Schwede Frank, Chepurny Oleg G, Kaufholz Melanie, Bertinetti Daniela, Leech Colin A, Cabrera Over, Zhu Yingmin, Mei Fang, Cheng Xiaodong, Manning Fox Jocelyn E, MacDonald Patrick E, Genieser Hans-G, Herberg Friedrich W, Holz George G
BIOLOG Life Science Institute (F.S., H.-G.G.), 28199 Bremen, Germany; Departments of Medicine (O.G.C., C.A.L., G.G.H.) and Pharmacology (G.G.H.), State University of New York, Upstate Medical University, Syracuse, New York 13210; Department of Biochemistry (M.K., D.B., F.W.H.), University of Kassel, 34132 Kassel, Germany; Eli Lilly and Company (O.C.), Indianapolis, Indiana 46225; Department of Integrative Biology and Pharmacology (Y.Z., F.M., X.C.), Texas Therapeutics Institute, The Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center, Houston, Texas 77030; Department of Pharmacology and the Alberta Diabetes Institute (J.E.M.F., P.E.M.), University of Alberta, Edmonton, Canada AB T6G 2E1.
Mol Endocrinol. 2015 Jul;29(7):988-1005. doi: 10.1210/me.2014-1330. Epub 2015 Jun 10.
cAMP-elevating agents such as the incretin hormone glucagon-like peptide-1 potentiate glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells. However, a debate has existed since the 1970s concerning whether or not cAMP signaling is essential for glucose alone to stimulate insulin secretion. Here, we report that the first-phase kinetic component of GSIS is cAMP-dependent, as revealed through the use of a novel highly membrane permeable para-acetoxybenzyl (pAB) ester prodrug that is a bioactivatable derivative of the cAMP antagonist adenosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-cAMPS). In dynamic perifusion assays of human or rat islets, a step-wise increase of glucose concentration leads to biphasic insulin secretion, and under these conditions, 8-bromoadenosine-3',5'-cyclic monophosphorothioate, Rp-isomer, 4-acetoxybenzyl ester (Rp-8-Br-cAMPS-pAB) inhibits first-phase GSIS by up to 80%. Surprisingly, second-phase GSIS is inhibited to a much smaller extent (≤20%). Using luciferase, fluorescence resonance energy transfer, and bioluminescence resonance energy transfer assays performed in living cells, we validate that Rp-8-Br-cAMPS-pAB does in fact block cAMP-dependent protein kinase activation. Novel effects of Rp-8-Br-cAMPS-pAB to block the activation of cAMP-regulated guanine nucleotide exchange factors (Epac1, Epac2) are also validated using genetically encoded Epac biosensors, and are independently confirmed in an in vitro Rap1 activation assay using Rp-cAMPS and Rp-8-Br-cAMPS. Thus, in addition to revealing the cAMP dependence of first-phase GSIS from human and rat islets, these findings establish a pAB-based chemistry for the synthesis of highly membrane permeable prodrug derivatives of Rp-cAMPS that act with micromolar or even nanomolar potency to inhibit cAMP signaling in living cells.
诸如肠促胰岛素激素胰高血糖素样肽-1等提高环磷酸腺苷（cAMP）的药物可增强胰腺β细胞对葡萄糖刺激的胰岛素分泌（GSIS）。然而，自20世纪70年代以来，关于cAMP信号传导对于葡萄糖单独刺激胰岛素分泌是否必不可少一直存在争议。在此，我们报告称，GSIS的第一阶段动力学成分是cAMP依赖性的，这是通过使用一种新型的高膜渗透性对乙酰氧基苄基（pAB）酯前药揭示的，该前药是cAMP拮抗剂腺苷-3',5'-环磷硫酰酯Rp异构体（Rp-cAMPS）的生物可活化衍生物。在人或大鼠胰岛的动态灌流试验中，葡萄糖浓度的逐步增加会导致双相胰岛素分泌，在这些条件下，8-溴腺苷-3',5'-环磷硫酰酯Rp异构体4-乙酰氧基苄基酯（Rp-8-Br-cAMPS-pAB）可将第一阶段GSIS抑制高达80%。令人惊讶的是，第二阶段GSIS的抑制程度要小得多（≤20%）。通过在活细胞中进行的荧光素酶、荧光共振能量转移和生物发光共振能量转移试验，我们验证了Rp-8-Br-cAMPS-pAB实际上确实阻断了cAMP依赖性蛋白激酶的激活。使用基因编码的Epac生物传感器也验证了Rp-8-Br-cAMPS-pAB阻断cAMP调节的鸟嘌呤核苷酸交换因子（Epac1、Epac2）激活的新作用，并在使用Rp-cAMPS和Rp-8-Br-cAMPS的体外Rap1激活试验中得到独立证实。因此，除了揭示人及大鼠胰岛第一阶段GSIS的cAMP依赖性外，这些发现还建立了一种基于pAB的化学方法，用于合成Rp-cAMPS的高膜渗透性前药衍生物，这些衍生物以微摩尔甚至纳摩尔的效力抑制活细胞中的cAMP信号传导。
