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.

PROTACs: great opportunities for academia and industry

“…Dario R. Alessi and coworkers first designed a PROTAC conjugate of the 308-R SGK inhibitor with the VH032 VHL binding ligand, targeting SGK3 for degradation 314 (Fig. 38).…”

PROteolysis TArgetting Chimeras (PROTACs) Strategy Applied to Kinases: Recent Advances

“…Thus, their inhibition could offer new ways to treat inflammatory or autoimmune diseases. Recently, several STKs were targeted using the PROTAC technology: the interleukin‐1 receptor‐associated kinase 4 (IRAK4), [ 94 ] the serum and glucocorticoid‐induced protein kinase 3 (SGK3), [ 95 ] the polo‐like kinase 1 (PLK1), [ 96 ] several members of the CDK family, [ 97–102 ] and the B isoform of the rapidly accelerated fribrosarcoma (BRAF). [ 103 ]…”

“…described in their study the design of one VHL‐based PROTAC using the Sanofi‐308 compound [ 116 ] as the POI‐binding warhead. This small molecule, in its R configuration, is able to inhibit SGK3 (IC 50 = 5 × 10 −9 m ), [ 95 ] and it was relatively selective on a panel of 140 kinases, even at a concentration of 1 × 10 −6 m . Two other kinases, the isoform SGK1 and the off‐target kinase S6K1, were also inhibited in a 1 × 10 −9 to 10 × 10 −9 m IC 50 range.…”

“…However, no effect on cell growth was observed. [ 95 ] This study stresses that an increase in selectivity could be promoted by the conversion of inhibitors into PROTACs. It also offers to the community a selective and efficient chemical probe to further investigations on the SGK3 cell role.…”

Significance of Selective Protein Degradation in the Development of Novel Targeted Drugs and Its Implications in Cancer Therapy

“…[ 136 ] Moreover, SGK3‐PROTAC1, which was constructed by the SGK inhibitor 308R and the VHL ligand VH032, was shown to selectively degrade SGK3 at submicromolar concentrations. [ 137 ] Furthermore, other kinases, such as Bruton's tyrosine kinase, [ 138 ] anaplastic lymphoma kinase, [ 139 ] Fms‐like tyrosine kinase 3, [ 140 ] serine/threonine kinase TANK‐binding kinase 1 (TBK1), [ 141 ] CDK6, [ 142 ] and CDK9, [ 143 ] have been designed as effective PROTACs using kinase inhibitors and E3 ligands such as VHL, CRBN, and MDM2, respectively.…”