HK40038482B - Method for adjusting ras ubiquitination - Google Patents

Description

Methods to regulate Ras ubiquitination

Technical Field

This invention belongs to the field of biotechnology and relates to a method for regulating Ras ubiquitination via the E3 ubiquitin ligase Nedd4-1.

Background Technology

Ras protein acts as a molecular switch within the cell, switching between an inactive state bound to GDP (guanosine diphosphate) and an active state bound to GTP (guanosine triphosphate). It plays a crucial role in numerous physiological and biochemical regulatory processes within the cell, including the regulation of gene expression, cell growth and differentiation, cell cycle, cytoskeleton and cell migration, vesicle transport, and transport between the nucleus and cytoplasm. GTP binding causes multiple residues of the Ras protein (primarily residues 30-40 in the sWitch I region and residues 60-70 in the sWitch II region) to form a conformation that allows Ras effector proteins to bind. The switching between the active and inactive states of Ras protein is regulated by GAPs (GTPase-activating proteins) and GEFs (guanosine monophosphate exchange factors). Mutations in the Ras gene can impair the inherent GAPase activity of the Ras protein or prevent GAP binding, leading to the sustained activation of downstream signaling pathways and promoting tumorigenesis and development. Meanwhile, Ras proteins (including H-Ras, K-Ras, and N-Ras) are also the most common proto-oncoproteins in humans, closely related to tumor growth and transformation activity. Mutations in the Ras gene have been found in 20%-30% of all human tumors, including point mutations in the Ras gene in 90% of pancreatic cancers, 50% of colon cancers, 30% of lung cancers, 50% of thyroid cancers, and 30% of myeloid leukemias. Studies have shown that various tumors depend on the persistent expression of Ras proteins at the cellular level and in animal models. Therefore, research on Ras family proteins has received considerable attention from researchers. Nedd4-1 is an E3 ubiquitin ligase belonging to the HECT family, structurally comprising an N-terminal C2 domain, a middle WW domain, and a C-terminal HECT domain, with the amino acid sequence shown in the NCBI protein database Accession: NP_006145. This factor can ubiquitinate and degrade Ras proteins.

Ubiquitination of protein molecules is an important covalent modification after protein translation in cells, regulating many vital biological functions, including protein degradation, DNA repair, gene transcription, cell cycle, and endocytosis. Ubiquitination-mediated protein degradation plays a crucial role in maintaining normal cellular biological functions; abnormal protein degradation can lead to many diseases, including cancer. Recent studies have shown that inhibiting the activity of proteasome complexes has therapeutic effects on some diseases, such as cancer, autoimmune diseases, inflammation, and cardiovascular diseases. Currently, proteasome inhibitors such as Bortezomib have been approved by the FDA for clinical oncology treatment, including multiple myeloma and other malignant tumors. However, because proteasome complex inhibitors nonspecifically affect the degradation of almost all proteins, they inevitably cause strong toxic side effects, greatly limiting their application.

Summary of the Invention

In one aspect, the present invention relates to a method for identifying compounds that regulate Ras levels, the method comprising:

a. Contact the test compound with cells expressing Ras or in vitro ubiquitination systems;

b. Select compounds that can increase or decrease the Ras level in cells or systems from step a.

Optionally, the method further includes:

c. Determine whether the compounds obtained in step b regulate Ras levels in a manner dependent on the presence of Nedd4-1 protein;

d. Select compounds that regulate Ras levels in dependence on the presence of Nedd4-1 protein.

According to the aforementioned method, the cells expressing Ras are cells expressing exogenous Ras or endogenous Ras or both.

According to any of the preceding methods, in step b, a compound that can reduce the Ras level in the cells or system in step a is selected.

In another aspect, the present invention relates to compounds obtained by screening using any of the foregoing methods.

According to the aforementioned compounds, the compounds are able to promote Ras ubiquitination degradation in a Nedd4-1 protein-dependent manner (i.e., the compounds can promote Ras protein degradation in the presence of Nedd4-1 protein, while the ability of the compounds to promote Ras protein degradation is significantly weakened when Nedd4-1 protein is not present).

According to the aforementioned compounds, the compounds are selected from:

Preferred options are:

In one aspect, the present invention relates to a method for regulating intracellular Ras levels, the method comprising:

a. Contact the compound that regulates Ras ubiquitination degradation with cells expressing Ras or in vitro ubiquitination systems.

b. Optionally, it also regulates the level of Nedd4-1 in cells or systems.

According to the aforementioned method, the regulation of Nedd4-1 levels includes: a) gene editing, b) overexpression, and c) RNA interference.

According to any of the preceding methods, the Ras is H-Ras, K-Ras, or N-Ras and their various mutants.

According to any of the preceding methods, the compound that regulates Ras ubiquitination degradation is a compound according to any of the preceding methods.

According to any of the preceding methods, the activity of the compound in regulating Ras ubiquitination degradation is Nedd4-1 dependent or independent.

In one aspect, the present invention relates to a method for regulating Ras levels, the method comprising:

a. Determine the state of Ras protein;

b. If the Ras protein is wild-type or inactive, the level of Ras is regulated by regulating the expression level of Nedd4-1;

c. If the Ras protein is a mutant protein or in an active state, the level of Ras can be regulated by adding compounds.

d. Optionally, the adjustment of Nedd4-1 expression level and the addition of the compound can be performed simultaneously.

In one aspect, the present invention relates to a method for regulating Ras levels, the method comprising:

a. Determine whether the Ras level can be adjusted by Nedd4-1;

b. If Ras levels can be modulated by Nedd4-1, then Ras levels are modulated by adjusting the expression levels of Nedd4-1.

c. If the Ras level cannot be regulated by Nedd4-1, the Ras level can be regulated by adding compounds.

d. Optionally, the adjustment of Nedd4-1 expression level and the addition of the compound can be performed simultaneously.

According to any of the preceding methods, the Ras level is reduced by increasing the level of Nedd4-1, or the Ras level is increased by decreasing the level of Nedd4-1.

According to any of the preceding methods, wherein the level of Ras protein is reduced by adding a compound, preferably reducing the level of Ras protein without affecting the level of Ras mRNA.

In one aspect, the present invention relates to a method for identifying compounds for the prevention or treatment of cancer or tumors, the method comprising:

a. Contacting a tumor cell line with the compound of the present invention in vitro, optionally, wherein the cancer or tumor has a Ras-positive genetic background.

b. Select compounds that can inhibit or reduce the ability of the cells to form colonies as candidate drugs.

In one embodiment of the present invention, if the drug to be tested can inhibit or reduce the ability of cancer cells to form colonies, it can be used as a candidate drug.

According to any of the preceding methods, the method is performed in vitro.

The method according to any of the foregoing methods is performed in vivo.

In one aspect, the present invention relates to a method for preventing or treating cancer or tumors, the method comprising administering a compound that promotes Ras ubiquitination degradation to a subject suffering from cancer or tumors or a subject at high risk of cancer or tumors, optionally screening and identifying compounds that promote Ras ubiquitination degradation using any of the foregoing methods of the present invention. Optionally, the cancer or tumor has a Ras-positive genetic background.

According to any of the preceding methods, the Ras is H-Ras, K-Ras, or N-Ras and its various active mutants, including but not limited to G12V, G12D, G13V, G13D, and Q61R. Preferably, the mutants are G12V, G13D, and Q61R of H-Ras; G12V, G12D, G13V, G13D, and Q61R of K-Ras; and G12V, G13V, G13D, and Q61R of N-Ras. Most preferably, the mutant is G12V of K-Ras.

In one aspect, the present invention relates to the use of compounds that regulate the expression level of Nedd4-1 in the preparation of medicaments for the prevention or treatment of cancer or tumors. The compounds that regulate the expression level of Nedd4-1 include, but are not limited to, nucleic acid constructs, small molecule compounds, antibodies, etc.

In one aspect, the present invention relates to the use of compounds that promote the ubiquitination and degradation of Ras proteins in the preparation of medicaments for the prevention or treatment of cancer or tumors, preferably said compounds that promote the ubiquitination and degradation of Ras proteins are Nedd4-1 dependent, and optionally, said compounds are compounds according to any of the foregoing.

In one aspect, the present invention relates to the use of compounds that promote the ubiquitination and degradation of Ras proteins in the preparation of medicaments for the prevention or treatment of Ras-related autoimmune diseases, inflammation, and cardiovascular diseases.

Attached Figure Description

Figure 1 shows the regulation of Ras protein levels by Nedd4-1. Figure 1(a) Interaction between endogenous Nedd4-1 and Ras protein in HeLa cells. Figure 1(b) Direct in vitro interaction between Nedd4-1 and Ras protein. Figure 1(c) Overexpression of Nedd4-1 protein reduces endogenous Ras protein levels in HeLa cells; statistical results are calculated using Mean ± SD from three independent experiments. Figure 1(d) Overexpression of Nedd4-1 protein reduces exogenous Ras protein levels. Figure 1(e) Nedd4-1-mediated Ras protein degradation occurs via the lysosomal pathway. Figure 1(f) Nedd4-1-mediated ubiquitination of H-Ras and N-Ras in vivo. Figure 1(g) Direct ubiquitination of H-Ras and N-Ras by Nedd4-1 under in vitro conditions. Figure 1(h) Nedd4-1 regulates the half-life of H-Ras protein. Figure 1(i) Nedd4-1 regulates the half-life of N-Ras protein. Figure 1(j) Overexpression of Nedd4-1 in HT-29 cells reduces the protein level of endogenous WT K-Ras, while knockdown of Nedd4-1 upregulates the protein level of endogenous WT K-Ras. Figure 1(k) The K-Ras G12V mutant does not show significant degradation when overexpressing F/Nedd4-1. Figure 1(l) The K-Ras G12V mutation weakens Nedd4-1-mediated in vivo ubiquitination of K-Ras. Figure 1(m) The K-Ras G12V mutation weakens Nedd4-1-mediated in vitro ubiquitination of K-Ras.

Figure 2 shows the compounds screened to reduce exogenous K-Ras-G12V protein levels. Figure 2(a) shows 28 compounds screened using dot blot high-throughput screening. Overexpression of Flag-K-Ras-G12V and Flag-Nedd4-1-WT in 293T cells, followed by drug treatment, revealed 7 compounds that reduced exogenous K-Ras-G12V protein levels. Figure 2(b) shows the 7 compounds that reduced exogenous K-Ras-G12V in SW620 cells, with 3 compounds selected to reduce endogenous K-Ras-G12V protein levels. Figure 2(c) shows the 3 compounds after endogenous and endogenous screening; whether the reduction in K-Ras-G12V protein levels by these three compounds depended on the presence of Nedd4-1 protein.

Figure 3 shows that the screened compounds exhibit a dose-response relationship in reducing the protein level of endogenous K-Ras-G12V in cells. Figure 3(a) shows that compound ZT-10-158-01 effectively reduced the protein level of endogenous K-Ras-G12V in SW620 cells, and the inhibitory effect was positively correlated with concentration. Figure 3(b) shows that compound ZT-10-158-01 effectively reduced the protein level of exogenous K-Ras-G12V in HEK293T cells, and the inhibitory effect was positively correlated with concentration.

Figure 4 shows that the screened compounds can selectively inhibit the colony formation of tumor cells with disordered Ras signaling.

Figure 4(a) shows that compound ZT-10-158-01 can effectively inhibit the colony formation ability of MCF-7 cells on agar.

Figure 4(b) shows that compound ZT-10-158-01 can effectively inhibit the colony formation ability of SW620 cells on agar.

Figure 4(c) shows that compound ZT-10-158-01 cannot inhibit the colony-forming ability of HT-29 cells on agar.

Detailed Implementation

The following are embodiments provided to assist those skilled in the art in practicing the invention. Modifications and variations can be made to the embodiments described herein without departing from the spirit or scope of this disclosure. All disclosures, patent applications, patents, figures, and other references mentioned herein are expressly incorporated herein by reference in their entirety.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this invention is for describing particular embodiments only and is not intended to limit the invention.

It should also be understood that in some methods described herein that include more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions of the method are listed, unless the context otherwise indicates.

As used herein, the term Ras refers to H-Ras (e.g., having the amino acid sequence shown in the NCBI protein database Accession: NP_001123914), K-Ras (e.g., having the amino acid sequence shown in the NCBI protein database Accession: NP_203524), or N-Ras (e.g., having the amino acid sequence shown in the NCBI protein database Accession: EAW56611), as well as their various isoforms and their various active mutants. These mutants include, but are not limited to, G12V, G12D, G13V, G13D, and Q61R. Preferably, the mutants are G12V, G13D, and Q61R of H-Ras; G12V, G12D, G13V, G13D, and Q61R of K-Ras; and G12V, G13V, G13D, and Q61R of N-Ras. Most preferably, the mutant is G12V of K-Ras. For example, an exemplary K-Ras protein sequence could be:

An example H-Ras protein sequence could be:

An exemplary N-Ras protein sequence could be:

As used herein, the term "Nedd4-1" is an E3 ubiquitin ligase belonging to the HECT family, structurally comprising an N-terminal C2 domain, a middle WW domain, and a C-terminal HECT domain, having an amino acid sequence as shown in the NCBI protein database Accession: NP_006145, for example, the following amino acid sequence:

As used in this article, cancers or tumors with a Ras-positive genetic background refer to a type of tumor in which the Ras protein is in a state of mutation or persistent activation due to upstream signals.

In one aspect, the present invention relates to a method for identifying compounds that regulate Ras levels, the method comprising:

a. Contact the test compound with cells expressing Ras or in vitro ubiquitination systems;

b. Select compounds that can increase or decrease the Ras level in cells or systems from step a.

Optionally, the method further includes:

c. Determine whether the compounds obtained in step b regulate Ras levels in a manner dependent on the presence of Nedd4-1 protein;

d. Select compounds that regulate Ras levels in dependence on the presence of Nedd4-1 protein.

The detection method can be a conventional method for detecting molecules in the art, including but not limited to microarrays, ELISA, multiplex technology, Luminex technology, mass spectrometry, flow cytometry, RNA blotting, DNA blotting, protein blotting, PCR, and RIA.

In one aspect, the present invention relates to a method for regulating Ras levels, the method comprising:

a. Determine the state of Ras protein;

b. If the Ras protein is wild-type or inactive, the level of Ras is regulated by regulating the expression level of Nedd4-1;

c. If the Ras protein is a mutant protein or in an active state, the level of Ras can be regulated by adding compounds.

d. Optionally, the adjustment of Nedd4-1 expression level and the addition of the compound can be performed simultaneously.

According to the aforementioned method, Ras level is reduced by increasing Nedd4-1 level, or Ras level is increased by decreasing Nedd4-1 level.

According to the aforementioned method, the level of Ras protein is reduced by adding a compound without affecting the level of Ras mRNA.

According to the method of the present invention, the cells expressing Ras are cells expressing exogenous Ras, endogenous Ras, or both. Exogenous Ras refers to Ras that is not originally present in the cell but is introduced into the cell from the outside for expression through molecular biology techniques. Endogenous Ras refers to Ras that is originally present in the cell and stably inherited. The in vitro ubiquitination system is based on in vitro ubiquitination experimental methods conventionally used in the art. This system may not require intact cells; ubiquitination is carried out by adding purified ubiquitin proteins E1 ubiquitin activator, _E2 ubiquitin conjugate, E3 ubiquitin ligase, and the corresponding substrate proteins to a reaction system of 50 mM Tris-hydrochloric acid (pH 7.5), 5 mM ATP, 10 mM MgCl2, and 50 μM DTT. Those skilled in the art may also add or reduce specific components as needed.

According to the method of the present invention, in step b, a compound is selected that can reduce the Ras level of cells in step a.

According to the method of the present invention, the Ras is H-Ras, K-Ras, or N-Ras and its various mutants, including but not limited to G12V, G12D, G13V, G13D, and Q61R. Preferably, the mutants are G12V, G13D, and Q61R of H-Ras; G12V, G12D, G13V, G13D, and Q61R of K-Ras; and G12V, G13V, G13D, and Q61R of N-Ras. Most preferably, the mutant is G12V of K-Ras.

According to the method of the present invention, the Ras level can be the basal level or state of molecules in the sample, or the level or state of molecules after a portion of the sample has been contacted with the regulator. In one embodiment, the molecule is a protein or nucleic acid molecule. In another embodiment, the molecule includes protein, nucleic acid, lipid, sugar, carbohydrate, or metabolite molecules. Still in another embodiment, the molecules are analyzed using a method selected from: microarray, ELISA, multiplex technology, Luminex technology, mass spectrometry, flow cytometry, RNA blotting, DNA blotting, protein blotting, PCR, and RIA.

According to the method of the present invention, the interaction can be detected by immunoassay, surface plasmon resonance (SPR) (Richand Myszka (2000) Curr. Opin. Biotechnol 11:54; Englebienne (1998) Analyst. 123:1599), isothermal titration calorimetry (ITC) or other kinetic interaction assays known in the art (see, for example, Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989); also see U.S. Patent No. 7,229,619, which describes exemplary SPR and ITC methods for calculating antibody binding affinity). Instruments and methods for real-time detection and monitoring of binding rates are known and commercially available (see BiaCore 2000, Biacore AB, Upsala, Sweden and GE Healthcare Life Sciences; Malmqvist (2000) Biochem. Soc. Trans. 27: 335). Those skilled in the art will understand that the methods described above for detecting molecular-level interactions and methods for detecting molecular interactions are sometimes interchangeable. For example, immunoblotting methods for detecting molecular-level interactions can also be used to detect interactions between proteins, interactions between proteins and RNA or DNA molecules, or interactions between antigens and antibodies, etc.

In another aspect, the present invention relates to compounds obtained by screening using any of the foregoing methods.

According to the aforementioned compounds, the compounds are able to promote Ras ubiquitination degradation in a Nedd4-1 protein-dependent manner (i.e., the compounds can promote Ras protein degradation in the presence of Nedd4-1 protein, while the ability of the compounds to promote Ras protein degradation is significantly weakened when Nedd4-1 protein is not present).

According to the aforementioned compounds, the compounds are selected from:

Preferred options are:

This invention also includes kits using the methods of this invention. The kits contain one or more reagents for detecting Ras or Nedd4-1 levels, optionally, an expression plasmid expressing Ras or Nedd4-1, optionally, host cells expressing Ras, optionally, a compound that promotes Ras ubiquitination degradation, optionally, said compound being a compound obtained using the methods described in any of the foregoing claims of this invention. The kits may also include suitable containers and instructions for use, etc. Detection of Ras or Nedd4-1 levels can be performed at the mRNA or protein level. For detection at the mRNA or protein level, other techniques and systems practiced in the art can be used, such as, for example, RT-PCR assays using one or more designed primers; immunoassays, such as enzyme-linked immunosorbent assays (ELISA); immunoblotting, such as Western blotting or in situ hybridization and similar techniques.

As used herein, unless otherwise indicated, the term "compound" as applicable in the context means any particular chemical compound disclosed herein and includes its tautomers, positional isomers, geometric isomers, and, where applicable, stereoisomers (including optical isomers (enantiomers) and other stereoisomers (diastereomers)) as well as its pharmaceutically acceptable salts and derivatives (including prodrug forms). In its context, the term "compound" refers generally to a single compound and may include other compounds such as stereoisomers, positional isomers, and/or optical isomers (including racemic mixtures) of the disclosed compounds, as well as specific enantiomers or mixtures enriched in enantiomers. In the context, the term also refers to a prodrug form of the compound that has been modified to facilitate the administration and delivery of the compound to its active site.

On the other hand, the present invention provides a pharmaceutical composition comprising the compound, its stereoisomer, its prodrug, or its pharmaceutically acceptable salt or pharmaceutically acceptable solvate, as described in any of the above-mentioned technical solutions, and a pharmaceutically acceptable carrier, diluent, or excipient. For example, the active ingredient of the pharmaceutical composition of the present invention may also comprise a compound obtained by the method of the present invention and a molecule that regulates the expression level of Nedd4-1, wherein the molecule regulating the expression level of Nedd4-1 includes, but is not limited to, small molecule compounds, Nedd4-1 nucleic acid constructs, Nedd4-1 protein, etc.

Methods for preparing various pharmaceutical compositions containing a certain amount of active ingredient are known, or will be obvious to those skilled in the art according to the disclosure of the present invention. As described in REMINGTON’S PHARMACEUTICAL SCIENCES, Martin, E.W., ed., Mack Publishing Company, 19th ed. (1995), the method for preparing the pharmaceutical composition includes incorporating appropriate pharmaceutical excipients, carriers, diluents, etc.

The pharmaceutical formulations of the present invention can be manufactured using known methods, including conventional mixing, dissolving, or lyophilizing methods. The compounds of the present invention can be formulated into pharmaceutical compositions and administered to patients via various routes suitable for a selected manner of administration, such as oral or parenteral (via intravenous, intramuscular, local, or subcutaneous routes).

As used herein, unless otherwise stated, the term "prodrug" refers to a derivative of a compound that can be hydrolyzed, oxidized, or otherwise reacted under biological conditions (in vitro or in vivo) to provide the compounds of the present invention. Prodrugs become active compounds only after undergoing this reaction under biological conditions, or they are active in their unreacted forms. Prodrugs can generally be prepared using well-known methods, such as those described in 1 Burger's Medicinal Chemistry and Drug Discovery (1995) 172-178, 949-982 (Manfred E. Wolff, ed., 5th edition).

As used herein, examples of the term "pharmaceutically acceptable salt" are organic acid adduct salts formed from organic acids that form pharmaceutically acceptable anions, including but not limited to formate, acetate, propionate, benzoate, maleate, fumarate, succinate, tartrate, citrate, ascorbate, α-ketoglutarate, α-glycerophosphate, alkyl sulfonates, or aryl sulfonates; preferably, the alkyl sulfonate is a methanesulfonate or ethyl sulfonate; and the aryl sulfonate is a benzenesulfonate or p-toluenesulfonate. Suitable inorganic salts may also be formed, including but not limited to hydrochlorides, hydrobromides, hydroiodates, nitrates, bicarbonates and carbonates, sulfates, or phosphates.

Pharmaceutically acceptable salts can be obtained using standard procedures well known in the art, for example, by reacting an adequate amount of a basic compound with a suitable acid that provides a pharmaceutically acceptable anion.

As used herein, the term "pharmaceuticalally acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delayers compatible with drug administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, the standard bibliography in the art, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, glucose solution, and 5% human serum albumin. Liposomes and non-aqueous carriers, such as immobilized oils, may also be used. The use of such media and reagents for pharmaceutically active substances is well known in the art. The use of any conventional media or reagent in the composition is envisioned, except that it is incompatible with the antibody.

As used herein, the term “treatment” generally refers to achieving the desired pharmacological and/or physiological effect. This effect may be preventative based on the complete or partial prevention of the disease or its symptoms; and/or therapeutic based on the partial or complete stabilization or cure of the disease and/or side effects resulting from the disease. As used herein, “treatment” encompasses any treatment of a patient’s disease, including: (a) prevention of the disease or symptoms occurring in a patient who is susceptible to the disease or its symptoms but has not yet been diagnosed with the disease; (b) suppression of the symptoms of the disease, i.e., prevention of its progression; or (c) relief of the symptoms of the disease, i.e., causing the disease or its symptoms to regress.

In another aspect, the present invention provides a method for treating or improving a disease, condition or symptom of a subject or patient (e.g., an animal, such as a human), the method comprising administering to a subject in need of a composition comprising an effective amount (e.g., a therapeutically effective amount) of a compound as described herein or a salt thereof; and a pharmaceutically acceptable carrier, wherein the composition is effective in treating or improving the disease or condition or symptom of the subject.

As used herein, the term "nucleic acid construct" is defined as a single-stranded or double-stranded nucleic acid molecule, preferably an artificially constructed nucleic acid molecule. Optionally, the nucleic acid construct may further comprise one or more operatively linked regulatory sequences.

As used herein, the term "operationally linked" refers to a functional spatial arrangement of two or more nucleotide regions or nucleic acid sequences. This "operationally linked" arrangement can be achieved through genetic recombination.

As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide that inhibits a protein can be inserted. Examples of vectors include: plasmids; phage particles; Cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus and cytomegalovirus (CMV)), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression.

As used herein, the term “host cell” refers to the cell into which the vector is introduced, including many cell types such as prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as HeLa cells, SW620 cells, SW480 cells, HT-29 cells, and HEK 293T cells.

As used herein, the term "effective dose" refers to a dose that can achieve therapeutic, preventive, ameliorative, and/or symptom-relieving effects on the disease or condition described in this invention in a subject.

As used herein, the term "disease and/or symptom" refers to a physical condition of the subject that relates to the disease and/or symptom described in this invention.

As used herein, the term "subject" may refer to a patient or other animal, particularly a mammal, such as a human, dog, monkey, cow, horse, etc., that receives the pharmaceutical composition of the present invention to treat, prevent, reduce and/or alleviate the disease or condition described in the present invention.

Example

The embodiments of the present invention will be described in detail below with reference to examples. Those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the examples, they should be performed according to the techniques or conditions described in the literature in the art (e.g., refer to J. Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Science Press, translated by Huang Peitang et al.) or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

Example 1: Nedd4-1 will ubiquitinate Ras protein to degrade it.

1. Experimental materials and main reagents

Cell lines:

Human cervical cancer cells HeLa (cat.CCL-2) were purchased from ATCC. Human embryonic kidney cells HEK293T (cat.CRL-3216) were purchased from ATCC. Human colon cancer cells HT-29 (cat.HTB-38) were purchased from ATCC.

The vector pCMV6 (cat.PS100001) was purchased from origene.

Transfection reagent: PEI (cat. 11668-027) was purchased from Invitrogen.

Dulbecco modified medium (DMEM, Gibco, cat. 11965) was purchased from ThermoFisher.

Primary antibody used for protein blotting:

Anti-c-Myc antibody, purchased from Santa Cruz; anti-rat-HA antibody, purchased from Roche; anti-Ub antibody, purchased from Santa Cruz; anti-Flag antibody, purchased from Sigma; anti-K-Ras antibody, purchased from Abcam; anti-H-Ras antibody, purchased from Abcam; anti-N-Ras antibody, purchased from Abcam; anti-Ras antibody, purchased from Abcam; anti-Nedd4-1 antibody, purchased from Millipore; anti-β-actin antibody, purchased from Santa Cruz.

Secondary antibody used in Western blotting:

Peroxidase-conjugated goat anti-mouse IgG (H+L) and peroxidase-conjugated goat anti-rabbit IgG (H+L) were both purchased from Thermo Fisher.

2. Experimental Methods

2.1 In vitro GST-pull-down experiment of proteins

GST, GST-Nedd4-1WT, and GST-Nedd4-1CA fusion proteins were expressed and purified in vitro using a glutathione S-transferase (GST) gene fusion vector. Simultaneously, GST-Flag-Ras protein was purified in vitro. Following the in vitro GST pull-down reaction system, the appropriate amount of purified protein was added to 0.5% TNTE reaction buffer. The mixture was incubated on a rotary shaker at 4°C for 1–2 hours, centrifuged at 2400 rpm for 1 minute at 4°C, and the supernatant was discarded. 0.5% TNTE reaction buffer was added, mixed, and centrifuged at 2400 rpm for 1 minute at 4°C, and the supernatant was discarded. This process was repeated 4–6 times. The reaction was terminated by adding 20 μL of 4× loading buffer and boiling in water for 5 minutes. Samples were analyzed by SDS-PAGE and Western blot.

2.2 In vitro ubiquitination experiment

In vitro ubiquitination experiments were performed according to standard experimental methods in the field. Specifically, GST-UBE1, His-UbcH7, His-Ub, GST-Nedd4-1 WT, GST-Nedd4-1 CA, and GST-Flag-Ras fusion proteins were expressed and purified in vitro. Appropriate amounts of purified protein were added according to the in vitro ubiquitination reaction system. During the reaction, E3 was first mixed with the substrate and incubated on ice for 30-45 minutes. Then, water, E2, Ub, ATP, and 5x buffer were added, and finally, E1 was added simultaneously to initiate the ubiquitination reaction. The reaction was incubated at 25°C for 50-60 minutes. After the reaction, 1% SDS was added and the mixture was boiled in water for 5 minutes. For each reaction group, 0.5% TNTE was added to expand the volume, and Flag antibody and protein A/G were added. The mixture was incubated overnight on a rotating shaker at 4°C. The mixture was centrifuged at 2400 rpm for 1 minute at 4°C, and the supernatant was discarded. Add 0.5% TNTE reaction buffer, mix well, centrifuge at 2400 rpm for 1 minute at 4°C, discard the supernatant, and repeat the above steps 3-4 times. Add 20 μL of 4× loading buffer and boil in water for 5 minutes. Perform SDS-PAGE and Western blot analysis on the sample.

2.3 Preparation of Cell-Expressed Protein Samples

1. After removing the cell culture plate from the 37°C CO2 incubator, aspirate the supernatant and wash once with warm PBS.

2. Add an appropriate amount of ice-cold lysis buffer (PMSF at a ratio of 1:100), place on a decolorizing shaker and shake for 20 minutes to lyse the cells.

3. Collect all the lysate into centrifuge tubes and centrifuge at 12,000 rpm for 10 minutes at 4°C.

4. Transfer the supernatant to a new tube, add 4× loading buffer, and mix well.

5. Heat in a 100℃ water bath for 5 minutes, then place in a -20℃ refrigerator for use in protein blot analysis.

2.4 Immunoprecipitation assay

1. Take 200 μL of protein A/G agarose beads (1:1 mixture), wash 3 times with TNTE 0.5% solution, centrifuge at 3000 rpm for 30 seconds each time, add 1% BSA to block the beads for 2 hours, wash 3 times with TNTE 0.5% solution again, and store at 4℃ for later use.

2. Mix 500 μL of cell lysis buffer with 0.5 μL of antibody or IgG and incubate at 4°C for 2 hours by rotation.

3. Add 20 μL of protein A/G agarose beads and incubate at 4°C for 12 hours to immunoprecipitate the labeled protein from the lysis buffer.

4. Centrifuge at 3000 rpm for 1 minute at 4℃ to transfer the agarose beads/antibody/antigen complex to the bottom of the tube.

5. Discard the supernatant, wash the agarose beads with 1 mL of TNTE 0.5% solution, then centrifuge at 3000 rpm for 1 minute at 4℃, discard the supernatant, and repeat this process 3 times.

6. Resuspend the agarose beads/antibody/antigen complex in 20 μL of SDS loading buffer, heat in a 100°C water bath for 5 minutes, and use the resulting sample for Western blot analysis.

3. Experimental Results

The experimental results are shown in Figure 1. Figure 1(a) shows the interaction between endogenous Nedd4-1 and Ras proteins in HeLa cells. Figure 1(b) shows the direct interaction between Nedd4-1 and Ras proteins in vitro. Figure 1(c) shows that overexpression of Nedd4-1 protein can reduce the level of endogenous Ras protein in HeLa cells; the statistical results are the mean ± SD of three independent experiments. Figure 1(d) shows that overexpression of Nedd4-1 protein can reduce the level of exogenous Ras protein. HEK293T cells were transfected with the corresponding plasmid combinations: Flag-tagged K-Ras (F/K-Ras), H-Ras (F/H-Ras), or N-Ras (F/N-Ras), and Flag-tagged Nedd4-1 (F/Nedd4-1) or Nedd4-2 (F/Nedd4-2), wild-type (WT), or the catalytically inactive mutants C867A or C942A. Whole-cell lysates were used for Western blotting to detect protein levels, with GAPDH protein levels serving as a control. Results were calculated as Mean ± SD from five independent experiments. Figure 1(e) shows that Nedd4-1-mediated Ras protein degradation occurs via the lysosomal pathway. In HEK293T cells, cis-transfected plasmid F/Nedd4-1 (WT or C867A) and different Ras proteins were used. Ras protein levels were measured after treatment with or without 20 μM proteasome inhibitor MG-132 or 120 μM lysosome inhibitor chloroquine for 12 hours. The statistical results were calculated as Mean ± SD of three independent experiments. Figure 1(f) shows Nedd4-1-mediated in vivo ubiquitination of H-Ras and N-Ras. In vivo ubiquitination experiments were performed in HEK293T cells using cis-transfected F/H-Ras or F/N-Ras, Myc/Nedd4-1 (WT or C867A), and HA/Ub. Figure 1(g) shows Nedd4-1 directly ubiquitinizing H-Ras and N-Ras under in vitro conditions. In vitro ubiquitination experiments were performed using purified GST/Nedd4-1 (WT or C867A) and F/H-Ras or F/N-Ras proteins. The ubiquitin-bound H-Ras or N-Ras proteins were detected using a Ub antibody. Figure 1(h) shows the regulation of the H-Ras protein half-life by Nedd4-1. HEK293T cells were treated with F/H-Ras, F/Nedd4-1, control shRNA, and Nedd4-1 shRNA plasmids, and cycloheximide (CHX) at different time points. The F/H-Ras protein level in whole-cell lysates was then detected. Protein quantification was performed using Image Lab software (Bio-Rad) with GAPDH as an internal control. The F/H-Ras protein level at each time point relative to zero was plotted as a line graph. Figure 1(i) shows the regulation of the N-Ras protein half-life by Nedd4-1. HEK293T cells were treated with F/N-Ras, F/Nedd4-1, control shRNA, and Nedd4-1 shRNA plasmids and cycloheximide (CHX) at different time points, as shown in the figure. The F/N-Ras protein level in whole-cell lysates was then detected. Protein quantification was performed using Image Lab software (Bio-Rad) with GAPDH as an internal control. The F/N-Ras protein level at each time point relative to zero was plotted as a line graph. Figure 1(j) shows that overexpression of Nedd4-1 in HT-29 cells reduces the endogenous WT K-Ras protein level, while knockdown of Nedd4-1 upregulates the endogenous WT K-Ras protein level. Figure 1(k) shows that the K-Ras G12V mutant does not show significant degradation when overexpressing F/Nedd4-1. Figure 1(l) shows that the K-Ras G12V mutation attenuates Nedd4-1-mediated K-Ras ubiquitination in vivo. F/K-Ras (WT or V12), Myc/Nedd4-1 (WT or C867A), and HA/Ub were cis-transferred into HEK293T cells for in vivo ubiquitination experiments. Figure 1(m) shows that the K-Ras G12V mutation attenuates Nedd4-1-mediated K-Ras ubiquitination in vitro. GST/Nedd4-1 (WT or C867A) and F/K-Ras (WT or V12) proteins were purified in vitro for ubiquitination experiments. K-Ras bound to ubiquitin was detected using a Ub antibody.

Example 2: Dot blotting high-throughput screening of compounds that promote K-Ras-G12V protein degradation The protein was further detected by Western blotting and finally obtained Nedd4-1-dependent K-Ras-G12V. The target compound to be degraded.

1. Experimental materials and main reagents

Cell lines:

Human colon cancer cells SW620 (cat.CCL-227) were purchased from ATCC. Human embryonic kidney cells HEK293T (cat.CRL-3216) were purchased from ATCC. Vector pCMV6 (cat.PS100001) was purchased from Origene.

Transfection reagent: PEI (cat.11668-027), purchased from Invitrogen.

Dulbecco modified medium (DMEM, Gibco, cat. 11965) was purchased from Thermo Fisher. RPMI medium 1640 (Gibco, cat. 31800022) was purchased from Thermo Fisher.

The primary antibody used in Western blotting:

Anti-Flag antibody, purchased from Sigma; anti-K-Ras antibody, purchased from Abcam; anti-Nedd4-1 antibody, purchased from Millipore; anti-β-actin antibody, purchased from Santa Cruz.

Secondary antibody used in Western blotting:

Peroxidase-conjugated goat anti-mouse IgG (H+L) and peroxidase-conjugated goat anti-rabbit IgG (H+L) were both purchased from Thermo Fisher.

2. Experimental Methods

dot blot high-throughput screening of compounds that promote the degradation of K-Ras-G12V protein

1) Cell transfection: Prepare HEK293T cells at a density of 70%-90%. Dilute the plasmids to be transfected (the plasmid expressing K-Ras-G12V with an N-terminal Flag tag (amino acid sequence DYKDDDDK) was constructed by cloning the K-Ras-G12V gene using a commercially available pCMV6 expression vector (e.g., the pCMV6 universal vector from Origene) and conventional PCR technology; the plasmid expressing Nedd4-1 with an N-terminal Flag tag was constructed by cloning the Nedd4-1 gene using a commercially available pCMV6 expression vector and conventional PCR technology) to 0.1 μg/μL. Prepare the transfection buffer according to the transfection system. Vortex for 10 seconds and incubate at room temperature for 30 minutes. During this time, replace the cells to be transfected with FBS-free DMEM medium. After 30 minutes, add the medium dropwise to the cells. Change the medium after 6-10 hours.

2) Cell plating: After transfecting HEK293T cells were digested and suspended, they were seeded into 96-well plates and cultured in a 37°C, 5% _CO2 incubator. The seeding amount was such that the cell density was about 70% when the drug was added the next day.

3) Cell drug delivery: After culturing for 24 hours, the various drug stock solutions were diluted with DMEM medium containing 10% FBS to obtain a final concentration of 20 μM. The original solution was discarded from the 96-well plate, and the prepared drug solution was added. The DMSO group was used as a blank control. After the drug was added, the plate was transferred to a 37℃ _CO2 incubator for further culture.

4) Detection of K-Ras protein levels: After culturing for 24 h, the liquid in the wells was discarded, cells were collected routinely, and total cell protein was extracted by adding cell lysis buffer. The protein concentration was determined by the BCA method. 30 μg of protein sample was spotted onto the nitrocellulose membrane. After the sample was completely adsorbed onto the membrane, the membrane was blocked sequentially. The membrane was incubated overnight at 4 °C with Flag antibody (1:2000) and internal control antibody β-actin (1:2000). After washing with TBST for 5 min x 3 times, the membrane was incubated with the corresponding horseradish peroxidase-labeled secondary antibody (1:5000) for 1-2 h. The membrane was washed with TBST for 5 min x 3 times. Chemiluminescence imaging was performed using ImageLab software.

5) Calculation of single-point K-Ras protein degradation rate: Flag-K-Ras and β-actin were quantified and calibrated using Image-Lab software, and then the intracellular Flag-K-Ras degradation rate was calculated using the following formula: Single-point Flag-K-Ras protein degradation rate (%) = (control group - drug group) / control group × 100%.

Compounds that can reduce exogenous K-Ras-G12V levels were screened in HEK293T cells.

1) Cell seeding: HEK293T cells in logarithmic growth phase are seeded into 12-well plates and cultured in a 37°C, 5% _CO2 incubator. The seeding amount is best when the cell density is 70%-90% on the second day after transfection.

2) Cell transfection and drug administration: After 24 hours of culture, the plasmids to be transfected (the plasmid expressing K-Ras-G12V with an N-terminal Flag tag (amino acid sequence DYKDDDDK) was constructed by cloning and inserting the K-Ras-G12V gene using a commercially available pCMV6 expression vector and conventional PCR technology; the plasmid expressing Nedd4-1 with an N-terminal Flag tag was constructed by cloning and inserting the Nedd4-1 gene using a commercially available pCMV6 expression vector and conventional PCR technology) were diluted. Prepare transfection buffer according to the transfection system, dilute to 0.1 μg/μL, vortex for 10 seconds and let stand at room temperature for 30 minutes. During this time, replace the cells to be transfected with DMEM medium without FBS. After 30 minutes, add the buffer dropwise to the cells. Change the medium after 6-10 hours. When changing the medium, dilute the various drug stock solutions with DMEM medium containing 10% FBS to obtain a final concentration of 20 μM. Discard the original solution in a 12-well plate and add the prepared drug solution. Use the DMSO group as a blank control. After the drug addition is complete, transfer the plate to a 37℃ _CO2 incubator for further culture.

3) Detection of K-Ras protein levels: After culturing for 24 h, the liquid in the wells was discarded, cells were collected routinely, washed twice with 1X PBS, and total protein was extracted by adding cell lysis buffer. The protein concentration was determined by the BCA method. 30 μg of protein sample was transferred to an SDS-polyacrylamide gel electrophoresis membrane. The membrane was blocked sequentially and incubated overnight at 4℃ with Flag antibody (1:2000) and internal control antibody β-actin (1:2000). After washing with TBST for 5 min x 3 times, the membrane was incubated with the corresponding horseradish peroxidase-labeled secondary antibody (1:5000) for 1-2 h. After washing with TBST for 5 min x 3 times, chemiluminescence imaging was performed using ImageLab software.

4) Calculation of single-point K-Ras protein degradation rate: Flag-K-Ras and β-actin were quantified and calibrated using Image-Lab software, and then the intracellular Flag-K-Ras degradation rate was calculated using the following formula: Single-point Flag-K-Ras protein degradation rate (%) = (control group - drug group) / control group × 100%.

Compounds that can reduce endogenous K-Ras-G12V levels were screened in SW620 cells.

1) Cell seeding: Logarithmic growth phase SW620 cells were seeded at 2× ^10⁴ /well in 24-well plates and cultured in a 37℃, 5% _CO₂ incubator.

2) Cell drug delivery: After culturing for 24 hours, the various drug stock solutions were diluted with RPMI-1640 medium containing 10% FBS to obtain a final concentration of 20 μM. The original solution was discarded from the 24-well plate, and the prepared drug solution was added. The DMSO group was used as a blank control. After the drug was added, the plate was transferred to a 37℃ _CO2 incubator for further culture.

3) Detection of K-Ras protein levels: After culturing for 24 h, the liquid in the wells was discarded, cells were collected routinely, washed twice with 1X PBS, and total protein was extracted by adding cell lysis buffer. The protein concentration was determined by the BCA method. 30 μg of protein sample was transferred to an SDS-polyacrylamide gel electrophoresis membrane. The membrane was blocked sequentially and incubated overnight at 4℃ with K-Ras antibody (1:1000) and internal control antibody β-actin (1:2000). After washing with TBST for 5 min x 3 times, the membrane was incubated with the corresponding horseradish peroxidase-labeled secondary antibody (1:5000) for 1-2 h. After washing with TBST for 5 min x 3 times, chemiluminescence imaging was performed using ImageLab software.

4) Calculation of single-point K-Ras protein degradation rate: K-Ras and β-actin were quantified and calibrated using Image-Lab software, and then the intracellular K-Ras degradation rate was calculated using the following formula: Degradation rate of single-point K-Ras protein (%) = (control group - drug group) / control group × 100%.

3. Experimental Results

The results are shown in Figure 2. Figure 2(a) shows that 28 compounds were screened out after high-throughput screening using dot blot. After overexpressing Flag-K-Ras-G12V and Flag-Nedd4-1-WT in 293T cells, 7 compounds that could reduce exogenous K-Ras-G12V protein levels were screened after drug treatment. Figure 2(b) shows that the 7 compounds that could reduce exogenous K-Ras-G12V (structures shown in the table below) were further screened in SW620 cells, and 3 compounds that could reduce endogenous K-Ras-G12V protein levels were selected. Figure 2(c) shows the 3 compounds after endogenous and exogenous screening. The determination of whether the reduction of K-Ras-G12V protein levels by these three compounds depended on the presence of Nedd4-1 protein was examined. The compound with the final compound number 3 (compound code: ZT-10-158-01) was selected for the next stage of experiments.

Example 3: The small molecule compound ZT-10-158-01 inhibits K-Ras-G12V protein levels in a positive concentration-dependent manner. sex

1. Experimental materials and main reagents

Cell lines:

Human colon cancer cells SW620 (cat. CCL-227) were purchased from ATCC. Human embryonic kidney cells HEK293T (cat. CRL-3216) were purchased from ATCC.

The vector pCMV6 (cat.PS100001) was purchased from origene.

Transfection reagent: PEI (cat.11668-027), purchased from Invitrogen.

Dulbecco modified medium (DMEM, Gibco, cat. 11965) was purchased from ThermoFisher. RPMI 1640 medium (Gibco, cat. 31800022) was purchased from ThermoFisher.

Primary antibody used for protein blotting:

Anti-Flag antibody, purchased from Sigma; anti-K-Ras antibody, purchased from Abcam; anti-Nedd4-1 antibody, purchased from Millipore; anti-β-actin antibody, purchased from Santa Cruz.

Secondary antibody used in Western blotting:

Peroxidase-conjugated goat anti-mouse IgG (H+L) and peroxidase-conjugated goat anti-rabbit IgG (H+L) were both purchased from Thermo Fisher.

2. Experimental Methods

Inhibition of endogenous K-Ras-G12V levels in SW620 cells

1) Cell seeding: Logarithmic growth phase SW620 cells were seeded at 2× ^10⁴ /well in 24-well plates and cultured in a 37℃, 5% _CO₂ incubator.

2) Cell drug delivery: After culturing for 24 h, the various drug stock solutions were diluted with RPMI-1640 medium containing 10% FBS to obtain drug solutions with final concentrations of 2.5 μM, 5 μM, 10 μM, 15 μM and 20 μM, respectively; the original solution was discarded in the 24-well plate, and the prepared drug solution was added, with the DMSO group as a blank control; after the drug addition was completed, the plate was transferred to a 37℃ _CO2 incubator for further culture.

3) Detection of K-Ras protein levels: After culturing for 24 h, the liquid in the wells was discarded, cells were collected routinely, washed twice with 1X PBS, and total protein was extracted by adding cell lysis buffer. The protein concentration was determined by the BCA method. 30 μg of protein sample was transferred to an SDS-polyacrylamide gel electrophoresis membrane. The membrane was blocked sequentially and incubated overnight at 4℃ with K-Ras antibody (1:1000) and β-actin internal control antibody (1:2000). After washing with TBST for 5 min x 3 times, the membrane was incubated with the corresponding horseradish peroxidase-labeled secondary antibody (1:5000) for 1-2 h. After washing with TBST for 5 min x 3 times, chemiluminescence imaging was performed using ImageLab software.

4) Calculation of single-site K-Ras protein degradation rate: K-Ras and β-actin were quantified and calibrated using Image-Lab software, and then the intracellular K-Ras degradation rate was calculated using the following formula: Degradation rate of single-site K-Ras protein (%)

= (Control group - Medication group) / Control group × 100%.

In HEK293T cells, the level of exogenous K-Ras-G12V is inhibited.

1) Cell seeding: HEK293T cells in logarithmic growth phase are seeded into 12-well plates and cultured in a 37°C, 5% _CO2 incubator. The seeding amount is best when the cell density is 70%-90% on the second day after transfection.

2) Cell transfection and drug administration: After culturing for 24 h, the plasmids to be transfected (all plasmids were constructed using commercially available plasmid vectors and conventional molecular biology methods) were diluted to 0.1 μg/μL. Transfection buffer was prepared according to the transfection system. The cells were vortexed for 10 s and then incubated at room temperature for 30 minutes. During this period, the cells to be transfected were replaced with DMEM medium without FBS. After 30 minutes, the medium was added dropwise to the cells. The medium was changed after 6-10 hours. During the medium change, the various drug stock solutions were diluted with DMEM medium containing 10% FBS to obtain drug solutions with final concentrations of 25 μM, 5 μM, 10 μM, 15 μM, and 20 μM, respectively. The original solution was discarded in the 12-well plate and the prepared drug solution was added. The DMSO group was used as a blank control. After the drug was added, the plate was transferred to a 37℃ _CO2 incubator for further culture.

3) Detection of K-Ras protein levels: After culturing for 24 h, the liquid in the wells was discarded, cells were collected routinely, washed twice with 1X PBS, and total protein was extracted by adding cell lysis buffer. The protein concentration was determined by the BCA method. 30 μg of protein sample was transferred to an SDS-polyacrylamide gel electrophoresis membrane. The membrane was blocked sequentially and incubated overnight at 4℃ with Flag antibody (1:2000) and β-actin internal control antibody (1:2000). After washing with TBST for 5 min x 3 times, the membrane was incubated with the corresponding horseradish peroxidase-labeled secondary antibody (1:5000) for 1-2 h. After washing with TBST for 5 min x 3 times, chemiluminescence imaging was performed using ImageLab software.

4) Calculation of single-point K-Ras protein degradation rate: Flag-K-Ras and β-actin were quantified and calibrated using Image-Lab software, and then the intracellular Flag-K-Ras degradation rate was calculated using the following formula: Single-point Flag-K-Ras protein degradation rate (%) = (control group - drug group) / control group × 100%.

3. Experimental Results

As shown in Figure 3, the experimental results show that compound ZT-10-158-01 can effectively reduce the level of K-Ras protein in cells in a positive concentration-dependent manner.

Example 4: Treatment of the small molecule compound ZT-10-158-01 can effectively inhibit the aggregation of cancer cells on soft agar. The ability to form a falling object.

1. Experimental materials and main reagents

Cell lines:

Human colon cancer cells SW620 (cat.CCL-227) were purchased from ATCC.

Human colon cancer cells HT-29 (cat.HTB-38), purchased from ATCC.

Human breast cancer cells MCF-7 (cat.HTB-22) were purchased from ATCC.

Agar powder (cat.A100637) was purchased from Sangon Biotech.

Dulbecco modified medium (DMEM, Gibco, cat. 11965) was purchased from ThermoFisher.

2. Experimental Methods

1) Bottom layer agar

i. Prepare 1% agar, autoclave, and before use, heat the 1% agar in a boiling water bath to melt it, and then cool it in a 40°C water bath (let it stand for at least 30 minutes to allow the agar temperature to cool to 40°C); at the same time, add 2×DMEM/FBS to the warm bath.

ii. Prepare a 0.5% bottom layer agar by mixing 1% agar with 2×DMEM/FBS in a sterile tube at a volume ratio of 1:1. Quickly add the mixture to each well of a 6-well plate, gently shake to mix, and allow to solidify at room temperature (after solidification, it can be stored at 4°C for one week. Before use, allow it to return to room temperature for at least 30 minutes).

2) Upper agar

Prepare a 0.3% agar + 1×DMEM/FBS mixture in a sterile tube using 1% agar, 2×DMEM/FBS, and sterile water at 40°C. Place the prepared mixture in a 40°C water bath until ready for use.

ii. Digest cells with trypsin and count the cells, adjusting the cell density according to experimental needs;

3) Take out the prepared 0.3% agar + 1×DMEM/FBS mixture from the 40℃ water bath, add the corresponding number of cells and a certain amount of DMSO/ZT-10-158-01, gently blow it evenly with a pipette, and add it to the solidified bottom layer of agar, 1.5mL per well.

4) Place the cell culture plates in a cell culture incubator at 37°C and 5% _CO2 for 10-30 days. Add fresh culture medium every 3-6 days, 200 μL per well.

5) Observe the formation of cell clones in the agar every day. When the cell clones are large enough to be photographed, take pictures and record them. Observe and compare the differences in the number and size of cell clones formed between the DMSO group and the experimental group treated with ZT-10-158-01.

3. Experimental Results

As shown in Figure 4, the experimental results show that compound ZT-10-158-01 can selectively inhibit the colony formation of tumor cells that require the Ras protein activation signaling pathway, such as MCF-7 cells and SW620 cells, but does not have a significant inhibitory effect on the colony formation of HT-29 cells, which have mutated Raf kinase downstream of the Ras signaling pathway and no longer require the Ras protein to activate this signaling pathway.

Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of this invention. The full scope of this invention is given by the appended claims and any equivalents thereof.

Claims (3)

1. A method for identifying compounds that regulate Ras levels, the method comprising: a. Contact the test compound with cells expressing Ras or in vitro ubiquitination systems; b. Select compounds that can reduce the Ras level in cells or systems in step a; c. Determine whether the compounds obtained in step b regulate Ras levels in a manner dependent on the presence of Nedd4-1 protein; d. Select compounds that regulate Ras levels in dependence on the presence of Nedd4-1 protein; The structural formula of the compound that regulates Ras levels in dependence on the presence of Nedd4-1 protein is as follows: . 2. The method of claim 1, wherein the cell or system expressing Ras is a cell or system expressing exogenous Ras or endogenous Ras or both. 3. The method according to claim 1, wherein the Ras is selected from H-Ras, K-Ras, or N-Ras or mutants thereof; The mutant has a glycine at position 12 mutated to valine.