Identification of A Novel Class of Benzofuran Oxoacetic Acid-Derived Ligands that Selectively Activate Cellular EPAC1

Elizabeth M. Beck, Euan Parnell, Angela Cowley, Alison Porter, Jonathan Gillespie, John Robinson, Lindsay Robinson, Andrew D Pannifer, Veronique Hamon, Philip Jones, Angus Morrison, Stuart McElroy, Martin Timmerman, Helma Rutjes, Pravin Mahajan, Jolanta Wiejak, Urszula Luchowska-Stańska, David Morgan, Graeme Barker, Holger RehmannStephen J. Yarwood

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Abstract

Cyclic AMP promotes EPAC1 and EPAC2 activation through direct binding to a specific cyclic nucleotide-binding domain (CNBD) within each protein, leading to activation of Rap GTPases, which control multiple cell responses, including cell proliferation, adhesion, morphology, exocytosis, and gene expression. As a result, it has become apparent that directed activation of EPAC1 and EPAC2 with synthetic agonists may also be useful for the future treatment of diabetes and cardiovascular diseases. To identify new EPAC agonists we have developed a fluorescent-based, ultra-high-throughput screening (uHTS) assay that measures the displacement of binding of the fluorescent cAMP analogue, 8-NBD-cAMP to the EPAC1 CNBD. Triage of the output of an approximately 350,000 compound screens using this assay identified a benzofuran oxaloacetic acid EPAC1 binder (SY000) that displayed moderate potency using orthogonal assays (competition binding and microscale thermophoresis). We next generated a limited library of 91 analogues of SY000 and identified SY009, with modifications to the benzofuran ring associated with a 10-fold increase in potency towards EPAC1 over SY000 in binding assays. In vitro EPAC1 activity assays confirmed the agonist potential of these molecules in comparison with the known EPAC1 non-cyclic nucleotide (NCN) partial agonist, I942. Rap1 GTPase activation assays further demonstrated that SY009 selectively activates EPAC1 over EPAC2 in cells. SY009 therefore represents a novel class of NCN EPAC1 activators that selectively activate EPAC1 in cellulae.

Original languageEnglish
Article number1468
Pages (from-to)1-18
Number of pages18
JournalCells
Volume8
Issue number11
Early online date12 Nov 2019
DOIs
Publication statusPublished - Nov 2019

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GTP Phosphohydrolases
Cyclic Nucleotides
Assays
Nucleotides
High-Throughput Screening Assays
Ligands
Oxaloacetic Acid
Acids
Triage
Exocytosis
Chemical activation
Cell Adhesion
Cyclic AMP
Cardiovascular Diseases
Cell Proliferation
Gene Expression
Thermophoresis
Proteins
Cell proliferation
Medical problems

Keywords

  • cyclic AMP
  • EPAC
  • uHTS
  • Rap1
  • guanine nucleotide exchange factor (GEF) activity
  • agonists

Cite this

Beck, E. M., Parnell, E., Cowley, A., Porter, A., Gillespie, J., Robinson, J., ... Yarwood, S. J. (2019). Identification of A Novel Class of Benzofuran Oxoacetic Acid-Derived Ligands that Selectively Activate Cellular EPAC1. Cells, 8(11), 1-18. [1468]. https://doi.org/10.3390/cells8111425
Beck, Elizabeth M. ; Parnell, Euan ; Cowley, Angela ; Porter, Alison ; Gillespie, Jonathan ; Robinson, John ; Robinson, Lindsay ; Pannifer, Andrew D ; Hamon, Veronique ; Jones, Philip ; Morrison, Angus ; McElroy, Stuart ; Timmerman, Martin ; Rutjes, Helma ; Mahajan, Pravin ; Wiejak, Jolanta ; Luchowska-Stańska, Urszula ; Morgan, David ; Barker, Graeme ; Rehmann, Holger ; Yarwood, Stephen J. / Identification of A Novel Class of Benzofuran Oxoacetic Acid-Derived Ligands that Selectively Activate Cellular EPAC1. In: Cells. 2019 ; Vol. 8, No. 11. pp. 1-18.
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abstract = "Cyclic AMP promotes EPAC1 and EPAC2 activation through direct binding to a specific cyclic nucleotide-binding domain (CNBD) within each protein, leading to activation of Rap GTPases, which control multiple cell responses, including cell proliferation, adhesion, morphology, exocytosis, and gene expression. As a result, it has become apparent that directed activation of EPAC1 and EPAC2 with synthetic agonists may also be useful for the future treatment of diabetes and cardiovascular diseases. To identify new EPAC agonists we have developed a fluorescent-based, ultra-high-throughput screening (uHTS) assay that measures the displacement of binding of the fluorescent cAMP analogue, 8-NBD-cAMP to the EPAC1 CNBD. Triage of the output of an approximately 350,000 compound screens using this assay identified a benzofuran oxaloacetic acid EPAC1 binder (SY000) that displayed moderate potency using orthogonal assays (competition binding and microscale thermophoresis). We next generated a limited library of 91 analogues of SY000 and identified SY009, with modifications to the benzofuran ring associated with a 10-fold increase in potency towards EPAC1 over SY000 in binding assays. In vitro EPAC1 activity assays confirmed the agonist potential of these molecules in comparison with the known EPAC1 non-cyclic nucleotide (NCN) partial agonist, I942. Rap1 GTPase activation assays further demonstrated that SY009 selectively activates EPAC1 over EPAC2 in cells. SY009 therefore represents a novel class of NCN EPAC1 activators that selectively activate EPAC1 in cellulae.",
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Beck, EM, Parnell, E, Cowley, A, Porter, A, Gillespie, J, Robinson, J, Robinson, L, Pannifer, AD, Hamon, V, Jones, P, Morrison, A, McElroy, S, Timmerman, M, Rutjes, H, Mahajan, P, Wiejak, J, Luchowska-Stańska, U, Morgan, D, Barker, G, Rehmann, H & Yarwood, SJ 2019, 'Identification of A Novel Class of Benzofuran Oxoacetic Acid-Derived Ligands that Selectively Activate Cellular EPAC1', Cells, vol. 8, no. 11, 1468, pp. 1-18. https://doi.org/10.3390/cells8111425

Identification of A Novel Class of Benzofuran Oxoacetic Acid-Derived Ligands that Selectively Activate Cellular EPAC1. / Beck, Elizabeth M.; Parnell, Euan; Cowley, Angela; Porter, Alison; Gillespie, Jonathan; Robinson, John; Robinson, Lindsay; Pannifer, Andrew D; Hamon, Veronique; Jones, Philip; Morrison, Angus; McElroy, Stuart; Timmerman, Martin; Rutjes, Helma; Mahajan, Pravin; Wiejak, Jolanta; Luchowska-Stańska, Urszula; Morgan, David; Barker, Graeme; Rehmann, Holger; Yarwood, Stephen J. (Lead / Corresponding author).

In: Cells, Vol. 8, No. 11, 1468, 11.2019, p. 1-18.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Identification of A Novel Class of Benzofuran Oxoacetic Acid-Derived Ligands that Selectively Activate Cellular EPAC1

AU - Beck, Elizabeth M.

AU - Parnell, Euan

AU - Cowley, Angela

AU - Porter, Alison

AU - Gillespie, Jonathan

AU - Robinson, John

AU - Robinson, Lindsay

AU - Pannifer, Andrew D

AU - Hamon, Veronique

AU - Jones, Philip

AU - Morrison, Angus

AU - McElroy, Stuart

AU - Timmerman, Martin

AU - Rutjes, Helma

AU - Mahajan, Pravin

AU - Wiejak, Jolanta

AU - Luchowska-Stańska, Urszula

AU - Morgan, David

AU - Barker, Graeme

AU - Rehmann, Holger

AU - Yarwood, Stephen J.

PY - 2019/11

Y1 - 2019/11

N2 - Cyclic AMP promotes EPAC1 and EPAC2 activation through direct binding to a specific cyclic nucleotide-binding domain (CNBD) within each protein, leading to activation of Rap GTPases, which control multiple cell responses, including cell proliferation, adhesion, morphology, exocytosis, and gene expression. As a result, it has become apparent that directed activation of EPAC1 and EPAC2 with synthetic agonists may also be useful for the future treatment of diabetes and cardiovascular diseases. To identify new EPAC agonists we have developed a fluorescent-based, ultra-high-throughput screening (uHTS) assay that measures the displacement of binding of the fluorescent cAMP analogue, 8-NBD-cAMP to the EPAC1 CNBD. Triage of the output of an approximately 350,000 compound screens using this assay identified a benzofuran oxaloacetic acid EPAC1 binder (SY000) that displayed moderate potency using orthogonal assays (competition binding and microscale thermophoresis). We next generated a limited library of 91 analogues of SY000 and identified SY009, with modifications to the benzofuran ring associated with a 10-fold increase in potency towards EPAC1 over SY000 in binding assays. In vitro EPAC1 activity assays confirmed the agonist potential of these molecules in comparison with the known EPAC1 non-cyclic nucleotide (NCN) partial agonist, I942. Rap1 GTPase activation assays further demonstrated that SY009 selectively activates EPAC1 over EPAC2 in cells. SY009 therefore represents a novel class of NCN EPAC1 activators that selectively activate EPAC1 in cellulae.

AB - Cyclic AMP promotes EPAC1 and EPAC2 activation through direct binding to a specific cyclic nucleotide-binding domain (CNBD) within each protein, leading to activation of Rap GTPases, which control multiple cell responses, including cell proliferation, adhesion, morphology, exocytosis, and gene expression. As a result, it has become apparent that directed activation of EPAC1 and EPAC2 with synthetic agonists may also be useful for the future treatment of diabetes and cardiovascular diseases. To identify new EPAC agonists we have developed a fluorescent-based, ultra-high-throughput screening (uHTS) assay that measures the displacement of binding of the fluorescent cAMP analogue, 8-NBD-cAMP to the EPAC1 CNBD. Triage of the output of an approximately 350,000 compound screens using this assay identified a benzofuran oxaloacetic acid EPAC1 binder (SY000) that displayed moderate potency using orthogonal assays (competition binding and microscale thermophoresis). We next generated a limited library of 91 analogues of SY000 and identified SY009, with modifications to the benzofuran ring associated with a 10-fold increase in potency towards EPAC1 over SY000 in binding assays. In vitro EPAC1 activity assays confirmed the agonist potential of these molecules in comparison with the known EPAC1 non-cyclic nucleotide (NCN) partial agonist, I942. Rap1 GTPase activation assays further demonstrated that SY009 selectively activates EPAC1 over EPAC2 in cells. SY009 therefore represents a novel class of NCN EPAC1 activators that selectively activate EPAC1 in cellulae.

KW - cyclic AMP

KW - EPAC

KW - uHTS

KW - Rap1

KW - guanine nucleotide exchange factor (GEF) activity

KW - agonists

U2 - 10.3390/cells8111425

DO - 10.3390/cells8111425

M3 - Article

C2 - 31726720

VL - 8

SP - 1

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JO - Cells

JF - Cells

SN - 2073-4409

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