Inhibitors of mTORC2

[0108] Example 1. Identification and structural characterization of M342

[0109] In this example, the inventors unexpectedly found that a longer peptide containing the M342 sequence may interact with Sin1 by using protein affinity purification and mass spectrometry identification.

[0110] (1) Protein purification and mass spectrometry identification

[0111] The peptide GST-M342-619 or GST-empty vector was expressed in HEK293T cells, purified by GSH-agarose beads, and the protein strips specifically pulled down by GST-M342-619 were found by SDS-PAGE electrophoresis and silver staining. These bands were excised and analyzed by mass spectrometry (HPLC / MS / MS), and four segments of Sin1 amino acid sequence (LLPMTVVTMASAR) were identified, suggesting that Sin1 may be its binding protein.

[0112] (2) Pull down experiment

[0113] COS-1 cells were used to co-express GST-Sin1 and HA-labeled peptides of different lengths containing the M342 sequence, and 40 hours later, whole cell...

[0121] Example 2. Identification of the precise binding region of M342 and Sin1

[0122] By analyzing M342 by cross-linking mass spectrometry, the inventors found that the Sin1-N terminus is a hot spot for protein binding, and the binding of multiple subunits such as Rictor and mLST8 is mediated by this region. Mutations in amino acid residues R81 or T86 in this region have been associated with disease and mTORC2 dysfunction. On the one hand, the flexibility of Sin1-N-terminus makes it unresolved in the cryo-EM structure, and on the other hand, it also implies the variability of its configuration, making it possible to serve as the interface for Sin1 to bind other proteins (such as Figure 4 ).

[0123] (1) In vitro co-expression and co-immunoprecipitation of HEK293 cell line

[0124] HEK293 cells were transfected with pcDNA3-NF-M342, pLVX-CS-Sin1 alone, or both, and 1x10 was collected 18 hours later 6 Cells, cell lysates were co-incubated with 10 μl of Flag-conjugated agar...

[0130] Example 3. Detection of M342 inhibition of mTORC2 activity

[0131] In order to test the effect of M342 on mTORC2 function, HEK293 cells were transformed into Flag-M342 expression vector pcDNA3-NF-M342 (recombinant expression vector of SEQ ID NO. 4), and at different time points, 1×10^6 cells were collected to extract whole cells The lysate was detected by western blot immunoprecipitation method to detect the expression of Flag-M342, the expression of endogenous Sin1 and the phosphorylation level of S473 at the activation site of Akt, a specific downstream kinase of mTORC2. The experimental results are shown in Image 6 , GAPDH is the internal reference.

[0132] HEK293 cells were transfected with pcDNA3-NF-M342, pLVX-CS-Sin1N or pLVX-CS-Sin1NR81T. Whole cell lysates were collected and detected by western blotting for M342 expression, Sin1-N and Akt S473 phosphorylation levels. The experimental results are shown in Figure 7 , GAPDH is the internal reference.

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