Abstract：
OBJECTIVE: Activation of CB1 by exogenous agonists causes adverse effects in vivo. Positive allosteric modulation may offer improved therapeutic potential and a reduced on-target adverse effect profile compared with orthosteric agonists, due to reduced desensitisation/tolerance, but this has not been directly tested. This study investigated the ability of PAMs/ago-PAMs to induce receptor regulation pathways, including desensitisation and receptor internalisation.
METHODS: Bioluminescence resonance energy transfer (BRET) assays in HEK293 cells were performed to investigate G protein dissociation, ERK1/2 phosphorylation and β-arrestin 2 translocation, while immunocytochemistry was performed to measure internalisation of CB1 in response to the PAMs ZCZ011, GAT229 and ABD1236 alone and in combination with the orthosteric agonists AEA, 2-AG, and AMB-FUBINACA.
RESULTS: ZCZ011, GAT229 and ABD1236 were allosteric agonists in all pathways tested. The ago-PAM ZCZ011 induced a biphasic ERK1/2 phosphorylation time course compared to transient activation by orthosteric agonists. In combination with 2-AG but not AEA or AMB-FUBINACA, ZCZ011 and ABD1236 caused the transient peak of ERK1/2 phosphorylation to become sustained. All PAMs increased the potency and efficacy of AEA-induced signalling in all pathways tested; however, no notable potentiation of 2-AG or AMB-FUBINACA was observed.
CONCLUSIONS: Ago-PAMs can potentiate endocannabinoid CB1 agonism by AEA to a larger extent compared with 2-AG. However, all compounds were found to be allosteric agonists and induce activation of CB1 in the absence of endocannabinoid, including β-arrestin 2 recruitment and internalisation. Thus, the spatiotemporal signalling of endogenous cannabinoids will not be retained in vivo.
METHODS: Bioluminescence resonance energy transfer (BRET) assays in HEK293 cells were performed to investigate G protein dissociation, ERK1/2 phosphorylation and β-arrestin 2 translocation, while immunocytochemistry was performed to measure internalisation of CB1 in response to the PAMs ZCZ011, GAT229 and ABD1236 alone and in combination with the orthosteric agonists AEA, 2-AG, and AMB-FUBINACA.
RESULTS: ZCZ011, GAT229 and ABD1236 were allosteric agonists in all pathways tested. The ago-PAM ZCZ011 induced a biphasic ERK1/2 phosphorylation time course compared to transient activation by orthosteric agonists. In combination with 2-AG but not AEA or AMB-FUBINACA, ZCZ011 and ABD1236 caused the transient peak of ERK1/2 phosphorylation to become sustained. All PAMs increased the potency and efficacy of AEA-induced signalling in all pathways tested; however, no notable potentiation of 2-AG or AMB-FUBINACA was observed.
CONCLUSIONS: Ago-PAMs can potentiate endocannabinoid CB1 agonism by AEA to a larger extent compared with 2-AG. However, all compounds were found to be allosteric agonists and induce activation of CB1 in the absence of endocannabinoid, including β-arrestin 2 recruitment and internalisation. Thus, the spatiotemporal signalling of endogenous cannabinoids will not be retained in vivo.
摘要：
目的：外源性激动剂对CB1的激活在体内引起不良反应。与正构激动剂相比，正变构调制可以提供改善的治疗潜力和减少的目标不良反应。由于脱敏/耐受性降低，但这还没有直接测试。这项研究调查了PAMs/ago-PAMs诱导受体调节途径的能力，包括脱敏和受体内化。
方法：在HEK293细胞中进行生物发光共振能量转移（BRET）测定以研究G蛋白解离，ERK1/2磷酸化和β-抑制蛋白2易位，虽然进行了免疫细胞化学来测量CB1对PAMsZCZ011，GAT229和ABD1236的反应以及与正构激动剂AEA的组合的内在化，2-AG,AMB-FUBINACA.
结果：ZCZ011，GAT229和ABD1236在所有测试途径中均为变构激动剂。与正构激动剂的瞬时激活相比，前PAMZCZ011诱导了双相ERK1/2磷酸化时程。与2-AG组合，但不与AEA或AMB-FUBINACA组合，ZCZ011和ABD1236引起ERK1/2磷酸化的瞬时峰变得持续。所有PAMs都增加了AEA诱导的信号在所有测试途径中的效力和功效；然而，未观察到2-AG或AMB-FUBINACA的显著增强作用.
结论：与2-AG相比，Ago-PAMs可以在更大程度上增强AEA对内源性大麻素CB1的激动作用。然而,发现所有化合物都是变构激动剂，并在不存在内源性大麻素的情况下诱导CB1活化，包括β-抑制蛋白2的招募和内部化。因此，内源性大麻素的时空信号不会在体内保留。
方法：在HEK293细胞中进行生物发光共振能量转移（BRET）测定以研究G蛋白解离，ERK1/2磷酸化和β-抑制蛋白2易位，虽然进行了免疫细胞化学来测量CB1对PAMsZCZ011，GAT229和ABD1236的反应以及与正构激动剂AEA的组合的内在化，2-AG,AMB-FUBINACA.
结果：ZCZ011，GAT229和ABD1236在所有测试途径中均为变构激动剂。与正构激动剂的瞬时激活相比，前PAMZCZ011诱导了双相ERK1/2磷酸化时程。与2-AG组合，但不与AEA或AMB-FUBINACA组合，ZCZ011和ABD1236引起ERK1/2磷酸化的瞬时峰变得持续。所有PAMs都增加了AEA诱导的信号在所有测试途径中的效力和功效；然而，未观察到2-AG或AMB-FUBINACA的显著增强作用.
结论：与2-AG相比，Ago-PAMs可以在更大程度上增强AEA对内源性大麻素CB1的激动作用。然而,发现所有化合物都是变构激动剂，并在不存在内源性大麻素的情况下诱导CB1活化，包括β-抑制蛋白2的招募和内部化。因此，内源性大麻素的时空信号不会在体内保留。
