Synthesis of the novel PARP-1 inhibitor AG-690/11026014 and its protective effects on angiotensin II-induced mouse cardiac remodeling

Article abstract of DOI:10.1038/aps.2016.159

We previously identified AG-690/11026014 (6014) as a novel poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor that effectively prevented angiotensin II (Ang II)-induced cardiomyocyte hypertrophy. In the present study, we reported a new synthesis route for 6014, and investigated its protective effects on Ang II-induced cardiac remodeling and cardiac dysfunction and the underlying mechanisms in mice. We designed a new synthesis route to obtain a sufficient quantity of 6014 for this in vivo study. C57BL/6J mice were infused with Ang II and treated with 6014 (10, 30, 90 mg·kg^-1 ·d^-1, ig) for 4 weeks. Then two-dimensional echocardiography was performed to assess the cardiac function and structure. Histological changes of the hearts were examined with HE staining and Masson's trichrome staining. The protein expression was evaluated by Western blot, immunohistochemistry and immunofluorescence assays. The activities of sirtuin-1 (SIRT-1) and the content of NAD+ were detected with the corresponding test kits. Treatment with 6014 dose-dependently improved cardiac function, including LVEF, CO and SV and reversed the changes of cardiac structure in Ang II-infused mice: it significantly ameliorated Ang II-induced cardiac hypertrophy evidenced by attenuating the enlargement of cardiomyocytes, decreased HW/BW and LVW/BW, and decreased expression of hypertrophic markers ANF, BNP and β-MHC; it also prevented Ang II-induced cardiac fibrosis, as implied by the decrease in excess accumulation of extracellular matrix (ECM) components collagen I, collagen III and FN. Further studies revealed that treatment with 6014 did not affect the expression levels of PARP-1, but dose-dependently inhibited the activity of PARP-1 and subsequently restored the activity of SIRT-1 in heart tissues due to the decreased consumption of NAD+ and attenuated Poly-ADP-ribosylation (PARylation) of SIRT-1. In conclusion, the novel PARP-1 inhibitor 6014 effectively protects mice against AngII-induced cardiac remodeling and improves cardiac function. Thus, 6014 might be a potential therapeutic agent for heart diseases.

Full text of DOI:10.1038/aps.2016.159

Acta Pharmacologica Sinica 2017: 1–13
© 2017 CPS and SIMM All rights reserved 1671-4083/17
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Original Article
Synthesis of the novel PARP-1 inhibitor AG-
690/11026014 and its protective effects on
angiotensin II-induced mouse cardiac remodeling
Guo-shuai FENG1, Cui-ge ZHU2, Zhuo-ming LI1, Pan-xia WANG1, Yi HUANG1, Min LIU3, Ping HE1, Lan-lan LOU2,
1,
Shao-rui CHEN1, , Pei-qing LIU
*
*
1Department of Pharmacology and Toxicology, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510006,
China; 2Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510006, China;
3Department of Pharmacology, Shandong University School of Medicine, Ji-nan 250012, China
Abstract
We previously identified AG-690/11026014 (6014) as a novel poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor that effectively
prevented angiotensin II (Ang II)-induced cardiomyocyte hypertrophy. In the present study, we reported a new synthesis route for 6014,
and investigated its protective effects on Ang II-induced cardiac remodeling and cardiac dysfunction and the underlying mechanisms
in mice. We designed a new synthesis route to obtain a sufficient quantity of 6014 for this in vivo study. C57BL/6J mice were infused
with Ang II and treated with 6014 (10, 30, 90 mg·kg-1·d-1, ig) for 4 weeks. Then two-dimensional echocardiography was performed to
assess the cardiac function and structure. Histological changes of the hearts were examined with HE staining and Masson’s trichrome
staining. The protein expression was evaluated by Western blot, immunohistochemistry and immunofluorescence assays. The activities
of sirtuin-1 (SIRT-1) and the content of NAD+ were detected with the corresponding test kits. Treatment with 6014 dose-dependently
improved cardiac function, including LVEF, CO and SV and reversed the changes of cardiac structure in Ang II-infused mice: it
significantly ameliorated Ang II-induced cardiac hypertrophy evidenced by attenuating the enlargement of cardiomyocytes, decreased
HW/BW and LVW/BW, and decreased expression of hypertrophic markers ANF, BNP and β-MHC; it also prevented Ang II-induced
cardiac fibrosis, as implied by the decrease in excess accumulation of extracellular matrix (ECM) components collagen I, collagen
III and FN. Further studies revealed that treatment with 6014 did not affect the expression levels of PARP-1, but dose-dependently
inhibited the activity of PARP-1 and subsequently restored the activity of SIRT-1 in heart tissues due to the decreased consumption of
NAD+ and attenuated Poly-ADP-ribosylation (PARylation) of SIRT-1. In conclusion, the novel PARP-1 inhibitor 6014 effectively protects
mice against AngII-induced cardiac remodeling and improves cardiac function. Thus, 6014 might be a potential therapeutic agent for
heart diseases..
Keywords: cardiac remodeling; PARP-1 inhibitor; 6014; PARylation; NAD+; SIRT-1
Acta Pharmacologica Sinica advance online publication, Feb 27 2017; doi: 10.1038/aps.2016.159
remodeling following sustained hypertension[2–6]
.
Introduction
PARylation, induced by poly(ADP-ribose) polymerases
(PARPs), is a type of reversible posttranslational modifica-
tion impacting numerous cellular processes, eg, DNA repair,
transcription, metabolism, or immune functions[7]. PARP-1,
the predominant member of the PARPs, accounts for approxi-
mately 90% of the cellular PARylation events[8]. Once acti-
vated, PARP-1 catalyzes protein PARylation, which results in
the formation of polymerized ADP-ribose (PAR) dependently
on NAD+ donor molecules, and subsequently the covalent
attachment of PAR polymers to target proteins[9].
Cardiac remodeling, manifested clinically as hypertrophy
(enlargement), fibrosis, and dysfunction of the heart resulting
from cardiac load or injury, is generally accepted as a deter-
minant of the pathogenesis of heart failure (HF)[1]. The renin-
angiotensin-aldosterone system (RAAS) plays a pivotal role in
the physiological and pathological processes of cardiac remod-
eling. It is also widely accepted that angiotensin II (Ang II),
an important component of the RAAS, could induce cardiac
*
To whom correspondence should be addressed.
E-mail chshaor@mail.sysu.edu.cn (Shao-rui CHEN);
liupq@mail.sysu.edu.cn (Pei-qing LIU)
Received 2016-09-26 Accepted 2016-11-24
PARP-1 is involved in pathological cardiac remodeling
and heart failure. Inhibition of PARP-1 prevents pathologi-

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Feng GS et al
cal cardiac remodeling induced by partial abdominal aortic detected at 254 nm; solvent A was CH3CN in double-deionized
constriction[10]. PARP-1-deficient mice are protected from Ang H2O. 1H NMR and 13C NMR spectra (AvanceIII, Burker) were
II-induced cardiac hypertrophy[11]. Moreover, PARP-1 activa- recorded using TMS as an internal standard with a Burker-
tion facilitates the pathogenesis of cardiac fibrosis[12] and the BioSpinUltrashield 400 NMR system. High-resolution mass
development of heart failure[13, 14]. These findings indicate that spectra (HRMS) (LCMS-IT-TOF, Shimadzu) were recorded on
PARP-1 could serve as a promising target for cardiac diseases, a Shimadzu LCMS-IT-TOF Instrument.
and the pharmacologic inhibition of PARP-1 may be a poten-
tial therapeutic strategy[15–17]
.
A previous study from our laboratory identified AG-690/ General procedure for the synthesis of 6014
11026014(6014) as a novel PARP-1 inhibitor from the Specs (1) 4-chlorophenol (77.8 mmol), 2-(chloromethyl)oxirane (306.9
database (www.specs.net), which contains approximately mmol) and K2CO3 (231.5 mmol) were refluxed in acetone
200,000 compounds based on virtual screening. We also (100 mL) overnight, cooled to room temperature and filtered.
reported that 6014 could significantly attenuate Ang II- The filtrate was concentrated and extracted with toluene and
induced cardiomyocyte hypertrophy[18]. To further deter- water, and then the 2-((4-chlorophenoxy)methyl)oxirane (M1)
mine the in vivo cardiac protective effect of 6014, the present in toluene was purified by column chromatography.
study used C57BL/6J mice infused with Ang II as the model,
(2) 3-methyl-1H-purine-2,6(3H,7H)-dione (30 mmol) and
and elucidated the effects and underlying mechanisms of AcONa (60 mmol) were dissolved in AcOH, and Br2 was
this novel PARP-1 inhibitor. This study aimed to determine added dropwise and reacted for 2 h at 65 °C. 8-Bromo-3-
whether 6014, as a PARP-1 inhibitor, has therapeutic potential methyl-1H-purine-2,6(3H,7H)-dione (M2) was generated by
for cardiac remodeling.
cooling, filtering, water washing and drying in a vacuum.
(3) M1 (31.0 mmol) and M2 (25.8 mmol) in ethanol (120 mL)
were combined with trimethylamine (2.6 mmol) and refluxed
for 3 h. 8-Bromo-7-(3-(4-chlorophenoxy)-2-hydroxypropyl)-
Materials and methods
Reagents
The compounds 3-aminobenzamide (3-AB), 4’,6-diamidino- 3-methyl-1H-purine-2,6(3H,7H)-dione (M3) was produced by
2-phenylindole (DAPI) and Ang II were purchased from cooling, filtering, ethanol washing and drying in a vacuum.
Sigma-Aldrich (St Louis, MO, USA). Anti-PARP-1, anti-
(4) M3 (8.15 mmol) and 2-mercaptoacetic acid (16.3 mmol) in
collagen I and anti-collagen III antibodies were provided DMF (35 mL) were combined with NaHCO3 (32.6 mmol) and
by Proteintech Group (Chicago, IL, USA). Anti-ANF and reacted for 2 h at 85°C. The reaction was stopped with water
anti-FN antibodies were purchased from Santa Cruz Bio- and then washed by methyl tertiary butyl ether. The products
technology (Santa Cruz, CA, USA). Anti-BNP antibody was in the water were combined with HCl (2.0 mmol) until the pH
obtained from Merck Millipore (Bedford, Ohio, USA). Anti- reached 3, and 2-((7-(3-(4-chlorophenoxy)-2-hydroxypropyl)-3-
PAR antibody was obtained by Trevigen (Gaithersburg, MD, methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)thio)acetic
USA). Anti-SIRT-1, anti-lamin B1, anti-GAPDH antibodies acid (M4) was yielded after filtering, water washing and dry-
and all secondary antibodies for Western blot experiments ing in a vacuum.
were purchased from Cell Signaling Technology (Boston,
(5) M4 (7.26 mmol) and ammonium chloride (21.7 mmol)
MA, USA). An hematoxylin and eosin (HE) staining kit was in DMF (40 mL) was added to HATU (8.71 mmol) and DIEA
purchased from Santa Cruz Biotechnology (Santa Cruz, CA, (21.8 mmol) on ice and reacted overnight at room tempera-
USA). A Masson’s staining kit and immunohistochemistry ture. The products were precipitated by adding water and
reagents including Cy3-conjugated goat anti-mouse IgG (H+L) then filtering. 2-((7-(3-(4-chlorophenoxy)-2-hydroxypropyl)-
were purchased from Wuhan Goodbio Technology Co, Ltd 3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)thio)
(Wuhan, China). RIPA lysis buffer and IP lysis buffer were acetamide (6014) was purified by preparative chromatography
purchased from Beyotime Biotechnology (Jiangsu, China). The (Varian, Agilent).
protease and phosphatase inhibitor mini tablets, BCA protein
The synthesized intermediates M1-M4 were structurally
assay kit, Bradford protein assay kit, and protein G-agarose identified by 1H NMR or MS, and 6014 was structurally identi-
beads were obtained from Pierce Biotechnology (Rockford, fied by 1H NMR, 13C NMR and HRMS.
AL, USA). A nuclear extract kit was purchased from ACTIVE
MOTIF (Carlsbad, NM, USA). The quick start Bradford dye 2-((4-chlorophenoxy)methyl)oxirane (M1)
reagent was obtained from Bio-Rad (Richmond, VA, USA). A Crystal oil, yield 62.0%; 1H NMR (400 MHz, CDCl3) δ 7.28–7.22
SIRT-1 deacetylase fluorometric assay kit was purchased from (m, 2H), 6.90–6.83 (m, 2H), 4.23 (dd, J=11.0, 3.0 Hz, 1H), 3.93
CycLex Ltd (Nagano, Japan).
(dd, J=11.0, 5.7 Hz, 1H), 3.39–3.32 (m, 1H), 2.92 (t, J=4.5 Hz,
The inhibitor 6014 was synthesized in our lab. The purities 1H), 2.76 (dd, J=4.8, 2.6 Hz, 1H).
of 6014 used for biological evaluation (99.61%) were deter-
mined on a DIONEX Ultimate 3000 HPLC system (Chrome- 8-bromo-3-methyl-1H-purine-2,6(3H,7H)-dione (M2)
1
leon SR9 Build 2673): column, Acclaim 120 C18, 5 mm, 4.6 White solid, yield 95%; H NMR (400 MHz, DMSO) δ 14.29
mm×250 mm; flow rate, 1 mL/min. The samples were (s, 1H), 11.18 (s, 1H), 3.32 (s, 3H). MS (ESI, neg ion) m/z: 242.9
eluted with a gradient of 5% to 95% solvent A for 25 min and
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Feng GS et al
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[M-H]–
The following parameters were measured and calculated: car-
diac output (CO), left ventricular fractional shortening (LVFS),
8-bromo-7-(3-(4-chlorophenoxy)-2-hydroxypropyl)-3-methyl-1H- left ventricular ejection fraction (LVEF), heart rate (HR), stroke
purine-2,6(3H,7H)-dione (M3)
White solid, yield 85.9%; MS (ESI, neg ion) m/z: 427.0 [M-H]–
volume (SV), left ventricular internal dimension (LVID) in
diastole and systole (LVIDd and LVIDs, respectively), left ven-
tricular posterior wall (LVPW) thickness in diastole and sys-
2-((7-(3-(4-chlorophenoxy)-2-hydroxypropyl)-3-methyl-2,6-dioxo- tole (LVPWd and LVPWs, respectively), and the interventricu-
2,3,6,7-tetrahydro-1H-purin-8-yl)thio)acetic acid (M4)
White solid, yield 90.5%; MS (ESI, neg ion) m/z: 439.0 [M-H]–
lar septum (IVS) dimension in diastole and systole (IVSd and
IVSs, respectively). All the parameters were obtained from
2-((7-(3-(4-chlorophenoxy)-2-hydroxypropyl)-3-methyl-2,6-dioxo- three cardiac cycles, and the data were analyzed by GraphPad
2,3,6,7-tetrahydro-1H-purin-8-yl)thio)acetamide (6014)
White solid, yield 30%; HPLC tR=11.24 min; H NMR (400
Prism 5 (GraphPad Inc, La Jolla, CA, USA).
1
MHz, DMSO) δ 11.11 (s, 1H), 7.65 (s, 1H), 7.32 (d, J=8.7 Hz, Collection of tissues
2H), 7.23 (s, 1H), 6.93 (d, J=8.7 Hz, 2H), 5.52 (d, J=4.9 Hz, 1H), After the echocardiography measurements were completed,
4.37 (d, J=9.7 Hz, 1H), 4.27–4.15 (m, 2H), 4.01 (dd, J=9.9, 3.3 all the mice were weighed and anesthetized by sodium pen-
Hz, 1H), 3.98–3.91 (m, 3H). 13C NMR (101 MHz, DMSO) δ tobarbital (40 mg/kg). The hearts of the mice were rapidly
168.66, 157.24, 153.93, 150.60, 150.53, 149.58, 129.14, 124.36, removed, imaged by photography, and weighed. Then, the
116.26, 108.11, 70.33, 67.65, 48.91, 36.68, 28.44. HRMS calcd. left ventricle was carefully trimmed and weighed. The ratio
for C17H18N5O5SCl [M+H]+: 440.0790, found 440.0763.
of heart weight to body weight (heart weight/body weight,
HW/BW) and left ventricular weight to body weight (left ven-
tricular weight/body weight, LVW/BW) were analyzed.
Half of the hearts from each group were stored in formalin
Animals
Healthy adult male C57BL/6J mice (10 weeks old, 20–25 g
weight) were raised in the animal center of Sun Yat-Sen Uni- and then embedded in 4% paraformaldehyde for later histo-
versity and kept under pathogen-free conditions at approxi- pathological analysis. For the other half, the left ventricles
mately 22°C. All experimental procedures were conducted in were isolated and weighed. The left ventricles of the heart
accordance with the Guidelines of Animal Experiments from were snap frozen in liquid nitrogen for Western blot analysis,
the Ethical Committee for Animal Research of Sun Yat-sen SIRT-1 activity measurement and NAD+ content detection.
University (No 44008500011624).
Histopathological analysis
Minipump implantation and administration
For immunohistochemical staining, the tissues were fixed in
As a commonly used PARP-1 inhibitor[19, 20], 3-aminobenza- 4% formalin and embedded in paraffin. Sections (5 mm) were
mide (3-AB) was used as a positive control in this study. The cut from the embedded blocks and floated onto a warm (42°C)
C57BL/6J male mice were randomly assigned to 6 groups, water bath from where they were placed on Superfrost plus
n=12/group: ① sham group; ② Ang II group; ③ Ang II+3-AB slides. The sections were deparaffinized and hydrated, and
(30); ④ Ang II+6014 (10); ⑤ Ang II+6014 (30); ⑥ Ang II+6014 the endogenous peroxidase activity was blocked with 3% H2O2
(90). 3-AB (30), 3-AB (30 mg·kg-1·d-1); 6014 (10), 6014 (10 in water for 10 min. Antigen retrieval was performed with 10
mg·kg-1·d-1); 6014 (30), 6014 (30 mg·kg-1·d-1); 6014 (90), 6014 mmol/L citrate buffer (pH 6.0) for 10 min. The slides were
(90 mg·kg-1·d-1).
incubated with blocking reagent (Thermo Scientific) for 10 min
Mice from all groups except the sham group were infused to block nonspecific binding. Then, they were incubated with
with Ang II (1000 ng·kg-1·min-1) via subcutaneously implanted anti-PAR antibodies overnight at 4 °C, and later with HRP-
minipumps (Alzet® pump model 2004, Cupertino, USA) for 4 conjugated secondary antibodies for 30 min at room tempera-
weeks. Control mice were infused with normal saline. After ture. Finally, the slides were incubated with diaminobenzi-
the minipumps were implanted, administration of 3-AB and dine and counterstained with HE.
6014 were carried out by intragastric administration twice a
For HE staining, after deparaffinization and hydration, the
day, and both 3-AB and 6014 were suspended in sodium car- sections were stained with HE. For Masson’s staining, after
boxymethylcellulose.
deparaffinization and hydration, the sections were stained
with hematoxylin and Ponceau, as well as with phosphomo-
lybdic acid and aniline blue successively in Masson’s staining.
For immunofluorescence, the frozen sections were fixed by
Echocardiography
At the end of the experiment, all the mice were anesthetized
with 1.5% isoflurane in oxygen (1 L/min). Two-dimensionally acetone. Bovine serum albumin (BSA) was used to block non-
guided M-mode echocardiography (ESAOTE, Italy) was per- specific binding after antigen retrieval. The tissue sections
formed using a Technos MPX ultrasound system equipped were incubated with anti-PAR antibody (1:100) overnight at
with a 21-MHz imaging transducer, as described previously[21]
.
4°C and incubated with Cy3-conjugated goat anti-mouse IgG
Ultrasound images of the heart were obtained by gently apply- (H+L) for 1 h. Subsequently, the nucleus was stained with
ing the probe to the chest. The M-mode images were obtained DAPI. All the stained sections were sealed with neutral resin
from the parasternal short-axis view at the papillary muscle. and photographed under an inverted fluorescence microscope
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Feng GS et al
(Olympus, Japan).
USA) at 4°C. The immunoprecipitated proteins were detected
by Western blot.
Western blotting assay
To extract the total protein, the mouse left ventricular tis- Detection of SIRT-1 activity
sue was homogenized in RIPA lysis buffer containing the To detect the activity of SIRT-1, a SIRT-1 deacetylase fluoro-
protease inhibitor PMSF, and the total protein contents were metric assay kit (CycLex Ltd) was used. In this method, the
determined with a BCA protein assay kit (Pierce). To extract reaction was initiated and the fluorescence intensity was mea-
the nucleoprotein, a nuclear extract kit (ACTIVE MOTIF) sured by mixing simultaneously fluorescence-labeled acety-
was used according to the manufacturer’s instructions, and lated peptide (substrate), NAD+, and developer with SIRT-1
the nucleoprotein contents were determined with a Bradford samples. SIRT-1 samples were obtained in the immunopre-
protein assay kit (Pierce). Then, equal amounts of protein cipitation assays using anti-SIRT-1 antibody. The fluorescence
samples were separated by SDS-PAGE and then transferred intensity was measured at 490±10 nm (excitation) and 530±10
to PVDF membranes (Millipore, MA, USA). After blocking nm (emitted light) by an automatic microplate reader (Tecan,
with 5% (w/v) nonfat milk (Bio-Rad) dissolved in Tris-Tween Switzerland).
20 (TBS-T) for 1 h at room temperature, the membranes were
incubated with anti-GAPDH, anti-lamin B1, anti-ANF, anti- Determination of NAD+ content
BNF, anti-β-MHC, anti-collagen I, anti-collagen III, anti-FN, The NAD+ levels were measured according to a method
anti-PARP-1, anti-SIRT-1 or anti-PAR antibodies overnight at described previously[22]. Briefly, the frozen crushed tissue was
4°C. Then, horseradish peroxidase (HRP)-labeled secondary suspended in perchloric acid before centrifugation. The super-
antibodies were added for incubation for 1 h at room tem- natant was neutralized with KOH and KH2PO4/K2HPO4, and
perature. Immunoreactive bands were visualized with Super- then added to the NAD+ reaction mixture containing ethanol,
Signal West Pico Chemiluminescent Substrate (Pierce). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide,
intensity of target protein bands was quantified with Image J phenazine ethosulfate, EDTA, BSA, and bicine. The reaction
software (National Institutes of Health, USA) and normalized was initiated by alcohol dehydrogenase and stopped by iodo-
to GAPDH or Lamin B1 as loading control.
acetate after incubation for 20 min at 37°C. The absorbance of
the reaction mixture was read at a wavelength of 570 nm. The
NAD+ content was calculated from the standard curve and
Co-Immunoprecipitation
To investigate whether there was an interaction between normalized to the protein content of the sample.
SIRT-1 and PARP-1, a co-immunoprecipitation (co-IP) assay
was performed. Nuclear proteins (800 µg) were incubated Statistical analysis
with 3 µg anti-SIRT-1 or anti-PARP-1 antibodies overnight Statistical analysis data are presented as the mean±SEM of
(rabbit normal IgG was used as a control), followed by a 4 h at least three independent experiments. Statistical analysis
incubation with protein G-agarose beads (Pierce, Rockford, IL, was performed using a two-tailed unpaired Student’s t-test
Scheme 1. Synthetic route of AG-690/11026014.
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Feng GS et al
5
between two groups and by one-way ANOVA followed by 6014 was comparable to that of 3-AB (6.4%±0.85%) (Figure 2C
Bonferroni’s test for multiple comparisons. The analyses were and 2F).
performed using GraphPad Prism 5.0 (GraphPad Inc, La Jolla,
CA, USA). P<0.05 was considered statistically significant.
Western blot analysis showed that 6014 dose-dependently
alleviated the expression of the hypertrophic marker pro-
teins (ANF, BNP and β-MHC) (Figure 3A–3C) and the fibro-
sis marker proteins (collagen I, collagen III and FN) (Figure
3D–3F), in contrast to Ang II. Taken together, these results
Results
Synthesis of 6014
To obtain a sufficient quantity of 6014 for the in vivo study, a suggested that 6014 could markedly prevent cardiac hypertro-
novel synthetic route of 6014 was designed that is illustrated phy and fibrosis induced by Ang II.
in Scheme 1. In brief, 6014 was synthesized through five steps,
three of which could be completed simply and independently Effects of 6014 on the expression and enzymatic activity of
on a column chromatograph to obtain pure product. This PARP-1
efficient synthetic route of 6014 was reported for the first time PARP-1 localizes mainly to the cell nucleus and to a lesser
herein, and could be worthy for large scale synthesis. Finally, extent in the mitochondria[23–25]; therefore, the expression of
an adequate amount of 6014 (2.0 g) was obtained and success- nuclear and total PARP-1 was detected by Western blot. As
fully used for animal experiments. The purity and structure of shown in Figure 4A, 4B, both the nuclear and total expression
6014 were confirmed by 1H NMR, 13C NMR, HRMS and HPLC of PARP-1 was up-regulated upon stimulation of Ang II but
(Figure S1–S4).
was unaltered by 3-AB and 6014. Target proteins undergo the
PARylation modification when PARP-1 is activated. Thus, the
6014 prevented cardiac dysfunction and changes of cardiac enzymatic activity of PARP-1 was evaluated by the expression
structure in Ang II-infused mice
of PARylated proteins using anti-PAR antibody. As shown
To investigate the protective effect of 6014 on cardiac func- in Figure 4C, Ang II clearly stimulated the PARylation of pro-
tion and structure, two-dimensional echocardiography was teins (3.20±0.30), whereas 6014 dose-dependently inhibited
performed (Table S1). The echocardiography results indi- the increased expression of PARylated proteins (3.10±0.26,
cated that the LVPWd, LVPWs, IVSd, IVSs were remarkably 1.53±0.06 and 1.20±0.10).
increased, while the LVIDd and LVIDs were decreased in Ang
Immunohistochemistry and immunofluorescence assays
II-infused mice. The inhibitor 6014 dose-dependently reversed were subsequently performed to further confirm the inhibition
these Ang II-induced changes, similarly as the well-known of 6014 on PARP-1 activity. Representative images of immu-
PARP-1 inhibitor 3-AB (Figure 1A–1G). The LVEF, LVFS, SV nohistochemistry are shown in Figure 5A, 5B. Ang II clearly
and CO have been most widely used to evaluate left ventricu- increased the expression of PARylated proteins, which were
lar systolic function. Uniquely, LVFS was not significantly labeled with the anti-PAR antibody (brown). The up-regula-
different among mice receiving different treatments (Table S1). tion of PARylated proteins was prominently inhibited by the
-1 -1
·
·
Treatment with 6014 attenuated the Ang II-induced increases treatment of 6014 at the doses of 30 and 90 mg kg d , compa-
-1 -1
·
·
of LVEF, SV and CO in a dose-dependent manner (Figure rable to 3-AB (30 mg kg d ). Likewise, the fluorescence inten-
1H–1J).
sity of PARylated proteins (red) was augmented in the hearts
of Ang II-infused mice but was suppressed by 6014 (30 and 90
6014 attenuated cardiac hypertrophy and fibrosis induced by Ang II mg·kg-1·d-1) and 3-AB (30 mg·kg-1·d-1) (Figure 5C). Therefore,
As shown in Figure 2A, Ang II treatment led to the enlarge- these observations were in line with the Western blot results
ment of heart size, which could be prevented by 6014 at the (Figure 4C), confirming that 6014 efficiently inhibited the acti-
-1 -1
·
·
doses of 30 and 90 mg kg d , as well as 3-AB at a dose of 30 vation of PARP-1 but did not affect its protein expression in
-1 -1
·
·
mg kg d . Statistical results demonstrated that the ratios of Ang II-infused mice.
HW/BW and LVW/BW were increased to 6.38±0.55 mg/g
and 3.70±0.43 mg/g respectively by stimulation with Ang II, 6014 inhibited PARylation of SIRT-1 and restored the activity of
-1 -1
·
·
SIRT-1 in Ang II-infused mice
while 6014 (and 10, 30 and 90 mg kg d ) dose-dependently
inhibited the increase of HW/BW (5.97±0.55, 5.72±0.53 and Ang II treatment significantly decreased the NAD+ content
5.46±0.61 mg/g) and LVW/BW (3.65±0.42, 3.26±0.23 and (80.3%±4.5%) and SIRT-1 activity (42.7%±5.0%) in left ven-
3.09±0.26 mg/g) (Figure 2D and 2E). HE staining images also tricles (Figure 6A and 6B). Treatment with 6014 did not retard
showed that 6014 significantly prevented the enlargement of the decrease of NAD at 10 and 30 mg kg d but did restore
cardiomyocytes induced by Ang II (Figure 2B).
Additionally, a significant increase of total collagen in the dose of 90 mg kg d (Figure 6A), which may due to the strong
hearts of the Ang II group was observed by the Masson’s inhibition of PARP-1 by 6014. SIRT-1 is an NAD+ dependent
staining assays, which represented the appearance of cardiac enzyme that can compete with PARP-1 in NAD+ consumption.
+
-1 -1
·
·
the NAD+ content to 95.3%±5.0% of the sham group at the high
-1 -1
·
·
fibrosis (Figure 2C). Treatment with 6014 remarkably and Interestingly, 6014 could strongly restore SIRT-1 activity at
-1 -1
·
·
dose-dependently prevented the accumulation of collagen the medium dose of 30 mg kg d (66.3%±9.1%) compared to
(10.3%±1.5%, 4.2%±0.76% and 3.0%±0.15%) compared to the the Ang II (42.7%±5.0%), even though the NAD+ content was
Ang II group (14.4%±2.0%) (Figure 2C and 2F). The effect of not increased by 6014 at the same dose (Figure 6A and 6B).
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Feng GS et al
Figure 1. 6014 reversed the cardiac dysfunction and changes of cardiac structure induced by Ang II. Representative echocardiographic graphs (A) were
shown and IVSd (B), LVPWd (C), LVIDd (D), IVSs (E), LVPWs (F), LVIDs (G), LVEF (H), SV (I), and CO (J) were measured and calculated from parameters of
echocardiography. Data were expressed as mean±SEM from three independent experiments. **P<0.01 vs Sham. ##P<0.01 vs Ang II.
The results indicated that other factors underlying the SIRT-1 alleviated the expression of SIRT-1 in contrast to Ang II (Fig-
activity regulation may exist in addition to the restoration of ure 6C), and the trend was consistent with that of SIRT-1
NAD+ content by 6014.
activity. SIRT-1 was precipitated by anti-PARP-1 (Figure 6D),
Western blot analysis showed that 6014 dose-dependently suggesting an interaction between SIRT-1 and PARP-1. Addi-
tionally, Ang II enhanced this interaction significantly, and
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Figure 2. 6014 attenuated the enlargement of the heart size, cardiomyocytes size and prevented the accumulation of collagen. (A) Representative
digram of mice hearts under different treatment. (B) Representative images diagram of HE staining of cardiomyocytes. (C) Representative images of
Masson’s staining of total collagen in tissue sections of heart. (D) Statistical results of HW/BW. (E) Statistical results of LVW/BW. (F) Statistical areas
**
of total collagen production versus areas of whole tissue. Data were expressed as mean±SEM from three independent experiments.
Sham. #P<0.05, ##P<0.01 vs Ang II.
P<0.01 vs
6014 attenuated the enhancement (Figure 6D). Moreover, the
Discussion
PARylation of SIRT-1 was detected using an anti-PAR anti- In this study, we first designed a novel route of synthesis and
body (Figure 6E). We observed two bands (approximately 170 successfully synthesized a sufficient amount of 6014. During
and 250 kDa), which were present at higher levels than the cardiac remodeling, the most apparent change is the enlarge-
SIRT-1 band (120 kDa) (Figure 6E), suggesting the PARylation ment of the heart, i.e., cardiac hypertrophy. In the cardiac
of SIRT-1. The results indicated 6014 significantly reduced the remodeling mouse model induced by Ang II, we observed
PARylation of SIRT-1, in contrast to Ang II, which might con- that 6014 ameliorated cardiac hypertrophy by attenuating
tribute to its activation of SIRT-1 activity.
the enlargement of cardiomyocytes and the increase of HW/
BW and LVW/BW and by decreasing the expression of fetal
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Feng GS et al
Figure 3. Effects of 6014 on the expression of hypertrophy marker proteins and fibrosis marker proteins. Protein expression of ANF (A), BNP (B), β-MHC
(C), FN (D), collagen I (E) and collagen III (F) were measured by Western blot. Data were expressed as mean±SEM from duplicates in three independent
experiments. **P<0.01 vs Sham. #P<0.05, ##P<0.01 vs Ang II.
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Figure 4. Effects of 6014 on the expression and enzymatic activity of PARP-1. The expression of PARP-1 in nucleus (A) and whole cell (B) as well as
PARylated proteins in whole cell (C). Data were expressed as mean±SEM from duplicates in three independent experiments. *P<0.05, **P<0.01 vs
Sham. #P<0.05, ##P<0.01 vs Ang II.
genes, including ANF, BNP and β-MHC. Cardiac hypertro- exerts cardiac protective effects against cardiac remodeling.
phy, only when accompanied by fibrosis during remodeling, Importantly, 6014 showed better cardiac protective effects
can lead to cardiac dysfunction[26]. It was also found that and lower cytotoxicity (Figure S5) than 3-AB, which proved
PARylation of PARP-1 is responsible for the pathogenesis it to be very meaningful to investigate its cardio-protection in
of tissue fibrosis[27, 28]. Some researchers have reported that vivo.
PARylation of c-Jun/c-Fos makes an important contribution
to Ang II-induced cardiac fibrosis[29], and the inhibition of tor with obvious inhibition of the activity of PARP-1 at the
PARP-1 prevented Ang II-induced aortic fibrosis in rats[30]
enzyme level and in a cardiomyocyte model. However, the
Our previous work identified 6014 as a novel PARP-1 inhibi-
.
Herein, we also found that 6014 prevented cardiac fibrosis underlying mechanisms of its cardiac protective effects in vivo
induced by Ang II, as implied by the decrease of excess accu- remained unexplored. Herein, we further demonstrated that
mulation of extracellular matrix (ECM) components, includ- the cardio-protective effects of 6014 depended on the inhibi-
ing collagen I, collagen III and FN. Moreover, 6014 improved tion of PARylation, which was shown to activate SIRT-1.
cardiac function, including LVEF, CO and SV and reversed
SIRT-1, a prototypical member of the sirtuin family, is an
the changes of cardiac structure in Ang II-infused mice. NAD+-dependent deacetylase enzyme[31]. Recently, SIRT-1 has
Above all, these results confirmed for the first time that 6014 gathered significant interest due to its cardiovascular protec-
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Feng GS et al
Figure 5. Effects of 6014 on enzymatic activity of PARP-1. (A, B) Representative images of PARylated proteins expression by immunohistochemistry.
PARylated proteins were stained with anti-PAR antibody (brown). (C) Representative images of PARylated proteins by immunofluorescence. PARylated
proteins were stained with anti-PAR antibody (red) and nuclei were stained with DAPI (blue).
tive role[32, 33]. Several studies focusing on the responses of res- substrate arose 10 years ago[36]. PARP-1 also has a higher
veratrol (RES) as an accepted SIRT-1 activator also indicated affinity for NAD+ than does SIRT-1 (Km 20–60 μmol/L vs 100–
that SIRT-1 may have a beneficial role in heart diseases and 300 μmol/L, respectively)[37, 38]. Therefore, over-activation of
that the up-regulation of endogenous SIRT-1 is a protective PARP-1 can easily limit the NAD+ availability for the activity
mechanism in cardiac diseases[34, 35]. Moreover, the cardio- of SIRT-1. In this study, overactivation of PARP-1 induced by
protection of RES by activating SIRT-1 was also detected in Ang II in C57BL/6J mice did lead to significant consumption
this paper (Figure S6, S7 and Table S2), which further con- of NAD+ and, subsequently, the down-regulation and inactiv-
firmed the beneficial roles of SIRT-1 in heart diseases. Since ity of SIRT-1, as expected. More importantly, these pheno-
both PARP-1 and SIRT-1 are present in the nuclear compart- types induced by Ang II were all reversed after the inhibition
ment, the idea that they may compete for the common NAD+ of PARP-1 activity by 6014. We confirmed that the decrease
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Feng GS et al
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Figure 6. Effects of 6014 on the regulation and activity of SIRT-1. NAD+ content analysis (A) and SIRT-1 activity (B) were analyzed. Protein expression
of SIRT-1 were measured by Western blot (C). The interaction between SIRT-1 and PARP-1 (D) and the PARylation of SIRT-1 (E) were measured by co-IP
assay. Data were expressed as mean±SEM from three independent experiments. *P<0.05, **P<0.01 vs Sham. #P<0.05, ##P<0.01 vs Ang II.
of NAD+ consumption from the strong inhibition of PARP-1’s might contribute to the decrease of SIRT-1 activity, and the
activity by 6014 was one reason for reversing the activity of decreased PARylation of SIRT-1 by 6014 may be another new
SIRT-1, which benefited the protection from cardiac remodel- reason for activating SIRT-1 under conditions of limited NAD+
ing and dysfunction.
content; we aim to investigate the novel interaction of SIRT-1
Interestingly, we still observed the restorative effects of and PARP-1 in our later work.
SIRT-1 in the case in which the activity of PARP-1 was clearly
In summary, our study confirms that PARP-1 inhibition may
inhibited but the NAD+ content was not reversed by 6014 at the suggest a therapeutic strategy for cardiac diseases. The novel
dose of 30 mg·kg-1·d-1. Notably, the interconnection between PARP-1 inhibitor 6014 protected mice against Ang II-induced
SIRT-1 and PARP-1 is more intricate[39], even though NAD+ cardiac remodeling, and ameliorated cardiac function. Treat-
was reported as one accepted crosslink between them. Our ment with 6014 might be a potential therapeutic strategy for
results also confirmed the direct interaction of SIRT-1 and heart diseases.
PARP-1 by co-IP assay and, surprisingly, detected the expres-
sion of PARylation of SIRT-1, which was greatly up-regulated
Acknowledgements
by Ang II but down-regulated by 6014 at the dose of 30 mg·kg- This work was supported by grants from the National Natural
1·d-1. Therefore, we deduced that the PARylation of SIRT-1 Science Foundation of China (No 81673433, No 81026548 and
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12
Feng GS et al
Poly (ADP-ribose) polymerase-1-deficient mice are protected from
angiotensin II-induced cardiac hypertrophy. Am J Physiol Heart Circ
Physiol 2006; 291: H1545–H53.
No 81273499 to Dr Pei-qing LIU), Team item of the Natural Sci-
ence Foundation of Guangdong Province (No S2011030003190
to Dr Pei-qing LIU), the Major Project of Guangdong Province
(No 2015B020232009, No 2014B020210003, 2013B090700010
to Dr Pei-qing LIU), the Fund of the Guangdong Provincial
Bureau of Traditional Chinese Medicine (No 20161049 to Dr
Shao-rui CHEN), and the Medical Scientific Research Foun-
dation of Guangdong Province (No A2015220 to Dr Shao-rui
CHEN).
12 Huang D, Yang C, Wang Y, Liao Y, Huang K. PARP-1 suppresses
adiponectin expression through poly (ADP-ribosyl) ation of PPARγ in
cardiac fibroblasts. Cardiovasc Res 2009; 81: 98–107.
13 Pacher P, Liaudet L, Bai P, Virag L, Mabley JG, Hasko G, et al.
Activation of poly(ADP-ribose) polymerase contributes to development
of doxorubicin-induced heart failure. J Pharmacol Exp Ther 2002;
300: 862–7.
14 Pillai JB, Russell HM, Raman J, Jeevanandam V, Gupta MP. Increased
expression of poly(ADP-ribose) polymerase-1 contributes to caspase-
independent myocyte cell death during heart failure. Am J Physiol
Heart Circ Physiol 2005; 288: H486–96.
15 Bartha E, Solti I, Kereskai L, Lantos J, Plozer E, Magyar K, et al. PARP
inhibition delays transition of hypertensive cardiopathy to heart failure in
spontaneously hypertensive rats. Cardiovasc Res 2009; 83: 501–10.
16 Palfi A, Toth A, Hanto K, Deres P, Szabados E, Szereday Z, et al. PARP
inhibition prevents postinfarction myocardial remodeling and heart
failure via the protein kinase C/glycogen synthase kinase-3β pathway.
J Mol Cell Cardiol 2006; 41: 149–59.
Author contribution
Pei-qing LIU and Shao-rui CHEN defined the research theme
and revised the manuscript critically; Guo-shuai FENG, Cui-ge
ZHU, Pan-xia WANG, Yi HUANG, Min LIU, Zhuo-ming LI,
Ping HE, and Lan-lan LOU performed the research; Guo-shuai
FENG, Cui-ge ZHU, Zhuo-ming LI, Shao-rui CHEN, and Pei-
qing LIU analyzed the data; and Guo-shuai FENG wrote the
paper.
17 Szabó G, Bährle S, Stumpf N, Sonnenberg K, Szabó É, Pacher P, et
al. Poly (ADP-ribose) polymerase inhibition reduces reperfusion injury
after heart transplantation. Circ Res 2002; 90: 100–6.
18 Liu M, Li Z, Chen GW, Li ZM, Wang LP, Ye JT, et al. AG-690/11026014,
a novel PARP-1 inhibitor, protects cardiomyocytes from Ang II-induced
hypertrophy. Mol Cell Endocrinol 2014; 392: 14–22.
19 Koh SH, Chang DI, Kim HT, Kim J, Kim MH, Kim KS, et al. Effect of
3-aminobenzamide, PARP inhibitor, on matrix metalloproteinase-9 level
in plasma and brain of ischemic stroke model. Toxicology 2005; 214:
131–9.
20 Nguewa PA, Fuertes MA, Cepeda V, Alonso C, Quevedo C, Soto M,
et al. Poly(ADP-ribose) polymerase-1 inhibitor 3-aminobenzamide
enhances apoptosis induction by platinum complexes in cisplatin-
resistant tumor cells. Med Chem 2006; 2: 47–53.
Supplementary information
Supplementary files are available at the website of Acta
Pharmacologica Sinica.
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