12I

Amino Acid Sequence

3D Structure (/structures/AF-Q9Y243-F1-model_v6.cif)

Function

AKT3 is one of 3 closely related serine/threonine-protein kinases (AKT1, AKT2 and AKT3) called the AKT kinase, and which regulate many processes including metabolism, proliferation, cell survival, growth and angiogenesis. This is mediated through serine and/or threonine phosphorylation of a range of downstream substrates. Over 100 substrate candidates have been reported so far, but for most of them, no isoform specificity has been reported. AKT3 is the least studied AKT isoform. It plays an important role in brain development and is crucial for the viability of malignant glioma cells. AKT3 isoform may also be the key molecule in up-regulation and down-regulation of MMP13 via IL13. Required for the coordination of mitochondrial biogenesis with growth factor-induced increases in cellular energy demands. Down-regulation by RNA interference reduces the expression of the phosphorylated form of BAD, resulting in the induction of caspase-dependent apoptosis.

Amino Acid Sequence

3D Structure (/structures/AF-P00533-F1-model_v6.cif)

Function

Receptor tyrosine kinase binding ligands of the EGF family and activating several signaling cascades to convert extracellular cues into appropriate cellular responses. Known ligands include EGF, TGFA/TGF-alpha, AREG, epigen/EPGN, BTC/betacellulin, epiregulin/EREG and HBEGF/heparin-binding EGF. Ligand binding triggers receptor homo- and/or heterodimerization and autophosphorylation on key cytoplasmic residues. The phosphorylated receptor recruits adapter proteins like GRB2 which in turn activates complex downstream signaling cascades. Activates at least 4 major downstream signaling cascades including the RAS-RAF-MEK-ERK, PI3 kinase-AKT, PLCgamma-PKC and STATs modules. May also activate the NF-kappa-B signaling cascade. Also directly phosphorylates other proteins like RGS16, activating its GTPase activity and probably coupling the EGF receptor signaling to the G protein-coupled receptor signaling. Also phosphorylates MUC1 and increases its interaction with SRC and CTNNB1/beta-catenin. Positively regulates cell migration via interaction with CCDC88A/GIV which retains EGFR at the cell membrane following ligand stimulation, promoting EGFR signaling which triggers cell migration. Plays a role in enhancing learning and memory performance (By similarity). Plays a role in mammalian pain signaling (long-lasting hypersensitivity) (By similarity). Isoform 2 may act as an antagonist of EGF action. (Microbial infection) Acts as a receptor for hepatitis C virus (HCV) in hepatocytes and facilitates its cell entry. Mediates HCV entry by promoting the formation of the CD81-CLDN1 receptor complexes that are essential for HCV entry and by enhancing membrane fusion of cells expressing HCV envelope glycoproteins.

Mechanism

In an ATP-dependent manner, the ubiquitin-activating enzyme (E1) activates ubiquitin (Ub) and conjugates it to a ubiquitin-conjugating enzyme (E2). E3 ligase subsequently catalyzes the transfer of Ub to the target protein, leading to the formation of a polyubiquitin chain that completes the ubiquitination mark. The 19S regulatory particle of the 26S proteasome recognizes this ubiquitination signal and, after deubiquitination, mediates the ATP-dependent delivery of the substrate into the 20S proteasome for degradation.

Description

To elucidate the mechanism of 12l-induced Akt3 degradation, we further investigated the contribution of proteasome in the protein degrading process. As shown in Figure 4B, Akt3 51 degradation could be triggered by compound 12l as early as 2 h after pre-treatment of a protein synthesis inhibitor cycloheximide. Whereas, the degradation of Akt3 could be prevented by the proteasome inhibitor MG132 (Figure 4C), suggesting involvement of the ubiquitin proteasome-mediated proteolysis process. This finding was further supported by an immunoprecipitation assay that demonstrated increased ubiquitination of Akt3 in H1975OR cells following 12l treatment (Figure 4D). These data collectively suggested that 12l-induced Akt3 degradation was mediated by the ubiquitin proteasome system.

Description

The resulting compounds 12k and 12l displayed DC50 values of 33 and 13nM against Akt3 in H1975 OR cells, where as their EGFR degrading potencies(DC50) were 4-and 44.5-fold less potent with DC50 values of 143.3 and 578.6 nM in H1975 cells, respectively.

Description

The resulting compounds 12k and 12l displayed DC50 values of 33 and 13nM against Akt3 in H1975 OR cells, where as their EGFR degrading potencies(DC50) were 4-and 44.5-fold less potent with DC50 values of 143.3 and 578.6 nM in H1975 cells, respectively.

Description

It was also shown that the selective Akt3 degrader 12l exhibited approximately 140-fold stronger antiproliferative activity than 8 in H1975OR cells, with IC 50 values of 0.007 ± 0.001 μM.

Description

The in vivo antitumor efficacy and selective Akt3 degrading potency of 12l were also evaluated in NOD-SCID-IL2Rg-/ (NSI) mice bearing the H1975OR xenograft model with different dosages and administration schedules [followed the in vivo pharmacokinetic (PK) properties of degrader (Figure S8A)]. The results showed that monotreatment of 1 did not show obvious antitumor activity in the xenograft model. Whereas, intraperitoneal administration of 12l with 10 or 20 mg/kg at one dose every 3 days for 5 weeks induced significant tumor growth inhibition (TGI) without causing obvious body weight loss or other signs of toxicity (Figures 6C,D and S8B). Moreover, almost complete TGI was achieved after 3−4 weekly dosages of 12l at 40 mg/kg, supporting the long duration of the degrader's efficacy. The results also showed that the TGI values were appoximately 33.2, 88.8, 87.6, and 79.8% at the indicated treatment conditions, respectively.

Description

The in vivo antitumor efficacy and selective Akt3 degrading potency of 12l were also validated in NOD-SCID-IL2Rg-/-(NSI) mice bearing the PC9 xenograft model (Figure S8C−E).The results also showed that 12l exhibited promising in vivo antitumor efficacy with an approximately TGI value of 75%,without causing obvious body weight loss.