title: Rp-cAMPS Prodrugs Reveal the cAMP Dependence of First-Phase Glucose-Stimulated Insulin Secretion.
abstract: 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.
authors.affiliation: 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.
authors.forename: Frank
authors.initials: F
authors.lastname: Schwede
authors.affiliation: 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.
authors.forename: Oleg G
authors.initials: OG
authors.lastname: Chepurny
authors.affiliation: 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.
authors.forename: Melanie
authors.initials: M
authors.lastname: Kaufholz
authors.affiliation: 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.
authors.forename: Daniela
authors.initials: D
authors.lastname: Bertinetti
authors.affiliation: 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.
authors.forename: Colin A
authors.initials: CA
authors.lastname: Leech
authors.affiliation: 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.
authors.forename: Over
authors.initials: O
authors.lastname: Cabrera
authors.affiliation: 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.
authors.forename: Yingmin
authors.initials: Y
authors.lastname: Zhu
authors.affiliation: 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.
authors.forename: Fang
authors.initials: F
authors.lastname: Mei
authors.affiliation: 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.
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Cheng
authors.affiliation: 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.
authors.forename: Jocelyn E
authors.initials: JE
authors.lastname: Manning Fox
authors.affiliation: 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.
authors.forename: Patrick E
authors.initials: PE
authors.lastname: MacDonald
authors.affiliation: 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.
authors.forename: Hans-G
authors.initials: HG
authors.lastname: Genieser
authors.affiliation: 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.
authors.forename: Friedrich W
authors.initials: FW
authors.lastname: Herberg
authors.affiliation: 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.
authors.forename: George G
authors.initials: GG
authors.lastname: Holz
mesh_terms: D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D019905:Benzyl Alcohol / Q000494:pharmacology; D002460:Cell Line; D000242:Cyclic AMP / Q000494:pharmacology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004950:Esterases / Q000378:metabolism; D005260:Female; D031541:Fluorescence Resonance Energy Transfer; D005786:Gene Expression Regulation / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D020035:Holoenzymes / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D019426:Integrases / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D011355:Prodrugs / Q000494:pharmacology*; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects; D013873:Thionucleotides / Q000494:pharmacology*
chemical_list: C105593:8-bromoadenosine-3',5'-cyclic monophosphorothioate; D020662:Guanine Nucleotide Exchange Factors; D020035:Holoenzymes; D007328:Insulin; D011355:Prodrugs; C116122:RAPGEF3 protein, human; C442864:RAPGEF4 protein, human; D013873:Thionucleotides; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000242:Cyclic AMP; D008156:Luciferases; D017868:Cyclic AMP-Dependent Protein Kinases; C045073:Cre recombinase; D019426:Integrases; D004950:Esterases; D005947:Glucose; D019905:Benzyl Alcohol