Lee, Jeong Hyun
(Therapeutics & Biotechnology Division, Korea Research Institute of Chemical Technology)
,
Seo, Ho Won
(Therapeutics & Biotechnology Division, Korea Research Institute of Chemical Technology)
,
Ryu, Jae Yong
(Therapeutics & Biotechnology Division, Korea Research Institute of Chemical Technology)
,
Lim, Chae Jo
(Therapeutics & Biotechnology Division, Korea Research Institute of Chemical Technology)
,
Yi, Kyu Yang
(Therapeutics & Biotechnology Division, Korea Research Institute of Chemical Technology)
,
Oh, Kwang-Seok
(Therapeutics & Biotechnology Division, Korea Research Institute of Chemical Technology)
,
Lee, Byung Ho
(Therapeutics & Biotechnology Division, Korea Research Institute of Chemical Technology)
G protein-coupled receptor kinase 5 (GRK5) has been considered as a potential target for the treatment of heart failure as it has been reported to be an important regulator of pathological cardiac hypertrophy. To discover novel scaffolds that selectively inhibit GRK5, we have identified a novel smal...
G protein-coupled receptor kinase 5 (GRK5) has been considered as a potential target for the treatment of heart failure as it has been reported to be an important regulator of pathological cardiac hypertrophy. To discover novel scaffolds that selectively inhibit GRK5, we have identified a novel small molecule inhibitor of GRK5, KR-39038 [7-((3-((4-((3-aminopropyl)amino)butyl)amino)propyl)amino)-2-(2-chlorophenyl)-6-fluoroquinazolin-4(3H)-one]. KR-39038 exhibited potent inhibitory activity (IC50 value=0.02 µM) against GRK5 and significantly inhibited angiotensin II-induced cellular hypertrophy and HDAC5 phosphorylation in neonatal cardiomyocytes. In the pressure overload-induced cardiac hypertrophy mouse model, the daily oral administration of KR-39038 (30 mg/kg) for 14 days showed a 43% reduction in the left ventricular weight. Besides, KR-39038 treatment (10 and 30 mg/kg/day, p.o.) showed significant preservation of cardiac function and attenuation of myocardial remodeling in a rat model of chronic heart failure following coronary artery ligation. These results suggest that potent GRK5 inhibitor could effectively attenuate both cardiac hypertrophy and dysfunction in experimental heart failure, and KR-39038 may be useful as an effective GRK5 inhibitor for pharmaceutical applications.
G protein-coupled receptor kinase 5 (GRK5) has been considered as a potential target for the treatment of heart failure as it has been reported to be an important regulator of pathological cardiac hypertrophy. To discover novel scaffolds that selectively inhibit GRK5, we have identified a novel small molecule inhibitor of GRK5, KR-39038 [7-((3-((4-((3-aminopropyl)amino)butyl)amino)propyl)amino)-2-(2-chlorophenyl)-6-fluoroquinazolin-4(3H)-one]. KR-39038 exhibited potent inhibitory activity (IC50 value=0.02 µM) against GRK5 and significantly inhibited angiotensin II-induced cellular hypertrophy and HDAC5 phosphorylation in neonatal cardiomyocytes. In the pressure overload-induced cardiac hypertrophy mouse model, the daily oral administration of KR-39038 (30 mg/kg) for 14 days showed a 43% reduction in the left ventricular weight. Besides, KR-39038 treatment (10 and 30 mg/kg/day, p.o.) showed significant preservation of cardiac function and attenuation of myocardial remodeling in a rat model of chronic heart failure following coronary artery ligation. These results suggest that potent GRK5 inhibitor could effectively attenuate both cardiac hypertrophy and dysfunction in experimental heart failure, and KR-39038 may be useful as an effective GRK5 inhibitor for pharmaceutical applications.
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제안 방법
The rats were randomly assigned to five experimental groups as follows: sham-operated group (n=7), MI+vehicle group (n=11), MI+10 mg/kg/day KR-39038 group (n=10), MI+30 mg/kg/day KR-39038 group (n=9) and MI+30 mg/kg/day captopril group (n=9). All treatments were administered orally once a day for 12 weeks starting from 24 h after surgery. To adjust the dose accordingly, body weights were measured once a week.
All treatments were administered orally once a day for 12 weeks starting from 24 h after surgery. To adjust the dose accordingly, body weights were measured once a week.
To assess the effects of KR-39038 on the phosphorylation of HDAC5 via GRK5 activation, we prepared extracts of angiotensin II-activated neonatal cardiomyocytes and determined the level of phosphorylated HDAC5 by immunoblotting (Fig. 2C). Treatment with 0.
After echocardiographic assessment, we performed hematoxylin and eosin and Masson’s trichrome staining with the left ventricles to measure the cross-sectional area of cardiomyocytes and interstitial fibrosis, respectively (Fig. 5A).
The survival rates were 53%, 67%, 80% and 73% in the vehicle-, captopril-, 10 and 30 mg/kg KR-39038-treated groups, respectively. The effect of KR-39038 on cardiac function was examined by echocardiography in myocardial infarction model in rats at 1, 2, 4, 8, and 12 weeks after coronary artery ligation. Representative echocardiographic images are shown in Fig.
대상 데이터
Human recombinant GRK5 was purchased from Invitrogen (Carlsbad, CA, USA). The Ulight-Histon H3 (Thr3) peptide (5Ulight-ARTKQTQRKSTG-COOH) and LANCE® timeresolved fluorescence resonance energy transfer (TR-FRET) assay kit was obtained from PerkinElmer (Waltham, MA, USA).
The LV fractional shortening (FS) and ejection fraction (EF) were measured before surgery (baseline) and after 1, 2, 4, 8, and 12 weeks of left anterior descending coronary artery ligation. The echocardiographic images were analyzed using VisualSonic analysis software by a blinded investigator.
데이터처리
Data were analyzed by one-way analysis of variance followed by Dunnett’s test for multiple comparisons (Sigma Stat, Jandel Co., San Rafael, CA, USA).
이론/모형
The inhibitory effect of KR-39038 on the activity of GRK5 was evaluated using a TR-FRET-based GRK5 kinase assay. The assay conditions of GRK5 kinase were verified with staurosporine and amlexanox as reference compounds (IC50 values: 0.
성능/효과
After 2 weeks of TAC (Fig. 3A), the left ventricular weight-totibia length ratio (6.7 ± 0.2 and 4.7 ± 0.1 mg/mm, respectively, p<0.05, Fig. 3B) and heart weight-to-tibia length ratio (8.2 ± 0.2 and 6.2 ± 0.1 mg/mm, respectively, p<0.05, Fig. 3C) were remarkably increased in the vehicle-treated group compared to the sham group.
At 12 weeks after coronary artery ligation, the vehicle-treated group was found to have significantly lower left ventricular ejection fraction (29.2 ± 2.1% and 69.9 ± 1.0%, respectively, p<0.05) and fractional shortening (14.6 ± 1.1% and 41.1 ± 0.9%, respectively, p<0.05) than that of the sham group.
There were no significant difference in the ratio of a left ventricular weight to body weight among the groups, but the myocyte cross-sectional area (504.6 ± 29.5 µm2 and 336.7 ± 9.9 µm2, respectively) and interstitial fibrosis (23.3 ± 2.9% and 3.7 ± 0.5%, respectively) were significantly increased in the vehicletreated group compared with the sham group at 12 weeks after MI (Fig. 5B, 5C).
The 10 and 30 mg/kg KR-39038-treated groups exhibited a prominent elevation of left ventricular ejection fraction (44.3 ± 2.6% and 43.4 ± 2.3%, respectively) compared to the vehicle-treated groups (p<0.05), and showed a significant increase in fractional shortening (23.4 ± 1.6% and 22.6 ± 1.3%, respectively).
The anti-hypertrophic and cardioprotective effects of KR-39038 were confirmed in another animal model of cardiac remodeling following the induction of chronic MI in rats. The survival rates were 53%, 67%, 80% and 73% in the vehicle-, captopril-, 10 and 30 mg/kg KR-39038-treated groups, respectively. The effect of KR-39038 on cardiac function was examined by echocardiography in myocardial infarction model in rats at 1, 2, 4, 8, and 12 weeks after coronary artery ligation.
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