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

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.

SGK3是一种含PX结构域（PX domain）的蛋白激酶，可在生长因子与致癌突变的作用下，于1类和3类磷脂酰肌醇3-激酶（PI3K）家族成员下游的内体处被激活。SGK3在介导乳腺癌细胞对1类PI3K或Akt抑制剂产生耐药性的过程中发挥关键作用：它可替代Akt活性的缺失，恢复mTORC1信号通路等增殖通路。因此，开发能够强效靶向SGK3、阻断其在耐药性中作用的工具至关重要。
本研究报道了SGK3-PROTAC1的开发过程——这是一种将308-R型SGK抑制剂与VH032型希佩尔-林道综合征（VHL）结合配体偶联而成的蛋白水解靶向嵌合体（PROTAC），可靶向SGK3并介导其降解。SGK3-PROTAC1（0.3 μM）可在2小时内使内源性SGK3降解50%，8小时内达到80%的最大降解效率，同时伴随SGK3底物NDRG1磷酸化水平的下降。
SGK3-PROTAC1不会降解与SGK3密切相关的SGK1和SGK2亚型，尽管这两种亚型可被308-R结合并抑制。蛋白质组学分析显示，经SGK3-PROTAC1处理后，SGK3是细胞内唯一水平发生显著降低的蛋白质。
低剂量的SGK3-PROTAC1（0.1~0.3 μM）可恢复依赖SGK3的ZR-75-1与CAMA-1乳腺癌细胞对Akt抑制剂（AZD5363）和PI3K抑制剂（GDC0941）的敏感性，而无法结合VHL型E3泛素连接酶的顺式异构体类似物则无此作用。
相较于传统SGK亚型抑制剂（14H），SGK3-PROTAC1可更有效地抑制经PI3K抑制剂（GDC0941）处理的ZR-75-1与CAMA-1癌细胞系的增殖。
本研究凸显了PROTAC策略相较于传统抑制剂，在靶向蛋白激酶信号通路时所具备的更高效力与选择性优势。SGK3-PROTAC1将成为探究SGK3通路功能的重要工具试剂。