Discovery Of Natural Compounds Against ?_1a And ?₂ Adrenergic Receptor Using Label-free Cell Phenotypic Assays

Objective:Cardio Vascular Disease?CDV?,also known as circulatory disease,is a series of diseases involving the circulatory system.The circulatory system refers to organs and tissues that transport blood in the human body,including the heart and blood vessels?arteries,veins,and microvessels?.Studies have shown that there are currently about 970 million cardiovascular patients worldwide,including about 290 million cardiovascular patients in China,with hypertension accounting for the majority.Adrenergic receptors are widely distributed in human tissues and organs,and are mainly divided into 9 subtypes of?_1A,?_1B,?_1D,?_2A,?_2B,?_2C,?₁, ?₂,and?₃.The ?₂-adrenoceptor? ?₂-AR?regulates functions of catecholamines by coupling to a stimulatory G protein?Gs?and subsequent activation of adenylate cyclase.The activated enzyme increases production of cyclic adenosine monophosphate?cAMP?,leading to the activation of protein kinase A.Inhibition of its signaling has potential benefits to patients with hypertension and heart failure.The?_1A-adrenoceptor(?_1A-AR)is also a member of the G-protein coupled receptor family and mediates a variety of physiological functions such as neurotransmission,vasoconstriction,cardiac variability,glycogenolysisetc,and has the function of regulating cardiovascular and central nervous system activities.In this study,new adrenergic receptor antagonists and new active compounds'skeleton were found from natural products by culturing A431 cells and HEK293-?_1AA cells.Methods:?1?Establishing a ?₂-adrenergic receptor screening model in the A431cells using adrenaline,propranolol and ICI 118,551 by a label-free cell pharmacological characterization technique;?2?Screening natural compounds against ?₂-AR using the ?₂-AR model;?3?Screening receptor specificity of compounds at the HCA-2 receptor and histamine H₁ receptor;?4?Screening the potency of compounds with specific activity on ?₂-AR;?5?Determining the kinetic binding characteristics of Palmatine on ?₂-AR using co-stimulation assays;?6?Examing the successful docking of palmatine on ?₂-AR employing LeDock program;?7?Obtaining high expression HEK293-?_1AA stable cell line of?_1AA receptor using plasmid transfection to,and establishing?_1AA adrenergic receptor model with epinephrine,A 61603,SNAP 5089;?8?Screening of natural compounds against?_1A-AR using the?_1A-AR model;?9?Screening the potency of compounds with specific activity on?_1A-AR.Results:?1?In the agonistic assay,?-?-epinephrine triggered a concentration-dependent saturable response.Its DMR signal consisted of an early negative DMR?N-DMR?event and a late prolonged positive DMR?P-DMR?,followed by an plateau,giving an EC₅₀0 value of 0.29±0.04 nM.In desensitization assay,addition of?-?-epinephrine at different concentrations followed by addition of 4 nM?-?-epinephrine revealed the desensitizing effect of early stimulation with an effective IC₅₀0 of 0.23±0.05 nM.In the antagonistic assay,the non-selective?-AR antagonist propranolol and the ?₂-AR-selective antagonist ICI 118,551 did not induce any detectable DMR in A431,but blocked the?-?-epinephrine DMR in a dose-dependently manner with an IC₅₀0 of 2.09±0.60 nM and 11.6±1.9 nM,respectively.?2?Nuciferine,Epiberberine,Harmaline,harmine,Palmatine and Columbamine suppressed?-?-epinephrine-induced DMR signals but had no effect on nicotinic acid-or histamine-induced DMR signals.However,emodin inhibited?-?-epinephrine,nicotinic acid and histamine-induced DMR signals;?3?Epiberberine,Nuciferine,Harmaline,Harmine suppressed?-?-epinephrine-induced DMR signals slightly on?₁-AR,but completely suppressed?-?-epinephrine-induced DMR signals on ?₂-AR.However,Columbamine,Palmatine inhibited?-?-epinephrine-induced DMR signals on both?₁-AR and ?₂-AR;?4?The IC₅₀0 value of epiberberine,palmatine,nuciferine,columbamine,harmaline,harmine are 2.3±0.2?M,2.6±0.3?M,15.8±2.6?M,17.0±4.0?M,⁶7?M,¹13?M.The Ki values of epiberberine,palmatine,nuciferine,columbamine,harmaline,harmine were 13.3?M,15.0?M,91.1?M,98.0?M,386.4?M,651.6?M,respectively.?5?Palmatine formed hydrogen bonding interactions with N312 and hydrophobic interactions with amino acid residues,such as D113 and V114.?6?The presence of Palmatine had little impact on the maximal response of?-?-epinephrine stimulation but concentration-dependently shifted the sigmoid curves rightward.Schild analysis suggested that Palmatine possessed an apparent pK_B value of-6.49 with a slope of 1.14;?7?In HEK293-?_1AA cells,Luteolin,Daurisoline,Baicalein,Quercetin and Tetrahydroberberine completely inhibited the DMR signal of A 61603 in a dose-dependent manner.Conclusion:?1?Endogenously expressed ?₂-AR in A431 is functional;?2?Emodin may act on ?₂-AR,HCA-2 and H₁-AR,these three GPCRs or their common downstream targets in the same cell line;?3?Nuciferine,Epiberberine,Harmaline,Harmine,Palmatine and Columbamine had antagonistic potency on?-AR,but the activity on?₁-AR was weaker than that on ?₂-AR;?4?The potency order was found to be Epiberberine?Palmatine>Nuciferine?Columbamine>Harmaline>Harmine;?5?Palmatine binds to ?₂-AR;?6?Palmatine acted as a competitive antagonist against ?₂-AR,but not an allosteric modulator;?7?Luteolin,Daurisoline,Baicalein,Quercetin and Tetrahydroberberine have relatively strong?_1AA receptor antagonistic activity,and their order of activity is:Baicalein>Daurisoline>Tetrahydroberberine>Luteolin>Quercetin.