G-protein coupled receptor kinase 4(GRK4)gene plays a core role in renal dopamine D1 receptor function for handing renal sodium excretion,the rs1801058 T variant has~50%frequency in the general East Asian population b...G-protein coupled receptor kinase 4(GRK4)gene plays a core role in renal dopamine D1 receptor function for handing renal sodium excretion,the rs1801058 T variant has~50%frequency in the general East Asian population but lacks functional evidence for salt-sensitivity.In this prospective study,we identified rs1801058 genotypes by sequencing in hypertensives patients and analyzed the variant's effects on blood pressure response to acute salt loading in 150 newly diagnosed hypertension patients.Our data indicated that there were no statistically significant difference in blood pressure changes between the overall rs1801058 genotype groups.Carriers of the C allele showed a significant rise in heart rate change according to acute saline loading.Among patients age≤40year,T allele carriers showed higher changes in systolic blood pressure(SBP),diastolic blood pressure(DBP),mean arterial pressure(MAP),and heart rate(HR)than C allele carriers when diuretic shrinkage.GRK4 single rs1801058 polymorphism cannot directly explain salt sensitivity in the Chinese hypertension patients.展开更多
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
Background G-protein–coupled receptor kinases (GRKs) play important roles in cardiac hypertrophy and heart failure. The role of GRK4 in hypertension has been well demonstrated, but little is known in cardiac ischemic...Background G-protein–coupled receptor kinases (GRKs) play important roles in cardiac hypertrophy and heart failure. The role of GRK4 in hypertension has been well demonstrated, but little is known in cardiac ischemic injury. In the present study, we explore if and how GRK4 regulates cardiomyocyte autophagy and influence the prognosis of myocardial infarction (MI).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
文摘G-protein coupled receptor kinase 4(GRK4)gene plays a core role in renal dopamine D1 receptor function for handing renal sodium excretion,the rs1801058 T variant has~50%frequency in the general East Asian population but lacks functional evidence for salt-sensitivity.In this prospective study,we identified rs1801058 genotypes by sequencing in hypertensives patients and analyzed the variant's effects on blood pressure response to acute salt loading in 150 newly diagnosed hypertension patients.Our data indicated that there were no statistically significant difference in blood pressure changes between the overall rs1801058 genotype groups.Carriers of the C allele showed a significant rise in heart rate change according to acute saline loading.Among patients age≤40year,T allele carriers showed higher changes in systolic blood pressure(SBP),diastolic blood pressure(DBP),mean arterial pressure(MAP),and heart rate(HR)than C allele carriers when diuretic shrinkage.GRK4 single rs1801058 polymorphism cannot directly explain salt sensitivity in the Chinese hypertension patients.
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·研究不同亚型G蛋白偶联受体激酶(G protein-coupled receptor kinase,GRK)在毒蕈碱型乙酰胆碱受体1(muscarinic acetylcholine receptor 1,M1受体)介导的偏向性信号转导过程中的作用机制,重点关注其调控M1受体与下游异源三聚体G蛋白(Gα_(q)-Gβ_(1)-Gγ_(2))及β-抑制蛋白2(β-arrestin 2,βarr2)结合的分子效应。方法·构建基于生物发光能量共振转移(bioluminescence resonance energy transfer,BRET)的高灵敏度蛋白互作检测系统,选取6种结构及功能各异的M1受体激动剂/变构调节剂,系统测定在不同激动剂/变构调节剂刺激下M1受体与4种GRK亚型(GRK2/3/5/6)、βarr2以及G蛋白之间的动态相互作用。所有BRET实验数据均采用时间-效应曲线的曲线下面积(area under the curve,AUC)进行量化统计。首先,通过梯度浓度激动剂/变构调节剂处理及AUC拟合,建立浓度-效应曲线,综合分析各激动剂/变构调节剂相较于内源性激动剂氯化乙酰胆碱(acetylcholine chloride,ACh),在促进M1受体与GRK3/5、βarr2及G蛋白相互作用方面的效能差异;然后,将GRK按亚型类别分为GRK2/3和GRK5/62组,分别计算高浓度条件下M1受体与2类GRK互作的最大AUC值,进而评估不同类型GRK对M1受体与βarr2或G蛋白结合强度的调控倾向。结果·6种激动剂/变构调节剂均能有效诱导M1受体与GRK3的结合,但是它们也均能引起M1受体与GRK5发生解离;变构调节剂BQCA不仅能单独激活M1受体并引发其与下游信号转导蛋白的结合,还在与ACh联合处理时,使M1-G蛋白和M1-βarr2体系的浓度-效应曲线显著左移,提示其对ACh增幅作用主要是通过减小半数效应浓度;7组药物诱导的M1-βarr2与M1-G蛋白互作最大AUC之间存在中度正相关(r=0.722),但无统计学意义(P=0.067);进一步分析表明,M1-GRK2/3与M1-GRK5/6互作最大AUC的比值,和M1-βarr2与M1-G蛋白互作最大AUC的比值同样呈正相关(r=0.760,P=0.047)。结论·M1受体可能在基础状态下即与GRK5/6预先结合,受体激动后两者解离,提示GRK5/6可能参与M1受体的失活或信号重编程;M1受体对不同GRK亚型的相对作用效率决定了其下游信号通路的偏好。
文摘Background G-protein–coupled receptor kinases (GRKs) play important roles in cardiac hypertrophy and heart failure. The role of GRK4 in hypertension has been well demonstrated, but little is known in cardiac ischemic injury. In the present study, we explore if and how GRK4 regulates cardiomyocyte autophagy and influence the prognosis of myocardial infarction (MI).
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。
