Characterization of sirtuin inhibitors in nematodes expressing a muscular dystrophy protein reveals muscle cell and behavioral protection by specific sirtinol analogues

Article abstract of DOI:10.1021/jm9013345

In oculopharyngeal muscular dystrophy (OPMD), a disease caused by polyalanine expansion in the nuclear protein PABPN1, the genetic inhibition of sirtuins and treatment with sirtuin inhibitors protect from mutant PABPN1 toxicity in transgenic nematodes. He

Full text of DOI:10.1021/jm9013345

J. Med. Chem. 2010, 53, 1407–1411 1407
DOI: 10.1021/jm9013345
Characterization of Sirtuin Inhibitors in Nematodes Expressing a Muscular Dystrophy Protein
Reveals Muscle Cell and Behavioral Protection by Specific Sirtinol Analogues
†
,‡,§
^
†,‡
Dante Rotili, Lucia Altucci, Francesca Farina, Guy A. Rouleau, Antonello Mai,* and
§
,
Matthieu Y. Pasco,
Christian N ꢀe ri*
,†,‡
†
‡
INSERM, Laboratory of Neuronal Cell Biology and Pathology, Center for Psychiatry and Neurosciences U894, Universit eꢀ Paris Descartes,
§
Equipe d’accueil 4059, 75014 Paris, France, University of Montreal, Department of Medicine and the Center for Excellence in Neuromics,
Montreal, Qu eꢀ bec, Canada H2L 4M1, Pasteur Institute;Cenci-Bolognetti Foundation, Dipartimento di Chimica e Tecnologie del Farmaco,
Sapienza University of Rome, 00185 Rome, Italy, and Dipartimento di Patologia Generale, Facolt aꢁ di Medicina e Chirurgia,
Seconda Universit aꢁ di Napoli, Vico L. De Crecchio 7, 80138 Napoli, Italy
^
Received September 8, 2009
In oculopharyngeal muscular dystrophy (OPMD), a disease caused by polyalanine expansion in
the nuclear protein PABPN1, the genetic inhibition of sirtuins and treatment with sirtuin inhibitors
protect from mutant PABPN1 toxicity in transgenic nematodes. Here, we tested the SIRT1/2 inhibitors
1-12, bearing different degrees of inhibition, for protection against mutant PABPN1 toxicity in
Caenorhabditis elegans. Compounds 2, 4, and 11 were the most efficient, revealing a potential
therapeutic application for muscle cell protection in OPMD.
Introduction
gene silencing in Fragile X mental retardation syndrome
under treatment with nicotinamide (NAM) or splitomicin
þ a
Sirtuins are NAD dependent deacetylases, also referred
to as type III histone deacetylases, that modify the acetylation
state of several intracellular messengers, thereby regulating
1
7, Figure 1). Sirtuin inhibition may also be involved in the
1
(
beneficial effect of NAM observed in the triple-transgenic
model of Alzheimer’s disease harboring PS1, APP, and tau
1
downstream mechanisms associated with these substrates.
1
2
mutations. Finally, the genetic (loss-of-function; LOF) and
pharmacological (sirtinol 1, Figure 1) inhibition of sir-2.1/
SIRT1 protects C. elegans transgenics from the toxicity of
polyalanine expansion in the oculopharyngeal muscular dys-
trophy (OPMD) protein PABPN1, an effect that requires the
transcriptional factor and key longevity protein daf-16/
These deacetylases are able to integrate environmental cues
and modulate several important physiological mechanisms
such as inflammation, apoptosis, glucose homeostasis, life
2
span, and neuroprotection. The pharmacological manipula-
tion of theses enzymes has therefore strong potential for the
therapy of many human diseases such as cancer, metabolic
1
3
3-5
FoxO, suggesting that sirtuin inhibitors may be protective
in OPMD through cell survival mechanisms.
disorders, and degenerative diseases.
Sirtuins (SIRT1-7 in mammals) are highly conserved
enzymes. The most studied member of the sirtuin family is
SIRT1. The SIRT1 protein is most related to the Caenorhab-
ditis elegans sirtuin sir-2.1. Transgenic nematodes with in-
Before evaluation of sirtuin modulators in mouse
models of disease, it is important to identify potent lead
compounds and to test whether they may be active in
physiological conditions. Because PABPN1 nematodes
show cellular (progressive loss of muscle cell nuclei) and
behavioral (defective motility) phenotypes amenable to
6
creased sir-2.1 dosage can live up to 50% longer. Recent
studies indicated that, depending on the cellular context and
type of sirtuin considered, either the activation or the inhibi-
7
13
tion of sirtuins may protect against cell/tissue injury.
Whereas sir-2.1/SIRT1 activation may protect against neuro-
drug screening, we hypothesized that these animals may
allow to select for sirtuin inhibitors that best rescue
the toxicity of mutant PABPN1 at the muscle cell and
behavioral levels.
8
nal dysfunction in simple models of HD pathogenesis and
9
models of ALS and Aβ toxicity, the pharmacological inhibi-
tion of SIRT2 may be neuroprotective against R-synuclein
toxicity. Additionally, SIRT1 inhibition may alleviate
In this study, we tested some known SIRT inhibitors,
14,15
1
0
15 15
meta-sirtinol 2, para-sirtinol 3, salermide
such as 1,
1
6
17
7, EX-527 11, and AGK-2 12, prepared by us
18
10
4
,
according to published procedures (Figure 1). In addition,
we tested two new synthetic sirtinol analogues (5 and 6), a
splitomicin derivative (the ketosplitomicin analogue 8),
and the benzodeazaoxaflavines (BDF4s) 9 and 10, two
totally different compounds recently identified in our lab
(Figure 1). Compounds 1-12 were tested for their ability
to inhibit the enzymatic activity of SIRT1 and SIRT2 in
vitro and to modulate mutant PABPN1 toxicity in trans-
genic nematodes.
*
To whom correspondence should be addressed. For C.N.: christian.
neri@inserm.fr. For chemistry, A.M.: phone, þ39 064 991 3392; fax,
þ39 064 91 491; E-mail, antonello.mai@uniroma1.it.
a
Abbreviations: ALS, amyotrophic lateral sclerosis; APP, amyloid
precusor protein; Aβ, β-amyloid peptide; FoxO, forkhead box class O;
GFP, green fluorescent protein; HD, Huntington’s disease; NAD ,
þ
nicotinamide adenine dinucleotide; NAM, nicotinamide; OPMD, ocu-
lopharyngeal muscular dystrophy; PABPN1, polyadenylate-binding
protein, nuclear, 1; PS1, presenilin-1 transmembrane protein; RNAi,
RNA interference; RT-PCR, reverse transcription-polymerase chain
reaction.
r
2
009 American Chemical Society
Published on Web 12/30/2009
pubs.acs.org/jmc

1
408 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Pasco et al.
Figure 1. Structures of sirtuin inhibitors described in this study.
1
3
Chemistry
of a nuclear collapse. Consistent with the finding that
sir-2.1/SIRT1 LOF is protective in these animals, with increased
sir-2.1/SIRT1 dosage being detrimental, we previously reported
that sir-2.1 RNAi rescues the loss of GFP signals in PABPN1-
1
5,16
The syntheses of 1-4 were previously reported by us.
Reaction between 4-nitrobenzenesulfonyl chloride and
-phenylethylamine in the presence of triethylamine
Et N) in dry dichloromethane furnished the 4-nitro-
1
13
A13 animals and that the sirtuin inhibitor 1 is able to rescue
13
mutant PABPN1 toxicity in C. elegans muscle cells. However,
shows a moderate rescue of the loss of GFP signals (ED50
above 1 μM) and defective motility (about 40% rescue com-
pared to untreated animals), suggesting that it may be poorly
effective in mouse models of OPMD pathogenesis.
To search for more promising sirtuin inhibitors, we synthe-
(
3
N-(1-phenylethyl)benzenesulfonamide 13, which was in
turn reduced with stannous chloride and 35% hydrochlo-
ric acid (HCl) in ethanol to the 4-amino analogue 14 and
then condensed with 2-hydroxy-1-naphthaldheyde in the
presence of glacial acetic acid to yield 5 (Scheme S1a
in Supporting Information (SI)). The meta-thioether ana-
logue of sirtinol, 6, was prepared by alkylation of
1
13
2
3
1
5,16
sized a series of sirtinol analogues
by shifting the ortho-
benzamide moiety to the meta or the para position and/or by
replacing the benzamide group with an anilide, sulfonamide,
or thioether function (compounds 2-6, Figure 1), and we
tested them for the inhibition of the enzymatic activity of
SIRT1 and SIRT2 in comparison to 1. We observed that
all compounds were able to inhibit SIRT1 and SIRT2
3-aminothiophenol with 1-bromo-2-phenylpropane in dry
N,N-dimethylformamide (DMF) in the presence of potassium
carbonate, followed by condensation of the obtained inter-
mediate 15 with 2-hydroxy-1-naphthaldheyde in acidic medium
(
Scheme S1b in SI).
A 7 sample was synthesized according to the literature,
while the ketosplitomicin derivative 8 was obtained by
1
9
(
Figure 2A). Compared to 1 (IC s: 123.3 and 45.9 μM against
50
2
0
SIRT1 and SIRT2, respectively), the 2-6 analogues were
more efficient in inhibiting both SIRT1 and SIRT2 (IC50
values: 40.3-58.6 μM (SIRT1) and 19.2-34.5 μM (SIRT2))
see SI). Upon evaluation in PABPN1 nematodes treated with
00-0.1 μM concentrations from the L1 larvae stage until
reaction of ethyl 3-(2-chlorophenyl)-3-oxopropanoate and
-hydroxy-1-naphthaldheyde in dry ethanol with catalytic
2
amounts of piperidine and glacial acetic acid. The 2-(2-chloro-
benzoyl)-3H-benzo[f]chromen-3-one, 16, thus obtained was
reduced with sodium borohydride in dry pyridine to afford
the 1H-benzo[f]chromen-3(2H)-one derivative 8 (Scheme S2
in SI).
The two BDF4 derivatives, 9 and 10, already known in
literature as organic oxidants, were both prepared by con-
densation of the appropriate barbituric acids and 2-chloro-
(
1
young adulthood, almost all the tested compounds were able
to modulate the loss of nuclear GFP signals in mutant
PABPN1-A13 nematodes, with no effect observed in normal
PABPN1-A10 nematodes (Figure 2B). Of note, 6 did not
showany effect on the lossof nuclearGFP signals (Figure2B).
This compound was thus not tested for effect on motility. The
inabilityof6 toreach musclecellsis likely toexplain the lack of
effect because all of the other related compounds were active
in nematodes (Figure 2B). These effects were unrelated to a
reduction in transgene expression as tested by RT-PCR and
Western blotting (Figure S1, SI). However, rescue by 5 was
accompanied by increased PABPN1 expression with no effect
detected in RT-PCR (Figure S1, SI), suggesting that 5 may
stabilize PABPN1. The para-substituted sirtinol analogue 3
enhancedmutant PABPN1 toxicity(Figure2B), it likely being
not specific of sir-2.1 (there are three other sirtuins in worms);
on the other hand, the para-substituted benzenesulfonamide 5
was one of the most effective compounds in rescuing the loss
of nuclear GFP signals (Figure 2B) and defective motility
(Figure 2C), with a Rmax value above 10% and EC50 lower
2
1
22
1
-arylaldheydes in ethanol in the presence of pyridine, as
2
2
described by Chen et al. for the synthesis of 10 (see Scheme
S3 in SI). Compound 11 was synthesized as previously
1
reported, while 12 was purchased from Sigma-Aldrich.
8
Experimental procedures, chemical and physical data
Table S1, SI), and elemental analyses (Table S2, SI) for
(
compounds 5, 6, 8, 13-16 are reported in SI.
Results and Discussion
Adult C. elegans transgenics that specifically coexpress
nuclear GFP and mutant PABPN1 (PABPN1-A13) in muscle
cells show two major phenotypes including defective moti-
lity as reflected by an increased number of body bends, an
effect accompanied by a loss of nuclear GFP signals indicative

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1409
Figure 2. Characterization of sirtinol analogues: (A) SIRT1 and -2 inhibitory activity, expressed as % of deacetylase activity. Compounds
were tested at 50 μM. (B) Ability to rescue mutant PABPN1 (PABPN1-A13) toxicity (loss of nuclear GFP nuclei) in transgenic nematodes in
comparison with that observed in normal PABPN1 (PABPN1-A10) nematodes. (C) Motility assay (number of body bends) in mutant and
normal nematodes. Data are represented as mean ( SEM *P < 0.05 and ***P < 0.01 compared to untreated animals (statistical effects were
resistant to one-way ANOVA test).
Figure 3. Characterization of 7-12: (A) SIRT1 and -2 inhibitory activity, expressed as % of remaining deacetylase activity. Compounds were
tested at 0.5 (11), 5 (12), and 50 (others) μM. (B) Ability to rescue mutant PABPN1 (PABPN1-A13) toxicity (loss of nuclear GFP nuclei) in
transgenic nematodes in comparison with that observed in normal PABPN1 (PABPN1-A10) nematodes. (C) Motility assay (number of body
bends) in mutant and normal nematodes. Data are represented as mean ( SEM *P < 0.05, **P < 0.01 and ***P < 0.001 compared to
untreated animals (statistical effects were resistant to one-way ANOVA test). For detail, see legend of Figure 2.
than 1 μM. This suggests that two parameters may influence
the sirtinol analogues’ efficiency in PABPN1 nematodes:
and (ii) the nature of the chemical link. The two meta-
substituted sirtinol analogues 2 and 4 were the most efficient
compounds for rescuing cellular phenotypes compared to 1,
(i) the position of the substituent at the benzimine portion,

1
410 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Pasco et al.
and they almost fully rescued defective motility back to the
level of normal PABPN1 animals when tested in the 1-4 μM
range.
Next, we compared 7 with the ketosplitomicin analogue 8,
carrying a 2-chlorobenzoyl moiety at the position 2 of the
compound activity in nematodes. The effect on muscle cell
phenotypes provide another criterion to assess compound
activity. In this respect, in the sirtinol analogue series the
comparison of 1 to 2, 4, and 5 at chemical (compound
structure), enzymatic (sirtuin inhibition), and biological
(effects in PABPN1 animals) level strongly suggests that a
better ability to protect from mutant PABPN1 toxicity corre-
lates with (i) a meta-substituted benzamide or anilide moiety or
para-substituted benzenesulfonamide moiety and (ii) an ability
to inhibit both SIRT1 and SIRT2 in vitro. Other sirtuin
inhibitors of interest raised by our study are 8, a ketosplito-
mycin analogue and highly active SIRT2 inhibitor, 10 and 11,
two SIRT1-selective inhibitors, and 12, a SIRT2-selective
inhibitor. Compared to the most effective sirtuin inhibitors
described herein (2, 4, and 11), compounds 8 and 10 showed a
lower ability to rescue behavioral phenotypes in transgenic
nematodes, and 12 displayed no specific effect in PABPN1
nematodes.
1
H-benzo[ f ]chromen-3(2H)-one nucleus (Figure 1). 8 was
SIRT2
found to strongly inhibit SIRT2 (IC50
limited ability to inhibit SIRT1 (IC50
= 9.4 μM), with a
SIRT1
= 89.8 μM)
Figure 3A). 7 was protective in mutant PABPN1 nematodes,
(
showing a profile of activity (Figure 3B,C) which is very
similar to that of 1. Compared to 7, 8 showed a greater ability
to rescue mutant PABPN1 cytotoxicity in nematodes with no
effect observed in normal PABPN1 animals (Figures 3B,C)
and no reduction of mutant PABPN1 expression (Figure S1, SI).
Thus, similarly to what was observed for sirtinol analogues, 8
provided a further example of the correlation between SIRT2
inhibition and rescue of mutant PABPN1 toxicity.
Afterward, we tested two novel SIRT inhibitors disclosed
by us and bearing a benzodeazaoxaflavine (BDF4) structure,
Future experiments will address the biochemical mecha-
nisms underlying the protection from mutant PABPN1 toxi-
city in cell system by sirtuin inhibitors such as 2, 4, and 11 and
their therapeutic potential in mouse models of OPMD.
9
two selective and highly potent SIRT1 inhibitors (9: IC
and 10 (Figure 1). Compounds 9 and 10 were found to be
SIRT1
5
0
SIRT1
=
8.4 μM; 10: IC50
= 5.3 μM), whereas they were much
less efficient against SIRT2 (Figure 3A). 9 rescued the loss of
nuclear GFP signals at high concentration 11.1 μM and
enhanced toxicity at low concentration 1.2 μM (Figure 3B).
The same was true for the modulation of defective motility
Experimental Section
SIRT Assay. Modulation of sirtuin activity by compounds
was assessed using the Flour de Lys fluorescent biochemical
assay available through BioMol International. In the first part
of a two-step reaction, an acetylated lysine side chain present on
the substrate was deacetylated during incubation at 37 ꢀC for 2 h
with active enzyme (SIRT1 or SIRT2), compounds 1-12, and
(
Figure 3C). In regard to 10, this compound showed at 3.7 μM
an efficient rescue of the loss of nuclear GFP signals
Figure 3B), and a moderate rescue of defective motility in
(
mutant PABPN1 animals (Figure 3C). These effects were
unrelated to a change in transgene expression (Figure S1, SI),
and both compounds showed no effect in normal PABPN1
animals.
Finally, we tested in our C. elegans system 11 as a sample of
potent, SIRT1-selective inhibitor, and 12, described as a
SIRT2-selective inhibitor. Our enzyme assays performed on
þ
NAD in white, 96-well polystyrene luminescence plates. The
latter half of the reaction produced a fluorophore upon treat-
ment with a developing reagent. The reaction was read by a
fluorometric reader (Inphinite 200 TECAN) with excitation set
at 360 nm and emission detection set at 460 nm. Experiments on
the SIRT1 and 2 inhibition were performed in quadruplicate.
Nematode Assay. C. elegans Strains. Transgenic nematodes
expressing either the wild-type or the mutant form of PABPN1
1
1 and 12 gave the expected results in terms of SIRT1 and
SIRT2 inhibition (Figure 3A). In normal (PABPN1-A10) and
mutant (PABPN1-A13) PABPN1 nematodes, 11 rescued the
loss of nuclear GFP signals also at low μM concentration
13
protein were used as previously described.
Drug Assays. For cellular assays, synchronized L1 animals
were incubated in OP-50.1 liquid culture with the drug as
previously described. The number of nuclear GFP signal was
8
(
(
Figure 3B) and fully rescued defective motility at 33.3 μM
Figure 3C). Compound 12, when tested on the loss of GFP
scored at the young adulthood stage using a ꢀ10 Zeiss fluore-
nuclei, showed a significant and dose-dependent rescue
max 20%) of loss of GFP nuclei as produced by mutant
scence microscope (Axioplan Imaging II) as previously de-
1
3
scribed. Percent rescue was calculated as ((test - control)/
(
(
control) ꢀ 100). With this formula, a negative value means
PABPN1 animals (Figure 3B). However, it also showed a
statistically significant and dose-dependent increase (max
enhancement of PABPN1 toxicity, and the maximal achievable
rescue is about 23%. A minimum of 100 worms/test were scored
per dose, and three independent assays performed. For motility
assays, day 3-5 old adults were laid on a thin layer of OP-50.1
bacteria on NGM plates and the number of bodybends
1
0%) in the number of GFP nuclei of normal PABPN1
animals, thus its effect was not specific for mutant PABPN1
nematodes.
Altogether, the evaluationof compounds1-12onSIRT1/2
inhibition in vitro and on modulation of mutant PABPN1
toxicity in transgenic nematodes indicated that (i) SIRT1/2
inhibitors such as 2 and 4 as well as the SIRT1-selective
inhibitor 11 are highly potent rescuers of mutant PABPN1
toxicity, (ii) SIRT1-selective inhibitors such as 9 and 10 may
also be active, however, showing a limited improvement of
defective motility, and (iii) the SIRT2-selective inhibitor 12
show no specific effect for mutant PABPN1 animals.
(
complete sinusoıds) immediately measured for 20 s as pre-
¨
13
viously described. A minimum of 60-100 worms/test were
scored per dose and three independent assays performed.
Statistical Analysis. Statistical analysis was performed using
the two-tailed Student’s t-test unless otherwise indicated in the
legend of the figures.
Acknowledgment. We thank Bernard Brais for providing
the human PABPN1 constructs, David Bear for providing the
human PABPN1 antibody, and the Caenorhabditis Genetics
Center for providing nematode strains. This work was sup-
ported by INSERM, FRM, the University of Paris Descartes,
and the Association Fran c- aise contre les Myopathies, Paris,
France. M.Y.P. is supported by the University of Montreal.
G.A.R. is supported by the Canadian Institutes of Health
Conclusion
The present study reveals chemical features that may support
the rescue of mutant PABPN1 toxicity by sirtuin inhibitors in
muscle cells. Motility is a strong criterion to discriminate

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1411
Research, the Muscular Dystrophy Association, and the
Foundation of Greater Philadelphia. A.M. is supported by
FIRB, RETI FIRB, and Fondazione Roma. L.A. is sup-
ported by AIRC; EU: “APO-SYS” HEALTH-F4-2007-
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