WO2023169125A1 - Inhibitor of mtorc2 - Google Patents

Abstract

Disclosed in the present application is an inhibitor of mTORC2. According to the present application, an inhibitor of mTORC2 is designed and developed on the basis of a protein region Sin1-N structure of an mTORC2 specific subunit Sin1, and the inhibitor of mTORC2 specifically inhibits activation of mTORC2 and specifically inhibits phosphorylation of a downstream kinase Akt activation site Ser473. Compared with existing small molecule inhibitors oriented to inhibit an mTOR active center, the present allosteric inhibitor has a stronger inhibitory effect and fewer side effects.

Description

mTORC2 inhibitors

Related application cross-references

This patent application claims priority to the Chinese patent application submitted on March 11, 2022, with application number 2022102420045 and the invention name "mTORC2 inhibitor". The full text of the above application is incorporated herein by reference.

Technical field

The present invention relates to the field of molecular biology, and in particular to mTORC2 inhibitors.

Background technique

The mTOR pathway is one of the key pathways for the body to sense external signals and regulate cell metabolism. It is involved in processes such as cell growth, proliferation, survival, and death. mTOR mainly works in the form of two complexes, mTORC1 and mTORC2. The mTORC1 complex is composed of mTOR, Raptor and mLST8 subunits. The main function of mTORC1 is to respond to external environmental signals such as oxygen or energy changes, and regulate protein synthesis, energy metabolism and autophagy through phosphorylation of downstream kinases such as S6K and 4E-BP1. . In addition to the core subunits mTOR and mLST8 shared with mTORC1, the newly discovered mTORC2 complex in recent years also contains specific subunits Rictor and Sin1. mTORC2 can sense signals such as insulin and growth factors, and plays an important role in metabolism and ion transport by regulating the phosphorylation of AKT, SGK and PKC respectively. Disturbance of the mTOR signaling pathway can cause a series of diseases including cancer, neuropathy, autoimmune diseases, etc., including acute leukemia, malignant glioma, breast cancer and other tumors. The mTOR signaling pathway is abnormally activated in patients with various tumors. Therefore, the mTOR pathway has always been one of the popular targets for tumor treatment.

Currently developed mTOR inhibitors are divided into two categories: one is a specific inhibitor of mTORC1 such as Sirolimus/Rapamycin, and the other is a pan-inhibitor that inhibits both mTORC1 and mTORC2 such as Omipalisib/KU-0063794. Through research, the inventor found that mTORC1-specific inhibitors not only inhibited mTORC1 activity but also inhibited the negative feedback mechanism of the mTORC1 pathway, which would instead lead to abnormal proliferation of tumor cells and the development of drug resistance; at the same time, because the mTOR signaling pathway plays an important role in cell physiological processes The key role of mTORC1 and mTORC2 pan-inhibitors often also produces major side effects, such as hyperlipidemia and bone marrow suppression. These shortcomings greatly affect the clinical application of mTOR inhibitors. There are no inhibitors specifically targeting mTORC2 in the prior art, although there have been reports of The RNAi nanodelivery system of mTORC2 subunit Rictor can inhibit the Akt kinase activity downstream of mTORC2, but the latest research shows that Rictor is also a component of the non-traditional mTOR complex. The metabolism of brown adipocytes mediated by SIRT6 does not rely on the conventional mTORC2 pathway, which may be Causing unexpected side effects. Therefore, there is still a need in this field to find an mTORC2 inhibitor with lower side effects based on the conventional mTORC2 pathway.

Contents of the invention

The object of the present invention is to provide an isolated polypeptide useful as an mTORC2 inhibitor.

Another object of the present invention is to provide polynucleotides encoding the above polypeptides.

Another object of the present invention is to provide a pharmaceutical composition containing the above polypeptide.

Another object of the present invention is to provide a method for preparing the above-mentioned polypeptide.

In order to solve the above technical problems, the first aspect of the present invention provides an isolated polypeptide, which includes an amino acid fragment that can specifically bind to the subunit Sin1 of mTORC2.

In some preferred embodiments, the polypeptide includes an amino acid fragment that specifically binds to the mTORC2 subunit Sin1-N.

In some preferred embodiments, the polypeptide includes a first peptide segment, which is identical to a partial fragment of the amino acid sequence shown in SEQ ID NO. 1, or has a homology greater than 50% (preferably greater than 60%, 70%, 80% or 90%).

In some preferred embodiments, the polypeptide includes a first peptide segment, which is identical to a partial fragment of the amino acid sequence shown in SEQ ID NO. 3, or has a homology greater than 50% (preferably greater than 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9%).

In some preferred embodiments, the amino acid sequence of at least a partial fragment of the polypeptide is selected from any of the following:

(i) The amino acid sequence shown in SEQ ID NO.1;

(ii) An amino acid sequence that has greater than 50% homology with the amino acid sequence shown in SEQ ID NO. 1, more preferably greater than 60%, more preferably greater than 70%, such as 90%;

(iii) The amino acid sequence shown in SEQ ID NO.2;

(iv) An amino acid sequence that has greater than 50% homology with the amino acid sequence shown in SEQ ID NO. 2, more preferably greater than 60%, more preferably greater than 70%, such as 90%;

(v) The amino acid sequence shown in SEQ ID NO.3;

(vi) An amino acid sequence that has greater than 50% homology with the amino acid sequence shown in SEQ ID NO. 3, more preferably greater than 60%, more preferably greater than 70%, such as 90%.

In some preferred embodiments, the polypeptide is selected from any of the following:

(i) The amino acid sequence shown in SEQ ID NO.1;

(ii) An amino acid sequence that has greater than 50% homology with the amino acid sequence shown in SEQ ID NO. 1, more preferably greater than 60%, more preferably greater than 70%, such as 90%;

(iii) The amino acid sequence shown in SEQ ID NO.2;

(iv) An amino acid sequence that has greater than 50% homology with the amino acid sequence shown in SEQ ID NO. 2, more preferably greater than 60%, more preferably greater than 70%, such as 90%;

(v) The amino acid sequence shown in SEQ ID NO.3;

(vi) An amino acid sequence that has greater than 50% homology with the amino acid sequence shown in SEQ ID NO. 3, more preferably greater than 60%, more preferably greater than 70%, such as 90%.

A second aspect of the present invention provides an isolated polynucleotide encoding a polypeptide having an amino acid sequence as shown in SEQ ID NO. 1.

In some preferred embodiments, the polynucleotide is selected from any of the following:

(a) A polynucleotide having the sequence shown in SEQ ID NO.4;

(b) A polynucleotide having greater than 90% homology with the sequence shown in SEQ ID NO.4;

(c) A polynucleotide having reverse complementarity to the polynucleotide sequence described in (a) or (b).

A third aspect of the present invention provides a vector, characterized in that the vector includes the polynucleotide according to the second aspect of the present invention.

A fourth aspect of the present invention provides a host cell, which includes the vector according to the third aspect of the present invention.

A fifth aspect of the present invention provides a pharmaceutical composition, which includes the polypeptide described in the first aspect of the present invention and a pharmaceutically acceptable excipient.

The sixth aspect of the present invention provides the use of the polypeptide described in the first aspect of the present invention or the pharmaceutical composition described in the fifth aspect of the present invention as an mTORC2 inhibitor.

The seventh aspect of the present invention provides the polypeptide described in the first aspect of the present invention, or the polypeptide of the present invention The pharmaceutical composition according to the fifth aspect is prepared for the treatment of cancer, neuropathy, and autoimmune diseases.

The above amino acid sequence is shown in the table below;

The above nucleic acid sequence is shown in the table below;

Compared with the prior art, the present invention has at least the following advantages:

(1) The present invention designs and develops an mTORC2 inhibitor based on the structure of the protein region Sin1-N of the mTORC2 specific subunit Sin1, which specifically inhibits mTORC2 activation and specifically inhibits the phosphorylation of the downstream kinase Akt activation site Ser473;

(2) The polypeptide designed in the present invention targets the specific subunit Sin1 of mTORC2 and has good specificity. Therefore, compared with the existing small molecule inhibitors oriented to inhibit the active center of mTOR, this allosteric inhibitor has a strong inhibitory effect. , and the side effects are small.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the following (truthfully The technical features specifically described in the embodiments can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described one by one here.

Description of the drawings

One or more embodiments are exemplified by the pictures in the corresponding drawings, and these exemplary illustrations do not constitute limitations to the embodiments.

Figure 1 is a three-level structural diagram of M342 flexible N-terminal according to an embodiment of the present invention;

Figure 2 is a C-terminal tertiary structure diagram of M342 helix according to an embodiment of the present invention;

Figure 3 is a diagram showing the co-immunoprecipitation and western blotting detection results of M342 and Sin1 binding protein according to the embodiment of the present invention;

Figure 4 is a diagram of the Sin1 N-terminal 3D structure prediction (PEP-FOLD 3.2) according to an embodiment of the present invention;

Figure 5 is a diagram showing the co-immunoprecipitation and western blotting detection results of M342 and Sin1-N binding protein according to the embodiment of the present invention;

Figure 6 is a schematic diagram of the phosphorylation levels of Flag-M342 expression, endogenous Sin1 expression and mTORC2-specific downstream kinase Akt activation site S473 at different time points according to the embodiment of the present invention;

Figure 7 is a schematic diagram of the phosphorylation level of Akt S473 in HEK293 cells transfected with pcDNA3-NF-M342, pLVX-CS-Sin1N or pLVX-CS-Sin1NR81T according to the embodiment of the present invention;

Figure 8 is a quantitative statistical diagram of the phosphorylation level of mTORC2-specific downstream kinase Akt activation site S473 in an embodiment of the present invention.

Detailed ways

In the existing technology, inhibitors targeting the mTOR pathway mainly target mTORC1 or both mTORC1 and mTORC2 complexes. They have strong toxic and side effects, and there are few studies on drugs targeting mTORC2. Some researchers have inhibited the Akt kinase activity downstream of mTORC2 by targeting the mTORC2 subunit Rictor. However, because the mTORC2 subunit Rictor may also be related to the mTOR complex, it has strong side effects and poor specificity. Therefore, the inventors focused on the structure of mTORC2-specific subunit Sin1 to obtain an mTORC2 inhibitor (short peptide M342) with strong specificity and low side effects. They further identified the short peptide M342 sequence that interacts with Sin1 through mass spectrometry. In vitro expression and co-immunoprecipitation confirmed the interaction between M342 and Sin1, and determined the interaction between Sin1 and M342. The combined protein region Sin1-N was confirmed in the HEK293 cell line that the inhibitory effect of M342 on mTORC2 is through the phosphorylation of Ser473, the activation site of the specific downstream kinase Akt.

At the same time, the inventor further optimized the short peptide sequence based on the 3D structure of M342, and obtained the isolated polypeptide provided by the first aspect of the present invention, which includes an amino acid fragment that can specifically bind to the mTORC2 subunit Sin1.

In some preferred embodiments, the polypeptide includes an amino acid fragment that specifically binds to the mTORC2 subunit Sin1-N.

In some preferred embodiments, the polypeptide includes a first peptide segment that is identical to a partial fragment of the amino acid sequence shown in SEQ ID NO. 1, or has a homology greater than 90%.

In some preferred embodiments, the polypeptide includes a first peptide segment that is identical to a partial fragment of the amino acid sequence shown in SEQ ID NO. 2, or has a homology greater than 90%.

In some more preferred solutions, the first peptide segment is identical to a partial fragment of the amino acid sequence shown in SEQ ID NO. 3, or has a homology greater than 90%.

In some preferred embodiments, the amino acid sequence of at least a partial fragment of the polypeptide is selected from any of the following:

(i) The amino acid sequence shown in SEQ ID NO.1;

(ii) An amino acid sequence that has greater than 50% homology with the amino acid sequence shown in SEQ ID NO. 1, more preferably greater than 60%, more preferably greater than 70%, such as 90%;

(iii) The amino acid sequence shown in SEQ ID NO.2;

(iv) An amino acid sequence that has greater than 50% homology with the amino acid sequence shown in SEQ ID NO. 2, more preferably greater than 60%, more preferably greater than 70%, such as 90%;

(v) The amino acid sequence shown in SEQ ID NO.3;

(vi) An amino acid sequence that has greater than 50% homology with the amino acid sequence shown in SEQ ID NO. 3, more preferably greater than 60%, more preferably greater than 70%, such as 90%.

In some preferred embodiments, the polypeptide is selected from any of the following:

(i) The amino acid sequence shown in SEQ ID NO.1;

(ii) An amino acid sequence that has greater than 50% homology with the amino acid sequence shown in SEQ ID NO. 1, more preferably greater than 60%, more preferably greater than 70%, such as 90%;

(iii) The amino acid sequence shown in SEQ ID NO.2;

(iv) An amino acid sequence that has greater than 50% homology with the amino acid sequence shown in SEQ ID NO. 2, more preferably greater than 60%, more preferably greater than 70%, such as 90%;

(v) The amino acid sequence shown in SEQ ID NO.3;

(vi) An amino acid sequence that has greater than 50% homology with the amino acid sequence shown in SEQ ID NO. 3, more preferably greater than 60%, more preferably greater than 70%, such as 90%.

Other embodiments of the invention provide an isolated polynucleotide encoding a polypeptide having the amino acid sequence shown in SEQ ID NO. 1.

In some preferred embodiments, the polynucleotide is selected from any of the following:

(a) A polynucleotide having the sequence shown in SEQ ID NO.4;

(b) A polynucleotide having greater than 95% homology with the sequence shown in SEQ ID NO.4;

(c) A polynucleotide having reverse complementarity to the polynucleotide sequence described in (a) or (b).

In other embodiments of the present invention, a vector is provided, characterized in that the vector includes the polynucleotide.

Other embodiments of the invention provide a host cell, which includes the vector.

In other embodiments, the present invention provides a pharmaceutical composition comprising the polypeptide and a pharmaceutically acceptable excipient.

Other embodiments of the present invention provide the use of the above-mentioned polypeptides as mTORC2 inhibitors.

Other embodiments of the present invention provide the use of the above-mentioned polypeptides in preparing drugs for treating diseases related to Akt kinase activation or Akt kinase phosphorylation.

In some preferred embodiments, the diseases related to Akt kinase activation or Akt kinase phosphorylation include: cancer, neuropathy or autoimmune diseases.

The use of any exemplary or exemplary wording (eg, "") provided with respect to certain embodiments herein is intended solely to better present the invention and does not limit the scope of the invention that is otherwise claimed. No words contained herein should be construed as indicating elements not recited in the claims as essential to the practice of the invention.

If a term in a cited document is defined or used differently than the term described in this article If there is any consistency or inconsistency, the definition of the term described in this article shall apply rather than the definition of the term in the cited document.

In this article, mTORC2 refers to the mammalian target of rapamycin protein complex 2, which includes the mammalian target of rapamycin protein mTOR and subunits Sin1, Rictor and mLST8. It is a mechanism for mammals to sense external signals and regulate cell metabolism. One of the key pathways, involved in cell growth, proliferation, survival, death and other processes. mTORC2 due to its role in activating Akt kinase activity, Akt drives promotes proliferative processes such as glucose uptake and glycolysis (Warburg effect), while also inhibiting apoptosis. By inhibiting the activation and phosphorylation of Akt kinase downstream of mTORC2, the Akt-driven proliferation process can be inhibited.

As used herein, the term "isolated" refers to a substance that has been separated from its original environment (which, in the case of a natural substance, is the natural environment). For example, polynucleotides and polypeptides in their natural state within living cells are not separated and purified. However, if the same polynucleotide or polypeptide is separated from other substances present in its natural state, it is separated and purified.

As used herein, sequence "homology," "identity," and "percent identity" refer to the percentage of identical (i.e., identical) nucleotides or amino acids between two or more polynucleotides or polypeptides . Sequence identity between two or more polynucleotides or polypeptides can be measured by the following methods. Align the nucleotide or amino acid sequences of a polynucleotide or polypeptide, score the number of positions in the aligned polynucleotide or polypeptide that contain the same nucleotide or amino acid residue, and compare this to the aligned polynucleotide or polypeptide Compare the number of positions containing different nucleotide or amino acid residues. A polynucleotide may differ at a position, for example, by the inclusion of a different nucleotide (i.e., a substitution or variation) or a deletion of a nucleotide (i.e., an insertion or deletion of one or two nucleotides in the polynucleotide). ). Polypeptides may differ at one position, for example, by the inclusion of an amino acid (ie, a substitution or mutation) or the deletion of an amino acid (ie, an amino acid inserted into one or both polypeptides or a deletion of an amino acid). Sequence identity can be calculated by dividing the number of positions containing identical nucleotides or amino acid residues by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing identical nucleotides or amino acid residues by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide, and then multiplying by 100.

As used herein, the term "polypeptide" is a protein that occurs naturally or is produced or altered by chemical or other means by recombination and is essentially conceived to function in the same manner as the native protein. Three-dimensional structure of post-translationally processed proteins.

The present invention also includes derivatives of "polypeptides", which are peptides that have substantially the same function or biological activity as the "polypeptide", for example, amino acid sequences that are more than 90% homologous to the polypeptide. composed of peptides. These peptides can be obtained from the sequence as shown by 1-40, preferably 1-30, preferably 1-20, preferably 1-10 amino acid residue mutations (substitutions), insertions or deletions, and these mutations Insertions or deletions alter the activity of the polypeptide itself.

As used herein, the term "nucleic acid sequence" or "polynucleotide sequence" refers to a sequence of nucleotides or nucleotide monomers formed from naturally occurring combinations of bases, sugars and sugars (hubs). The term also includes defined or substituted sequences containing monomers or portions thereof that do not occur in nature. The nucleic acid sequence of the present invention may be a deoxyribonucleic acid sequence (DNA) or a ribonucleic acid sequence (RNA), and may contain the natural bases of adenine, guanine, cytosine and uracil.

Nucleic acids can be isolated using techniques well known in the art. For example, nucleic acids may be isolated using any method, including, but not limited to, recombinant nucleic acid technology and/or polymerase chain reaction (PCR). Isolated nucleic acids can also be chemically synthesized, either as a single nucleic acid molecule or as a series of oligonucleotides.

As used herein, the term "Reverse Complementary Sequence" refers to a sequence that is in the opposite direction of the original polynucleotide sequence and is complementary to the original polynucleotide sequence. For example, if the original polynucleotide sequence is ACTGAAC, its reverse complement is GTTCAT.

As used herein, the term "vector" refers to a delivery vehicle for a polynucleotide. In some embodiments, in genetic engineering recombinant technology, the vector includes a polynucleotide sequence encoding a specific protein that is operably inserted to achieve expression of the protein. Vectors are used to transform, transduce or transfect host cells, and the genetic material elements delivered by the vector can be expressed in the host cells. A "vector" disclosed in this question may be any suitable vector, including chromosomal, non-chromosomal and synthetic nucleic acid vectors (nucleic acid sequences including a series of appropriate expression control elements). For example, the vector may be a recombinant plasmid vector, a recombinant eukaryotic virus vector, a recombinant bacteriophage vector, a recombinant yeast mini-chromosome vector, a recombinant bacterial artificial chromosome vector or a recombinant yeast plasmid vector.

As used herein, the term "host cell" is a eukaryotic host cell or a prokaryotic host cell. Among them, the eukaryotic host cell can be a mammalian host cell, an insect host cell, a plant host cell, a fungal host cell, a eukaryotic algal host cell, a nematode host cell, or a protozoan host. cells and fish host cells. Illustratively, the host cell in the present disclosure is a eukaryotic host cell, and the eukaryotic host cell is a mammalian host cell. Among them, the mammalian host cells are Chinese hamster ovary cells (CHO cells), COS cells, Vero cells, SP2/0 cells, NS/O myeloid cells, human fetal kidney cells, immature hamster kidney cells, HeLa cells, human B cells, cv-1/EBNA cells, L cells, 3T3 cells, HEPG2 cells, PerC6 cells.

Protein (polypeptide) expression system

The "protein/polypeptide expression system" disclosed in the present invention includes vectors containing host and foreign genes, and is a system capable of achieving the purpose of expressing foreign genes in the host. Protein expression systems generally include the following factors: (1) Expression host, that is, a living organism that can select proteins from bacteria, yeast, plant cells, animal cells, etc.; (2) Vectors corresponding to the host. Depending on the host, the vector It can be divided into prokaryotic (bacterial) expression vectors, yeast expression vectors, plant expression vectors, mammalian expression vectors and insect expression vectors, etc. The vector contains fragments of the foreign gene. Foreign genes can be expressed in the host through vectors. In some embodiments, the expressed protein product is secreted. In some embodiments, the vector is embedded in the DNA of the host cell.

An important step in protein expression is to select recombinant host cells that are successfully transfected with a vector containing a foreign gene encoding the protein of interest. Most commonly, selection markers are included in vectors. The selectable marker may be a gene or DNA sequence capable of distinguishing recombinant host cells that contain the marker from recombinant host cells that do not contain the marker. By combining a selection marker and a selection medium, while the proliferation of recombinant host cells transfected with the vector is made possible, the proliferation of host cells that cannot be successfully transfected is also hindered.

Proteins can be purified from natural sources (eg biological samples) by known methods such as DEAE ion exchange, gel filtration and hydroxyapatite chromatography. The protein can also be purified, for example, by expressing the nucleic acid in an expression vector. In addition, purified polypeptides can be obtained through chemical synthesis. The degree of purity of a polypeptide can be determined using any suitable method, such as column chromatography, polyacrylamide gel electrophoresis or HPLC analysis.

Antibodies can be used to detect proteins. Techniques for detecting proteins using antibodies include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, and immunofluorescence.

As used herein, a "pharmaceutical composition" contains an effective amount of a polypeptide as described herein, optionally combined with a pharmaceutically acceptable carrier, additive or excipient. Compounds according to the present disclosure The substance may be administered in immediate release, intermediate release, or sustained or controlled release form. Sustained or controlled release forms are preferably administered orally, but suppositories and transdermal or other topical forms may also be administered. Intramuscular injection in liposome form may also be used to control or maintain the release of the compound at the site of injection.

As used herein, the term "pharmaceutically acceptable" refers to molecular entities and compositions that do not generally produce allergic or other serious adverse reactions when administered using routes well known in the art. Approved by the State Food and Drug Administration of the People's Republic of China, approved by a federal or state government regulatory agency, or listed in the United States Pharmacopeia, Chinese Pharmacopoeia, or other recognized pharmacopeia for use in animals, and more particularly in humans. Molecular entities and compositions are considered "pharmaceutically acceptable".

As used herein, the term "pharmaceutically acceptable excipient" refers to an excipient whose administration can be tolerated by the patient to whom it is administered. Excipients that can be used include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersion or suspension aids, solubilizers, colorants, flavoring agents, coatings, disintegrants, lubricants, Sweeteners, preservatives, isotonic agents and combinations thereof. The selection and use of suitable excipients is taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003), and Gennaro, ed., Remington's Pharmaceutical Sciences (Mack Publishing) Company,19th ed.1995). The formulation may further include one or more carriers, diluents, preservatives, solubilizers, buffers, albumin to prevent protein loss from the vial surface, and the like.

As used herein, the term "neuropathy" refers to neurological dysfunction resulting from damage to nerve cells, including central neuropathy and peripheral neuropathy.

In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the invention and are not intended to limit the scope of the invention. Experimental methods without specifying specific conditions in the following examples usually follow conventional conditions or conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight. The experimental materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Unless otherwise specified, technical and scientific terms used herein have the same meanings as commonly understood by those of ordinary skill in the technical field to which this application belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the present invention. Exemplary embodiments of the application.

Example 1. Identification and structural characterization of M342

In this example, the inventors used protein affinity purification and mass spectrometry to unexpectedly discover that a longer peptide containing the M342 sequence may interact with Sin1.

(1) Protein purification and mass spectrometry identification

The peptide GST-M342-619 or GST-empty vector was expressed in HEK293T cells. After purification using GSH-Sepharose beads, the protein strips specifically pulled down by GST-M342-619 were found through SDS-PAGE electrophoresis and silver staining. These bands were cut out and analyzed by mass spectrometry (HPLC/MS/MS), and four Sin1 amino acid sequences including (LLPMTVVTMASAR) were identified, suggesting that Sin1 may be its binding protein.

(2) Pull down experiment

COS-1 cells were used to co-express GST-Sin1 and HA-tagged peptides of different lengths containing the M342 sequence. Whole cell lysis was performed 40 hours later (50mM HEPES, pH 7.6, 150mM NaCl, 1.5mM MgCl2, 1mM EDTA, 1% Triton ( Anti-HA antibodies were detected. The sequence of M342 is as SEQ ID NO.1.

(2) Sequence prediction

Using different amino acid sequence prediction software to analyze the high-level structure of M342, consistent results were obtained. Its amino acid sequence and PEP-FOLD 3.2 predicted tertiary structure are shown in Figures 1 and 2. Among them, M342 contains a flexible N-terminus (Figure 1 ) and the C-terminus of the helix (Figure 2).

The inventor designed truncated peptides M342-a and M342-b based on M342, whose sequences are shown in SEQ ID NO.2 and SEQ ID NO.3 respectively:

Example 2. Identification of the precise binding region between M342 and Sin1

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

(1) Co-expression and co-immunoprecipitation in HEK293 cell line in vitro

HEK293 cells were transfected with pcDNA3-NF-M342, pLVX-CS-Sin1 alone or both co-transfected. ^1x10 cells were collected after 18 hours, and the cell lysate was mixed with 10 μl Flag-coupled agarose gel beads at 4 °C for two hours, and after three washes, the eluted proteins were analyzed by SDS-PAGE electrophoresis, and the bound proteins were detected by western blotting with anti-strep or anti-Flag antibodies. The results are shown in Figure 3.

Figure 3A is a schematic diagram of the co-immunoprecipitation of M342 and Sin1, and Figure 3B is the result of detecting the bound protein using anti-strep or anti-Flag antibodies.

As shown in Figure 3, M342 interacts with full-length Sin1.

(2) The binding region between M342 and Sin1 is located at Sin1-N

The inventor constructed the Sin-N fragment and its mutant Sin1N-R81T, and used similar experimental steps (co-immunoprecipitation + binding protein detection) in (1) to prove that the binding region of M342 to Sin1 is located in Sin1-N, but M342 does not bind to Sin1-N. Mutated Sin1N-R81T binds (Figure 5).

Figure 5B is a schematic diagram of the co-immunoprecipitation of M342 and Sin1-N; Figure 5C is the result of detecting bound proteins using anti-strep or anti-Flag antibodies.

Example 3. Detection of M342 inhibiting mTORC2 activity

In order to test the effect of M342 on mTORC2 function, HEK293 cells were transferred into Flag-M342 expression vector pcDNA3-NF-M342 (SEQ ID NO.4 recombinant expression vector), and at different time points, 1x10^6 cells were collected to extract whole cells The lysate was used to detect Flag-M342 expression, endogenous Sin1 expression, and the phosphorylation level of mTORC2-specific downstream kinase Akt activation site S473 by using western Blot immunoprecipitation method. The experimental results are shown in Figure 6, and GAPDH was used as the internal control.

HEK293 cells were transfected with pcDNA3-NF-M342, pLVX-CS-Sin1N or pLVX-CS-Sin1NR81T. Whole cell lysates were collected and Western Blot was used to detect M342 expression, Sin1-N and Akt S473 phosphorylation levels. The experimental results are shown in Figure 7, and GAPDH was used as the internal control.

The results were quantified using GAPDH protein expression as the internal reference (Figure 8).

From Figure 6 to Figure 8, it can be seen that as the expression of M342 increases, the phosphorylation of Akt S473 shows a downward trend, and the level of endogenous Sin1 also decreases. This is because the free Sin1 located outside the mTORC2 complex will be quickly degraded, proving that M342 can inhibit the activity of mTORC2.

In addition, the inventors also tested M342-a and M342-b to inhibit mTORC2 activity.

Those of ordinary skill in the art can understand that the above-mentioned embodiments are specific examples for realizing the present invention, and in practical applications, various changes can be made in form and details without departing from the spirit and spirit of the present invention. scope.

Claims (10)

1. An isolated polypeptide, characterized in that the polypeptide includes an amino acid fragment that can specifically bind to the subunit Sin1 of mTORC2.
2. The polypeptide of claim 1, wherein the polypeptide includes an amino acid fragment that can specifically bind to the mTORC2 subunit Sin1-N.
3. The polypeptide according to claim 1, wherein the amino acid sequence of at least part of the fragment of the polypeptide is selected from any of the following:

   (i) The amino acid sequence shown in SEQ ID NO.1;

   (ii) An amino acid sequence that has greater than 50% homology with the amino acid sequence shown in SEQ ID NO.1;

   (iii) The amino acid sequence shown in SEQ ID NO.2;

   (iv) An amino acid sequence that has greater than 50% homology with the amino acid sequence shown in SEQ ID NO.2;

   (v) The amino acid sequence shown in SEQ ID NO.3;

   (vi) An amino acid sequence that has greater than 50% homology with the amino acid sequence shown in SEQ ID NO.3.
4. The polypeptide according to claim 1, characterized in that the polypeptide is selected from any of the following:

   (i) The amino acid sequence shown in SEQ ID NO.1;

   (ii) An amino acid sequence that has greater than 50% homology with the amino acid sequence shown in SEQ ID NO.1;

   (iii) The amino acid sequence shown in SEQ ID NO.2;

   (iv) An amino acid sequence that has greater than 50% homology with the amino acid sequence shown in SEQ ID NO.2;

   (v) The amino acid sequence shown in SEQ ID NO.3;

   (vi) An amino acid sequence that has greater than 50% homology with the amino acid sequence shown in SEQ ID NO.3.
5. An isolated polynucleotide, characterized in that the polynucleotide is used to encode a polypeptide having the amino acid sequence shown in SEQ ID NO.1.
6. The polynucleotide of claim 5, wherein the polynucleotide is selected from any of the following:

   (a) A polynucleotide having the sequence shown in SEQ ID NO.4;

   (b) A polynucleotide having greater than 90% homology with the sequence shown in SEQ ID NO.4;

   (c) A polynucleotide having reverse complementarity to the polynucleotide sequence described in (a) or (b).
7. A vector, characterized in that the vector includes the polynucleotide of claim 6.
8. A host cell, characterized in that the host cell includes the vector of claim 7.
9. A pharmaceutical composition, characterized in that the pharmaceutical composition includes the polypeptide and a pharmaceutically acceptable excipient.
10. Use of the polypeptide according to any one of claims 1 to 3 as an mTORC2 inhibitor.