G protein coupled receptor kinase 2 (GRK2) is a kinase that regulates cardiac signaling activity. Inhibiting GRK2 is a promising mechanism for the treatment of heart failure (HF). Further development and optimization ...G protein coupled receptor kinase 2 (GRK2) is a kinase that regulates cardiac signaling activity. Inhibiting GRK2 is a promising mechanism for the treatment of heart failure (HF). Further development and optimization of inhibitors targeting GRK2 are highly meaningful. Therefore, in order to design GRK2 inhibitors with better performance, the most active molecule was selected as a reference compound from a data set containing 4-pyridylhydrazone derivatives and triazole derivatives, and its scaffold was extracted as the initial scaffold. Then, a powerful optimization-based framework for de novo drug design, guided by binding affinity, was used to generate a virtual molecular library targeting GRK2. The binding affinity of each virtual compound in this dataset was predicted by our developed deep learning model, and the designed potential compound with high binding affinity was selected for molecular docking and molecular dynamics simulation. It was found that the designed potential molecule binds to the ATP site of GRK2, which consists of key amino acids including Arg199, Gly200, Phe202, Val205, Lys220, Met274 and Asp335. The scaffold of the molecule is stabilized mainly by H-bonding and hydrophobic contacts. Concurrently, the reference compound in the dataset was also simulated by docking. It was found that this molecule also binds to the ATP site of GRK2. In addition, its scaffold is stabilized mainly by H-bonding and π-cation stacking interactions with Lys220, as well as hydrophobic contacts. The above results show that the designed potential molecule has similar binding modes to the reference compound, supporting the effectiveness of our framework for activity-focused molecular design. Finally, we summarized the interaction characteristics of general GRK2 inhibitors and gained insight into their molecule-target binding mechanisms, thereby facilitating the expansion of lead to hit compound.展开更多
目的研究芍药苷-6-氧-苯磺酸酯(CP-25)通过抑制GRK2活性对骨关节炎(osteoarthritis,OA)小鼠膝关节软骨的保护作用。方法内侧半月板失稳(destabilization of the medial meniscus,DMM)手术诱导构建小鼠骨关节炎模型,实验分为假手术组、...目的研究芍药苷-6-氧-苯磺酸酯(CP-25)通过抑制GRK2活性对骨关节炎(osteoarthritis,OA)小鼠膝关节软骨的保护作用。方法内侧半月板失稳(destabilization of the medial meniscus,DMM)手术诱导构建小鼠骨关节炎模型,实验分为假手术组、模型组、CP-25给药组和帕罗西汀给药组。术后开始灌胃给药。给药12周处死动物,Micro-CT成像观察膝关节软骨退变、骨重塑异常等情况,番红固绿染色观察小鼠关节组织病理,免疫组化、免疫荧光检测软骨组织相关分子表达水平的影响。Western blot检测CP-25用药后软骨细胞的膜蛋白及总蛋白表达水平。结果模型小鼠关节软骨严重退变。CP-25可显著降低关节软骨骨赘数量及软骨下板厚度,促进软骨基质再生,减少软骨基质降解蛋白表达,对膝关节软骨有明显的保护作用。免疫组化和免疫荧光结果显示,CP-25治疗可显著降低膝关节组织中GRK2、ADAMTS5、MMP13的表达,并且升高膝关节组织中ColⅡ、Aggrecan表达。体外实验结果表明,CP-25给药可以显著降低GRK2的膜蛋白及总蛋白表达水平,升高EP4膜蛋白水平,降低MMP13水平。结论CP-25给药可显著促进OA小鼠关节软骨基质再生,减少软骨基质降解,对OA具有治疗作用,其机制与抑制GRK2介导的软骨基质代谢有关。展开更多
目的研究芍药苷-6′-O-苯磺酸酯(CP-25)在血管紧张素Ⅱ(AngⅡ)诱导的肾小球系膜细胞(MCs)增殖中的作用及相关机制。方法体外培养SV40 MES 13系膜细胞,以AngⅡ诱导MCs增殖,高内涵成像显微镜检测不同浓度CP-25(10、100、1000 nmol/L)对An...目的研究芍药苷-6′-O-苯磺酸酯(CP-25)在血管紧张素Ⅱ(AngⅡ)诱导的肾小球系膜细胞(MCs)增殖中的作用及相关机制。方法体外培养SV40 MES 13系膜细胞,以AngⅡ诱导MCs增殖,高内涵成像显微镜检测不同浓度CP-25(10、100、1000 nmol/L)对AngⅡ诱导的MCs增殖的影响;Western blot检测MCs中G蛋白偶联受体激酶-2(GRK2)和磷酸化p38(p-p38)蛋白表达水平;激光共聚焦显微镜检测GRK2和p-p38蛋白的荧光信号并分析二者共定位率;成像流式细胞仪检测MCs中GRK2入胞质的细胞比例。结果与对照组比较,AngⅡ可诱导MCs的增殖,增加GRK2和p-p38蛋白表达,上调GRK2和p-p38MAPK共定位率,提高GRK2入胞比例,差异有统计学意义(P<0.01)。CP-25(10、100、1000 nmol/L)可不同程度地抑制AngⅡ诱导的增殖,抑制GRK2、p-p38蛋白表达及GRK2和p-p38的共定位水平,降低GRK2入胞比例。结论CP-25可抑制AngⅡ诱导的MCs增殖,其机制与调节GRK2/p38信号有关。展开更多
OBJECTIVE To investigate the role and mechanism of G protein-coupled receptor kinase 2(GRK2)involving in hepatocel ular carcinoma(HCC)progression.METHODS Cel Counting Kit 8 and tumor colony formation assay were design...OBJECTIVE To investigate the role and mechanism of G protein-coupled receptor kinase 2(GRK2)involving in hepatocel ular carcinoma(HCC)progression.METHODS Cel Counting Kit 8 and tumor colony formation assay were designed to detect HCC cell proliferation,wound healing assay was to detect HCC migration.The correlation between GRK2 and early growth response-1(EGR1)were detected by RT-PCR and real-time PCR assays.Co-immunoprecipitation and Western blot assay were adopted to detect the relationship between GRK2and insulin-like growth factor 1 receptor(IGF-1R)signaling pathway.RESULTS In this study we find that GRK2plays an inhibition role in IGF1-induced HCC cell proliferation and migration.Overexpression of GRK2 causes a decrease in EGR1 expression,while knockdown of GRK2 leads to the dramatically increase in EGR1 expression in the treatment of IGF1.Through co-immunoprecipitation and Western blot assay,we confirm that GRK2can interact with IGF-1R and inhibiting IGF1-induced activation of IGF1R signaling pathway.Silencing EGR1attenuates GRK2 overexpression-caused inhibition of cell proliferation,tumor colony number and migrationactivity,while overexpressing of EGR1 restores the antiproliferative and migratory effect by GRK2 overexpression in HCCLM3 cells.CONCLUSION Taken together,these results suggest that GRK2 may inhibit IGF1-induced HCC cell growth and migration through down-regulation of EGR1 and indicate that enforced GRK2 may offer a potential therapeutic approach against HCC.展开更多
目的观察美托洛尔对高龄老年慢性心衰患者血清BNP和外周血淋巴细胞GRK2表达的影响。方法选取80岁以上慢性心衰患者60例,随机分为两组对照组和观察组各30例。对照组患者接受如利尿剂、血管紧张素拮抗剂、强心剂、β阻滞剂和紧张素转换抑...目的观察美托洛尔对高龄老年慢性心衰患者血清BNP和外周血淋巴细胞GRK2表达的影响。方法选取80岁以上慢性心衰患者60例,随机分为两组对照组和观察组各30例。对照组患者接受如利尿剂、血管紧张素拮抗剂、强心剂、β阻滞剂和紧张素转换抑制剂等常规治疗,观察组患者在常规治疗基础上加用美托洛尔。两组治疗前后均行心脏超声检查,观察对比两组治疗前后血清BNP水平和外周血淋巴细胞GRK2 m RNA表达。结果治疗前后两组心脏超声各项指标比较无明显差异(P>0.05),观察组BNP水平与外周血淋巴细胞GRK2 m RNA表达明显低于对照组,差异有统计学意义(P<0.05)。结论老年慢性心衰患者经美托洛尔短期治疗后,虽未能显著改善心脏射血分数等指标,但可明显降低血清BNP水平和外周血淋巴细胞GRK2 m RNA的表达。展开更多
OBJECTIVE Increased efficacy without increased toxicity is expected when treating rheumatoid arthritis(RA).However,there are many difficulties associated with currently available RA treatments.Reportedly,CP-25,a new c...OBJECTIVE Increased efficacy without increased toxicity is expected when treating rheumatoid arthritis(RA).However,there are many difficulties associated with currently available RA treatments.Reportedly,CP-25,a new compound developed by our group,significantly inhibits the progression of arthritis in animal models through reducing membrane expression of GRK2.This study observed CP-25 monotherapy and combined administration with MTX/LEF in treating animal arthritis model and investigated possible mechanisms.METHODS We set up AA rat and collageninduced arthritis(CIA)mice model.Experimental groups were divided into normal group,vehicle group,monotherapy groups and CP-25-combined MTX/LEF groups.We focused on the role of GRK2 on macrophage polarization and fibro⁃blast-like synoviocytes(FLS)proliferation.We measured cytokine levels,phosphorylation and protein expression,and interactions between proteins.Targeted disruption of GRK2 in FLS and macrophages through GRK2 siRNA and CRIS⁃PR/Cas9.RESULTS Equivalent therapeutic effects were observed between CP-25-combination groups and high-dose MTX/LEF groups.In the vehicle group,GRK2 membrane expression increased leading to the decreased GRK2-p-ERK interactions in FLS(leading to the phosphorylation of ERK related with FLS over-proliferation),and increased GRK2-EP4 interaction in macrophage(leading to the abnormal PGE2-EP4-cAMP-CREB signal related with imbalance of macro⁃phage polarization).CONCLUSION CP-25 monotherapy and combined administration with MTX/LEF ameliorate the progression of animal arthritis model.CP-25 inhibited p-ERK by reducing the membrane expression of GRK2 in FLS from AA rats.CP-25 restored normal PGE2-EP4-cAMP-CREB signal through inhibiting GRK2 transferring to membrane.These results highlighted CP-25 a kind of potential for treating patients with RA.展开更多
The adrenergic receptor kinase 1 (ADRBK1), thromboxane A2 receptor (TBXA2R) and vascular endothelial growth factor (VEGFA) regulatory (r) single nucleotide polymorphisms (SNPs) found in the potential stimulating prote...The adrenergic receptor kinase 1 (ADRBK1), thromboxane A2 receptor (TBXA2R) and vascular endothelial growth factor (VEGFA) regulatory (r) single nucleotide polymorphisms (SNPs) found in the potential stimulating protein-1 (SP1) and Kruppel-like factor-4 (KLF4) transcriptional factor binding sites (TFBS) within these genes are in linkage disequilibrium (LD). The LD may result from rSNP alleles that create TFBS for the KLF4 and SP1 transcriptional factors (TF) since the alternate rSNP alleles do not create these TFBS. Consequently, haplotypes carrying the rSNP alleles that create KLF4 and SP1 TFBS are essential for ADRBK1, TBXA2R and VEGFA gene regulation by these TFs.展开更多
基金supported by the National Natural Science Foundation of China Excellent Young Scientist Fund(22422801)the National Natural Science Foundation of China General Project(22278053)+1 种基金the National Natural Science Foundation of China General Project(22078041)Dalian High-level Talents Innovation Support Program(2023RQ059).
文摘G protein coupled receptor kinase 2 (GRK2) is a kinase that regulates cardiac signaling activity. Inhibiting GRK2 is a promising mechanism for the treatment of heart failure (HF). Further development and optimization of inhibitors targeting GRK2 are highly meaningful. Therefore, in order to design GRK2 inhibitors with better performance, the most active molecule was selected as a reference compound from a data set containing 4-pyridylhydrazone derivatives and triazole derivatives, and its scaffold was extracted as the initial scaffold. Then, a powerful optimization-based framework for de novo drug design, guided by binding affinity, was used to generate a virtual molecular library targeting GRK2. The binding affinity of each virtual compound in this dataset was predicted by our developed deep learning model, and the designed potential compound with high binding affinity was selected for molecular docking and molecular dynamics simulation. It was found that the designed potential molecule binds to the ATP site of GRK2, which consists of key amino acids including Arg199, Gly200, Phe202, Val205, Lys220, Met274 and Asp335. The scaffold of the molecule is stabilized mainly by H-bonding and hydrophobic contacts. Concurrently, the reference compound in the dataset was also simulated by docking. It was found that this molecule also binds to the ATP site of GRK2. In addition, its scaffold is stabilized mainly by H-bonding and π-cation stacking interactions with Lys220, as well as hydrophobic contacts. The above results show that the designed potential molecule has similar binding modes to the reference compound, supporting the effectiveness of our framework for activity-focused molecular design. Finally, we summarized the interaction characteristics of general GRK2 inhibitors and gained insight into their molecule-target binding mechanisms, thereby facilitating the expansion of lead to hit compound.
文摘目的研究芍药苷-6-氧-苯磺酸酯(CP-25)通过抑制GRK2活性对骨关节炎(osteoarthritis,OA)小鼠膝关节软骨的保护作用。方法内侧半月板失稳(destabilization of the medial meniscus,DMM)手术诱导构建小鼠骨关节炎模型,实验分为假手术组、模型组、CP-25给药组和帕罗西汀给药组。术后开始灌胃给药。给药12周处死动物,Micro-CT成像观察膝关节软骨退变、骨重塑异常等情况,番红固绿染色观察小鼠关节组织病理,免疫组化、免疫荧光检测软骨组织相关分子表达水平的影响。Western blot检测CP-25用药后软骨细胞的膜蛋白及总蛋白表达水平。结果模型小鼠关节软骨严重退变。CP-25可显著降低关节软骨骨赘数量及软骨下板厚度,促进软骨基质再生,减少软骨基质降解蛋白表达,对膝关节软骨有明显的保护作用。免疫组化和免疫荧光结果显示,CP-25治疗可显著降低膝关节组织中GRK2、ADAMTS5、MMP13的表达,并且升高膝关节组织中ColⅡ、Aggrecan表达。体外实验结果表明,CP-25给药可以显著降低GRK2的膜蛋白及总蛋白表达水平,升高EP4膜蛋白水平,降低MMP13水平。结论CP-25给药可显著促进OA小鼠关节软骨基质再生,减少软骨基质降解,对OA具有治疗作用,其机制与抑制GRK2介导的软骨基质代谢有关。
基金The project supported by National Natural Science Foundation of China(81502123,81330081,81202596)Natural Science Foundation of Anhui Province(1308085QH130)+3 种基金Anhui Province Natural Science Foundation in University(KJ2014A119)Grants for Scientific Research of BSKY from Anhui Medical University(XJ201212)Specialized Research Fund for the Doctoral Program of Higher Education(20113420120006,20123420110003)Program for Tackling Key Problems in Science and Technology by Anhui Province(1301042098)
文摘OBJECTIVE To investigate the role and mechanism of G protein-coupled receptor kinase 2(GRK2)involving in hepatocel ular carcinoma(HCC)progression.METHODS Cel Counting Kit 8 and tumor colony formation assay were designed to detect HCC cell proliferation,wound healing assay was to detect HCC migration.The correlation between GRK2 and early growth response-1(EGR1)were detected by RT-PCR and real-time PCR assays.Co-immunoprecipitation and Western blot assay were adopted to detect the relationship between GRK2and insulin-like growth factor 1 receptor(IGF-1R)signaling pathway.RESULTS In this study we find that GRK2plays an inhibition role in IGF1-induced HCC cell proliferation and migration.Overexpression of GRK2 causes a decrease in EGR1 expression,while knockdown of GRK2 leads to the dramatically increase in EGR1 expression in the treatment of IGF1.Through co-immunoprecipitation and Western blot assay,we confirm that GRK2can interact with IGF-1R and inhibiting IGF1-induced activation of IGF1R signaling pathway.Silencing EGR1attenuates GRK2 overexpression-caused inhibition of cell proliferation,tumor colony number and migrationactivity,while overexpressing of EGR1 restores the antiproliferative and migratory effect by GRK2 overexpression in HCCLM3 cells.CONCLUSION Taken together,these results suggest that GRK2 may inhibit IGF1-induced HCC cell growth and migration through down-regulation of EGR1 and indicate that enforced GRK2 may offer a potential therapeutic approach against HCC.
文摘目的观察美托洛尔对高龄老年慢性心衰患者血清BNP和外周血淋巴细胞GRK2表达的影响。方法选取80岁以上慢性心衰患者60例,随机分为两组对照组和观察组各30例。对照组患者接受如利尿剂、血管紧张素拮抗剂、强心剂、β阻滞剂和紧张素转换抑制剂等常规治疗,观察组患者在常规治疗基础上加用美托洛尔。两组治疗前后均行心脏超声检查,观察对比两组治疗前后血清BNP水平和外周血淋巴细胞GRK2 m RNA表达。结果治疗前后两组心脏超声各项指标比较无明显差异(P>0.05),观察组BNP水平与外周血淋巴细胞GRK2 m RNA表达明显低于对照组,差异有统计学意义(P<0.05)。结论老年慢性心衰患者经美托洛尔短期治疗后,虽未能显著改善心脏射血分数等指标,但可明显降低血清BNP水平和外周血淋巴细胞GRK2 m RNA的表达。
基金National Natural Science Foundation of China(8133008181673444+3 种基金3120067581573443)Anhui Province Natural Science Fund(outstanding youth)(170808J10)Key Projects of Outstanding young Talent Support Program in Colleges and Universities(gxyq ZD2016043)
文摘OBJECTIVE Increased efficacy without increased toxicity is expected when treating rheumatoid arthritis(RA).However,there are many difficulties associated with currently available RA treatments.Reportedly,CP-25,a new compound developed by our group,significantly inhibits the progression of arthritis in animal models through reducing membrane expression of GRK2.This study observed CP-25 monotherapy and combined administration with MTX/LEF in treating animal arthritis model and investigated possible mechanisms.METHODS We set up AA rat and collageninduced arthritis(CIA)mice model.Experimental groups were divided into normal group,vehicle group,monotherapy groups and CP-25-combined MTX/LEF groups.We focused on the role of GRK2 on macrophage polarization and fibro⁃blast-like synoviocytes(FLS)proliferation.We measured cytokine levels,phosphorylation and protein expression,and interactions between proteins.Targeted disruption of GRK2 in FLS and macrophages through GRK2 siRNA and CRIS⁃PR/Cas9.RESULTS Equivalent therapeutic effects were observed between CP-25-combination groups and high-dose MTX/LEF groups.In the vehicle group,GRK2 membrane expression increased leading to the decreased GRK2-p-ERK interactions in FLS(leading to the phosphorylation of ERK related with FLS over-proliferation),and increased GRK2-EP4 interaction in macrophage(leading to the abnormal PGE2-EP4-cAMP-CREB signal related with imbalance of macro⁃phage polarization).CONCLUSION CP-25 monotherapy and combined administration with MTX/LEF ameliorate the progression of animal arthritis model.CP-25 inhibited p-ERK by reducing the membrane expression of GRK2 in FLS from AA rats.CP-25 restored normal PGE2-EP4-cAMP-CREB signal through inhibiting GRK2 transferring to membrane.These results highlighted CP-25 a kind of potential for treating patients with RA.
文摘The adrenergic receptor kinase 1 (ADRBK1), thromboxane A2 receptor (TBXA2R) and vascular endothelial growth factor (VEGFA) regulatory (r) single nucleotide polymorphisms (SNPs) found in the potential stimulating protein-1 (SP1) and Kruppel-like factor-4 (KLF4) transcriptional factor binding sites (TFBS) within these genes are in linkage disequilibrium (LD). The LD may result from rSNP alleles that create TFBS for the KLF4 and SP1 transcriptional factors (TF) since the alternate rSNP alleles do not create these TFBS. Consequently, haplotypes carrying the rSNP alleles that create KLF4 and SP1 TFBS are essential for ADRBK1, TBXA2R and VEGFA gene regulation by these TFs.
