Design and Characterization of SGK3-PROTAC1, an Isoform Specific SGK3 Kinase PROTAC Degrader

Hannah Tovell, Andrea Testa, Houjiang Zhou, Natalia Shpiro, Claire Crafter, Alessio Ciulli (Lead / Corresponding author), Dario Alessi (Lead / Corresponding author)

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Abstract

SGK3 is a PX domain containing protein kinase activated at endosomes downstream of class 1 and 3 PI3K family members by growth factors and oncogenic mutations. SGK3 plays a key role in mediating resistance of breast cancer cells to class 1 PI3K or Akt inhibitors, by substituting for the loss of Akt activity and restoring proliferative pathways such as mTORC1 signaling. It is therefore critical to develop tools to potently target SGK3 and obstruct its role in inhibitor resistance. Here, we describe the development of SGK3-PROTAC1, a PROTAC conjugate of the 308-R SGK inhibitor with the VH032 VHL binding ligand, targeting SGK3 for degradation. SGK3-PROTAC1 (0.3 μM) induced 50% degradation of endogenous SGK3 within 2 h, with maximal 80% degradation observed within 8 h, accompanied by a loss of phosphorylation of NDRG1, an SGK3 substrate. SGK3-PROTAC1 did not degrade closely related SGK1 and SGK2 isoforms that are nevertheless engaged and inhibited by 308-R. Proteomic analysis revealed that SGK3 was the only cellular protein whose cellular levels were significantly reduced following treatment with SGK3-PROTAC1. Low doses of SGK3-PROTAC1 (0.1–0.3 μM) restored sensitivity of SGK3 dependent ZR-75-1 and CAMA-1 breast cancer cells to Akt (AZD5363) and PI3K (GDC0941) inhibitors, whereas the cis epimer analogue incapable of binding to the VHL E3 ligase had no impact. SGK3-PROTAC1 suppressed proliferation of ZR-75-1 and CAMA-1 cancer cell lines treated with a PI3K inhibitor (GDC0941) more effectively than could be achieved by a conventional SGK isoform inhibitor (14H). This work underscores the benefit of the PROTAC approach in targeting protein kinase signaling pathways with greater efficacy and selectivity than can be achieved with conventional inhibitors. SGK3-PROTAC1 will be an important reagent to explore the roles of the SGK3 pathway.
Original languageEnglish
Pages (from-to)2024-2034
Number of pages11
JournalACS Chemical Biology
Volume14
Issue number9
Early online date28 Aug 2019
DOIs
Publication statusPublished - 20 Sep 2019

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Phosphatidylinositol 3-Kinases
Protein Isoforms
Phosphotransferases
Cells
Degradation
Protein Kinases
Breast Neoplasms
Phosphorylation
Ubiquitin-Protein Ligases
Endosomes
Proteomics
Intercellular Signaling Peptides and Proteins
Ligands
Cell Line
Mutation
Substrates
Neoplasms
Proteins
2-(1H-indazol-4-yl)-6-(4-methanesulfonylpiperazin-1-ylmethyl)-4-morpholin-4-ylthieno(3,2-d)pyrimidine

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@article{745f638cacbf4630a11f1dc37b0d2f72,
title = "Design and Characterization of SGK3-PROTAC1, an Isoform Specific SGK3 Kinase PROTAC Degrader",
abstract = "SGK3 is a PX domain containing protein kinase activated at endosomes downstream of class 1 and 3 PI3K family members by growth factors and oncogenic mutations. SGK3 plays a key role in mediating resistance of breast cancer cells to class 1 PI3K or Akt inhibitors, by substituting for the loss of Akt activity and restoring proliferative pathways such as mTORC1 signaling. It is therefore critical to develop tools to potently target SGK3 and obstruct its role in inhibitor resistance. Here, we describe the development of SGK3-PROTAC1, a PROTAC conjugate of the 308-R SGK inhibitor with the VH032 VHL binding ligand, targeting SGK3 for degradation. SGK3-PROTAC1 (0.3 μM) induced 50{\%} degradation of endogenous SGK3 within 2 h, with maximal 80{\%} degradation observed within 8 h, accompanied by a loss of phosphorylation of NDRG1, an SGK3 substrate. SGK3-PROTAC1 did not degrade closely related SGK1 and SGK2 isoforms that are nevertheless engaged and inhibited by 308-R. Proteomic analysis revealed that SGK3 was the only cellular protein whose cellular levels were significantly reduced following treatment with SGK3-PROTAC1. Low doses of SGK3-PROTAC1 (0.1–0.3 μM) restored sensitivity of SGK3 dependent ZR-75-1 and CAMA-1 breast cancer cells to Akt (AZD5363) and PI3K (GDC0941) inhibitors, whereas the cis epimer analogue incapable of binding to the VHL E3 ligase had no impact. SGK3-PROTAC1 suppressed proliferation of ZR-75-1 and CAMA-1 cancer cell lines treated with a PI3K inhibitor (GDC0941) more effectively than could be achieved by a conventional SGK isoform inhibitor (14H). This work underscores the benefit of the PROTAC approach in targeting protein kinase signaling pathways with greater efficacy and selectivity than can be achieved with conventional inhibitors. SGK3-PROTAC1 will be an important reagent to explore the roles of the SGK3 pathway.",
author = "Hannah Tovell and Andrea Testa and Houjiang Zhou and Natalia Shpiro and Claire Crafter and Alessio Ciulli and Dario Alessi",
note = "This work was supported by the Medical Research Council [grant number MC_UU_12016/2 (to D.R.A.)]; the European Research Council (ERC) under the European Union’s Seventh Framework Programme (FP7/2007−2013) as a Starting Grant to A.C. [grant agreement No. ERC−2012−StG−311460 DrugE3CRLs]; and the pharmaceutical companies supporting the Division of Signal Transduction Therapy Unit (Boehringer Ingelheim, GlaxoSmithKline, and Merck KGaA, to D.R.A.). H.T. is supported by an AstraZeneca BBSRC Studentship.",
year = "2019",
month = "9",
day = "20",
doi = "10.1021/acschembio.9b00505",
language = "English",
volume = "14",
pages = "2024--2034",
journal = "ACS Chemical Biology",
issn = "1554-8929",
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T1 - Design and Characterization of SGK3-PROTAC1, an Isoform Specific SGK3 Kinase PROTAC Degrader

AU - Tovell, Hannah

AU - Testa, Andrea

AU - Zhou, Houjiang

AU - Shpiro, Natalia

AU - Crafter, Claire

AU - Ciulli, Alessio

AU - Alessi, Dario

N1 - This work was supported by the Medical Research Council [grant number MC_UU_12016/2 (to D.R.A.)]; the European Research Council (ERC) under the European Union’s Seventh Framework Programme (FP7/2007−2013) as a Starting Grant to A.C. [grant agreement No. ERC−2012−StG−311460 DrugE3CRLs]; and the pharmaceutical companies supporting the Division of Signal Transduction Therapy Unit (Boehringer Ingelheim, GlaxoSmithKline, and Merck KGaA, to D.R.A.). H.T. is supported by an AstraZeneca BBSRC Studentship.

PY - 2019/9/20

Y1 - 2019/9/20

N2 - SGK3 is a PX domain containing protein kinase activated at endosomes downstream of class 1 and 3 PI3K family members by growth factors and oncogenic mutations. SGK3 plays a key role in mediating resistance of breast cancer cells to class 1 PI3K or Akt inhibitors, by substituting for the loss of Akt activity and restoring proliferative pathways such as mTORC1 signaling. It is therefore critical to develop tools to potently target SGK3 and obstruct its role in inhibitor resistance. Here, we describe the development of SGK3-PROTAC1, a PROTAC conjugate of the 308-R SGK inhibitor with the VH032 VHL binding ligand, targeting SGK3 for degradation. SGK3-PROTAC1 (0.3 μM) induced 50% degradation of endogenous SGK3 within 2 h, with maximal 80% degradation observed within 8 h, accompanied by a loss of phosphorylation of NDRG1, an SGK3 substrate. SGK3-PROTAC1 did not degrade closely related SGK1 and SGK2 isoforms that are nevertheless engaged and inhibited by 308-R. Proteomic analysis revealed that SGK3 was the only cellular protein whose cellular levels were significantly reduced following treatment with SGK3-PROTAC1. Low doses of SGK3-PROTAC1 (0.1–0.3 μM) restored sensitivity of SGK3 dependent ZR-75-1 and CAMA-1 breast cancer cells to Akt (AZD5363) and PI3K (GDC0941) inhibitors, whereas the cis epimer analogue incapable of binding to the VHL E3 ligase had no impact. SGK3-PROTAC1 suppressed proliferation of ZR-75-1 and CAMA-1 cancer cell lines treated with a PI3K inhibitor (GDC0941) more effectively than could be achieved by a conventional SGK isoform inhibitor (14H). This work underscores the benefit of the PROTAC approach in targeting protein kinase signaling pathways with greater efficacy and selectivity than can be achieved with conventional inhibitors. SGK3-PROTAC1 will be an important reagent to explore the roles of the SGK3 pathway.

AB - SGK3 is a PX domain containing protein kinase activated at endosomes downstream of class 1 and 3 PI3K family members by growth factors and oncogenic mutations. SGK3 plays a key role in mediating resistance of breast cancer cells to class 1 PI3K or Akt inhibitors, by substituting for the loss of Akt activity and restoring proliferative pathways such as mTORC1 signaling. It is therefore critical to develop tools to potently target SGK3 and obstruct its role in inhibitor resistance. Here, we describe the development of SGK3-PROTAC1, a PROTAC conjugate of the 308-R SGK inhibitor with the VH032 VHL binding ligand, targeting SGK3 for degradation. SGK3-PROTAC1 (0.3 μM) induced 50% degradation of endogenous SGK3 within 2 h, with maximal 80% degradation observed within 8 h, accompanied by a loss of phosphorylation of NDRG1, an SGK3 substrate. SGK3-PROTAC1 did not degrade closely related SGK1 and SGK2 isoforms that are nevertheless engaged and inhibited by 308-R. Proteomic analysis revealed that SGK3 was the only cellular protein whose cellular levels were significantly reduced following treatment with SGK3-PROTAC1. Low doses of SGK3-PROTAC1 (0.1–0.3 μM) restored sensitivity of SGK3 dependent ZR-75-1 and CAMA-1 breast cancer cells to Akt (AZD5363) and PI3K (GDC0941) inhibitors, whereas the cis epimer analogue incapable of binding to the VHL E3 ligase had no impact. SGK3-PROTAC1 suppressed proliferation of ZR-75-1 and CAMA-1 cancer cell lines treated with a PI3K inhibitor (GDC0941) more effectively than could be achieved by a conventional SGK isoform inhibitor (14H). This work underscores the benefit of the PROTAC approach in targeting protein kinase signaling pathways with greater efficacy and selectivity than can be achieved with conventional inhibitors. SGK3-PROTAC1 will be an important reagent to explore the roles of the SGK3 pathway.

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U2 - 10.1021/acschembio.9b00505

DO - 10.1021/acschembio.9b00505

M3 - Article

VL - 14

SP - 2024

EP - 2034

JO - ACS Chemical Biology

JF - ACS Chemical Biology

SN - 1554-8929

IS - 9

ER -