1); 146.0 (C-3); 146.6 (C-4); F > 260 C.
Figure 5. Scheme of the synthesis of polyandrocarpamines.
4
.3. Biology
4
.3.1. Buffers
Homogenization buffer: 25 mM MOPS; 15 mM EGTA; 15 mM MgCl ; 60 mM
β
-glycerophosphate;
5 mM p-nitrophenylphosphate; 2 mM dithiothreitol (DTT); 1 mM Na3VO4; 1 mM NaF; 1 mM
di-sodium phenylphosphate; 1X protease inhibitor cocktail; 0.2% Nonidet P-40 substitute.
2
1
Buffer A: 10 mM MgCl ; 1 mM EGTA (MW 380.4); 1 mM DTT (MW 154.2); 25 mM Tris/HCl (MW
2
1
21.1), and; 50 µg/mL heparin.
Buffer C: 60 mM
β-glycerophosphate; 30 mM p-nitrophenylphosphate; 25 mM MOPS pH 7.0;
5
mM EGTA; 15 mM MgCl ; 1 mM DTT, and; 0.1 mM sodium vanadate.
2

Mar. Drugs 2017, 15, 316
10 of 15
All chemicals were purchased from Sigma Aldrich (St. Quentin Fallavier, France), unless otherwise
stated and the protease inhibitor cocktail was from Roche (Boulogne-Billancourt, France).
4
.3.2. Production and Purification of the Parasitic Kinases
The parasitic kinase genes (Supplementary Data, Table S3) were optimized for expression in
E. coli, synthesized, and cloned in pGEX-6P-1 (GE Healthcare, Sigma-Aldrich, St. Quentin Fallavier,
France) with the type II restriction enzymes BamHI and XhoI for glutathione S-transferase (GST)
fusion at the N-terminus (GenScript). Plasmids were transformed into chemically competent E. coli
◦
BL21(DE3), which were grown overnight at 37 C in 2
These cultures were used to inoculate 1 L volumes of 2
×
YT medium containing 100
µ
g/mL ampicillin.
×
YT medium (containing 100
µg/mL ampicillin)
◦
in 5 L flasks. The cultures were allowed to grow at 37 C before the temperature was decreased to
8 C. At an optical density at 600 nm (OD600) of about 1.0, protein expression was induced overnight
◦
1
◦
at 18 C with 0.1 mM isopropyl
β
-D-1-thiogalactopyranoside (IPTG). The bacteria were harvested by
◦
centrifugation and were frozen at −20 C.
Cells were resuspended in a lysis buffer (PBS pH 7.4, 1% NP40, 1 mM DTT, 1 mM EDTA, 1 mM
PMSF) in the presence of protease inhibitor cocktail (Roche), and lysozyme was added to 1 mg/mL
◦
and incubated 1 h at 4 C with gentle agitation. Cells were then sonicated on ice before 6 mM MgCl
2
and 25 U/mL benzonase (Novagen, Merck Millipore, Fontenay sous Bois, France) were added to
◦
degrade nucleic acids and decrease sample viscosity. After centrifugation at 4 C, the soluble fraction
was collected and proteins were bound to Glutathione sepharose 4B beads (GE Healthcare). Beads
were washed three times with lysis buffer and once with modified buffer C (25 mM MOPS pH 7.0,
6
1
0 mM
β-glycerophosphate, 15 mM p-nitrophenylphosphate, 15 mM EGTA, 15 mM MgCl , 2 mM DTT,
2
mM sodium vanadate). Proteins were eluted with elution buffer (buffer C containing 30 mM reduced
glutathione, 15% glycerol, and pH adjusted to 8.5).
4
.3.3. Protein Kinase Assays
Kinase activities were assayed in buffer A or C at 30 C at a final ATP concentration of 15
◦
µmol/L.
Blank values were subtracted and activities were expressed in percent of the maximal activity, i.e., in the
absence of inhibitors. Controls were performed with appropriate dilutions of DMSO.
The GS-1, CKS, CDK7/9 tide and RS peptide substrates were obtained from Proteogenix
(Oberhausbergen, France).
CDK1/cyclin B (M phase starfish oocytes, native), CDK2/cyclin A and CDK5/p25 (human,
recombinant) were prepared as previously described [6,7]. Their kinase activity was assayed in buffer
3
3
A, with 1 mg histone H1/mL, in the presence of 15
in a final volume of 30
µ
mol/L [
γ
- P] ATP (3000 Ci/mmol; 10 mCi/mL)
◦
µ
L. After 30 min incubation at 30 C, the reaction was stopped by harvesting
onto P81 phosphocellulose supernatant (Whatman, Dutscher SAS, Brumath, France) using a FilterMate
harvester (PerkinElmer, Courtaboeuf, France) and were washed in 1% phosphoric acid. Scintillation
fluid was added and the radioactivity measured in a Packard counter.
CDK9/cyclin T (human, recombinant, expressed in insect cells) was assayed as described
for CDK1/cyclin B, but using CDK7/9 tide (YSPTSPSYSPTSPSYSPTSPSKKKK) (8.1
a substrate.
µg/assay) as
GSK-3α/β (porcine brain, native) and PfGSK3 (plasmodium falciparum, recombinant, expressed in
E. coli as GST fusion proteins) was assayed, as described for CDK1 with 0.5 mg BSA/mL + 1 mM DTT,
using GS-1 (YRRAAVPPSPSLSRHSSPHQSpEDEEE) (pS stands for phosphorylated serine), a GSK-3
specific substrate [54].
CK1δ/ε (porcine brain, native) and LmCK1 (leishmania major, recombinant, expressed in E. coli as
HIS fusion proteins [34]) was assayed as described for CDK1 but in buffer C and using 25
peptide (RRKHAAIGpSAYSITA), a CK1-specific substrate [55].
µM CKS
DYRK1A, 1B, 2 and 3 (Human, recombinant, expressed in E. coli as GST fusion proteins) were
purified by affinity chromatography on glutathione-agarose and assayed as described for CDK1/cyclin

Mar. Drugs 2017, 15, 316
11 of 15
B with with 0.5 mg BSA/mL + 1 mM DTT and using Woodtide (KKISGRLSPIMTEQ) (1.5
a substrate, a residue of transcription factor FKHR.
µg/assay) as
CLK1, 2, 3 and 4 (mouse, recombinant, expressed in E. coli as GST fusion proteins) were assayed
as described for CDK1/cyclin B with 0.5 mg BSA/mL + 1 mM DTT and RS peptide (GRSRSRSRSRSR).
Protozoan DYRKs and CLKs (recombinant, expressed in E. coli) were assayed as described for
CDK1/cyclin B with 0.5 mg BSA/mL + 1 mM DTT and Woodtide or RS peptide for DYRKs or
CLKs isoforms.
4
.3.4. Kinase Interaction Panel (Ambit Biosciences/DiscoveRx)
Assays were performed essentially as described previously [35]. For most assays, kinase-tagged
T7 phage strains were grown in parallel in 24-well blocks in an E. coli host derived from the BL21 strain.
E. coli were grown to log-phase and infected with T7 phage from a frozen stock (multiplicity of infection
◦
~
0.1) and incubated with shaking at 32 C until lysis (~90 min). The lysates were centrifuged (6000
×
g)
and filtered (0.2 µm) to remove cell debris. The remaining kinases were produced in HEK-293 cells and
subsequently tagged with DNA for qPCR detection. Streptavidin-coated magnetic beads were treated
with biotinylated small molecule ligands for 30 min at room temperature (RT) to generate affinity resins
for kinase assays. The liganded beads were blocked with excess biotin and washed with blocking
buffer (SeaBlock (Pierce, ThermoFisher Scientific, Illkirch, France), 1% BSA, 0.05% Tween 20, 1 mM
DTT) to remove unbound ligand and to reduce non-specific phage binding. Binding reactions were
assembled by combining kinases, liganded affinity beads, and test compounds in 1
20% SeaBlock, 0.17 PBS, 0.05% Tween 20, 6 mM DTT). An 11-point threefold serial dilution of each
test compound was prepared in 100% DMSO at 100 final test concentration which and subsequently
diluted to 1 in the assay. All reactions were performed in polystyrene 96-well plates in a final volume
of 0.135 mL. The assay plates were incubated at RT with shaking for 1 h and the affinity beads were
washed four times with wash buffer (1 PBS, 0.05% Tween 20). The beads were then resuspended in
elution buffer (1 PBS, 0.05% Tween 20, 0.5 M non-biotinylated affinity ligand) and incubated at RT
with shaking for 30 min. The kinase concentration in the eluates was measured by qPCR.
×
binding buffer
(
×
×
×
×
×
µ
4
.3.5. Thr286-Cyclin D1 Phosphorylation in SH-SY5Y Cells
Cell treatments. SH-SY5Y cells conditionally expressing human DYRK1A [36], under the control of
doxycycline (gift from Dr. Walter Becker) were cultured Dulbecco’s Modified Eagle Medium (DMEM):
Nutrient Mixture F-12 (DMEM/F-12, Gibco, c/o Invitrogen, Saint Aubin, France) containing 1%
Penicillin Streptomycin mixture (Gibco) and 10% fetal bovine serum (FBS, Gibco) in a humidified, 5%
◦
CO incubator at 37 C. Cells were split routinely once a week. They were first rinsed with phosphate
2
◦
buffered saline (PBS, Gibco) and detached from the plate bottom using 2 mL Trypsin (Gibco) at 37 C
for 3–4 min. Eight mL of fresh medium were added to the cell suspension and the mix was centrifuged
1
for 3 min at 1000 rpm. The cell pellet was resuspended in fresh medium before seeding ( dilution) in
2
new 150 mm plates. First, 1
were incubated 24 h in a humidified, 5% CO incubator at 37 C. Drugs (0, 3 or 30
µ
g/mL doxycycline was used to induce DYRK1A expression and cells
◦
µM final) were then
2
added with a final concentration of 0.1% DMSO and cells were incubated for an additional 24 h in a
◦
humidified, 5% CO incubator at 37 C. Afterwards, cells were scrapped in medium and centrifuged
2
◦
for 5 min at 3000 rpm at 4 C. The pellets were washed twice with PBS, centrifuge for 5 min at 10,000 g
◦
◦
at 4 C, snap-frozen in liquid nitrogen and kept at −80 C.
Western blot analysis. Cell pellets were lysed in homogenization buffer. Protein extracts were
mixed (1:1 v/v) with sample buffer (2
×
NuPAGE LDS sample buffer, 200 mM DTT) and 20 µg of total
proteins were loaded on NuPAGE 4–12% Bis-Tris protein gels. Electrophoresis was run at 70 V in
MOPS buffer for 4 h. Rapid blot transfers were performed at 2.5 A/25 V for 7 min. Blotting membranes
were blocked in milk (5% Regilait in Tris Buffered Saline with 0.1% Tween (TBST)) for 1 h. Blotting
◦
membranes were incubated overnight at 4 C with anti-cyclin D1 antibody (1:2000 dilution; Cell
Signaling, Leiden, The Netherlands) or anti-phospho T286-cyclin D1 antibody (1:2000 dilution; Cell

Mar. Drugs 2017, 15, 316
12 of 15
Signaling) or for 1 h at RT with anti-DYRK1A antibody (1:2000 dilution; Abnova, Taoyuan City, Taiwan)
or anti-GAPDH antibody (1:30,000 dilution; Bio-Rad, Marnes-la-Coquette, France). Next, blotting
membranes were incubated for 1 h at RT with goat anti-rabbit or goat anti-mouse antibodies (Bio-Rad).
Chemiluminescent detection was achieved with homemade ECL-Tris buffer (100 mM Tris pH 8.5,
0
.009% H O , 0.225 mM p-coumaric acid, 1.25 mM luminol) with Fusion F × 7 camera software.
2
2
Supplementary Materials: This material is available free of charge via the internet at www.mdpi.com/1660-3397/
5/10/316/s1.
1
Acknowledgments: We are grateful to Walter Becker for providing the DYRK1A-expressing SH-SY5Y cell line.
This research was supported by grants from the “Fonds Unique Interministériel” (FUI) PHARMASEA and TRIAD
projects (Laurent Meijer, Jean-Pierre Bazureau, François Carreaux), “Fondation Jérôme Lejeune” (Laurent Meijer),
ANR grant TRANSLEISH and an EEC FP7-KBBE-2012 grant (BlueGenics) (Laurent Meijer). Morgane Cam is
recipient of a CIFRE/ManRos Therapeutics Ph.D. fellowship. M.K. is funded by a fellowship from the Brazilian
National Research Council (CNPq) and research grants from the CNPq and Coordination for the Improvement of
Higher Education Personnel (CAPES). Rohan A. Davis acknowledges the National Health and Medical Research
Council (NHMRC) for financial support (Grant APP1024314) and thanks the Australian Research Council (ARC)
for support toward NMR and MS equipment (Grants LE0668477 and LE0237908), as well as financial support
(Grant LP120200339).
Author Contributions: Laurent Meijer, Benoît Villiers, Tania Tahtouh conceived and designed the experiments;
Nadège Loaëc, Emilie Durieu and Tania Tahtouh ran the in vitro kinase assays; Benoît Villiers cloned and
expressed the unicellular kinases; Morgane Cam ran the SH-SY5Y experiments; Rohan A. Davis supplied the
synthesized polyandrocarpamine derivatives PAC1 to PAC11; Mélanie Roue, Marie-Lise Bourguet-Kondracki and
Peter Proksch provided clathridine, clathridimine and hymenialdisine, spongiacidin, respectively; Emmanuelle
Limanton, Solène Guiheneuf, François Carreaux and Jean-Pierre Bazureau synthesized and provided leucettamine
B, polyandrocarpamine A/B, aplysinopsine and dispacamide A; Aline Alencar and Michelle Klautau analyzed
the phylogeny of Leucetta and Clathrina sp. and provided a full description of the genera; Eletta Attanasio and
Marie-Lise Bourguet-Kondracki reviewed the 2-aminoimidazolone alkaloids; Laurent Meijer wrote the paper.
Conflicts of Interest: François Carreaux, Jean-Pierre Bazureau and Laurent Meijer are co-inventors on the
leucettine patent. Laurent Meijer is CEO and CSO of ManRos Therapeutics which develops leucettines as DS and
AD drug candidates.
Abbreviations
AD, Alzheimer’s disease; CDKs, cyclin-dependent kinases; CK1, casein kinase 1; CK2, casein kinase 2;
CLKs, cdc2-like kinases; DMEM, Dulbecco’s Modified Eagle Medium; DMSO, dimethylsulfoxide; DS, Down
syndrome; DTT, dithiothreitol; DYRKs, dual-specificity, tyrosine phosphorylation regulated kinases; GAPDH
glyceraldehydes-3-phosphate dehydrogenase; GSH, glutathione; GSK-3, glycogen synthase kinase-3; GST
glutathione-S-transferase; IC50, concentration inducing 50% inhibition of the maximal kinase activity; IPTG
isopropyl β-D-1-thiogalactopyranoside; PBS, phosphate-buffered saline; RT, room temperature.
,
,
,
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