Selective degradation of trimeric proteins via chemical induction of proximity to TRIM21

Abstract

A method for degrading a target protein in a cell via the TRIM21 E3 ligase ubiquitin-proteasome pathway, the method comprising contacting the target protein with an effective amount of a compound selected from compounds of formula (I), formula (II), or molecular gel compounds or PROTACs carrying compounds of formula (I) or (II) as ligands for recruiting TRIM21 ligases.

Description

Technical Field

[0001] This article discloses a method for degrading a target protein in a cell via the TRIM21 E3 ligase ubiquitin-proteasome pathway, the method comprising contacting the target protein with an effective amount of a compound selected from: compounds of formula (I) that serve as ligands for recruiting TRIM21 ligases, compounds of formula (II), or molecular gel compounds or PROTACs carrying compounds of formula (I) or (II). Background Technology

[0002] Targeted protein degradation (TPD), a novel therapeutic approach for treating proteins considered untreatable with traditional small-molecule inhibitors, is achieved through chemically induced proximity between a specific E3 ubiquitin ligase and a novel substrate protein. Two classes of chemicals have been used in TPD: monovalent molecular glue degraders and bifunctional PROTACs (proteolytic-targeting chimeras). Molecular glues are compounds that bind two proteins together by facilitating their interaction. PROTAC molecules consist of two binding units, one binding to the target protein and the other to the E3 ubiquitin ligase. This brings the target protein and the E3 ligase very close, leading to ubiquitination of the target protein and subsequent degradation by the proteasome.

[0003] IMiD (immunomodulatory imide drugs) highlights the effects of TPD, which acts as a molecular glue to induce the degradation of various novel substrates, such as zinc finger transcription factors Ikaros and Aiolos, to exert superior efficacy in the treatment of multiple myeloma. Recently, a large number of well-designed PROTACs targeting pathogenic proteins have entered clinical trials, providing proof-of-concept for the efficacy and safety of TPD in human patients.

[0004] First-generation TPD agents utilize E3 ligases with general expression patterns and constitutive activity, and are therefore unsuitable for disease-related proteins that have essential functions in healthy tissues. To overcome these limitations, several strategies are being actively explored to achieve selectivity for TPDs. First, by modifying the chemical structure and ternary complex stability, TPD agents can achieve selectivity in distinguishing highly similar targets. Second, the subcellular localization of E3 ubiquitin ligases can be used to confine TPDs to specific subcellular compartments. Third, tissue-specific expression of E3 ubiquitin ligases can be used to confine TPDs to specific tissue and cell types. In addition to their constitutive active counterparts, many E3 ligases are autorepressed in the basal state and activated upon binding to specific substrates. This mechanism enhances the specificity of these E3 ligases to their homologous substrates, but has not yet been utilized to improve TPD selectivity.

[0005] Selective TPD is an attractive area of ​​technology for therapeutic targeting of biomolecular condensates, which are membrane-free assemblies enriched with specific proteins and nucleic acids in a subcellular environment. Numerous recent studies have revealed the crucial functions of biomolecular condensates in both normal and diseased states. In particular, aberrant protein aggregation has been causally linked to neurodegeneration and cancer, thus revealing exciting therapeutic opportunities. However, the selective degradation of proteins within biomolecular condensates without affecting proteins in the dilution phase has proven technically challenging.

[0006] Although over 600 E3 ligases exist in human cells, current TPD strategies primarily utilize E3 ligase complexes containing cereblon (CRBN) or von Hippel−Lindau (VHL). Therefore, chemicals using alternative E3 ligases with different properties are highly sought after to avoid limitations and expand the application of TPD. TRIM21 is an E3 ubiquitin-protein ligase belonging to the family of ring-containing tripartite motif (TRIM) proteins. TRIM21 is a major autoantigen in autoimmune diseases and a regulator of innate immune signaling. TRIM21 uses antibodies as bridging molecules to guide invading pathogens to proteasome degradation as a mechanism of intracellular immunity. This ability of TRIM21 has recently been used in the Trim-Away technique to achieve acute and rapid degradation of endogenous cellular proteins. While Trim-Away has great potential, it requires electroporation or microinjection to deliver antibodies to cells, which is impractical in most therapeutic settings. The TRIM21-directed degrader shown here provides a chemical starting point for extending the TRIM-Away to therapeutic applications. TRIM21 forms an inactive dimer in which a central antiparallel coiled helix positions two RING domains at opposite ends of the dimer. Upon binding to a multimeric substrate, the RING domains from adjacent TRIM21 dimerize, leading to activation of its enzymatic activity.

[0007] Abnormal protein assemblies lead to several diseases. However, some of these abnormal protein assemblies are found to be untreatable targets. In the context of cancer, fusion transcription factors are often associated with chromosomal translocations, genetic abnormalities that can drive the development of certain types of cancer. Some of these fusions form aberrant multimers, such as EWS-FLI1 in Ewing sarcoma and NUP98-fusions in various hematologic malignancies, including acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), as well as some cases of myelodysplastic syndromes (MDS) and chronic myeloid leukemia (CML). Intracellular amyloid protein aggregation (such as Tau, α-synuclein, and mutant huntingtin) is a common cause of neurodegeneration. Furthermore, protein multimerization (such as cGAS and NLRP3) activates immune responses, which may be a potential cause of autoimmune diseases.

[0008] Therefore, there is a need for degradative agents based on the E3 ligase TRIM21 to selectively degrade the multimeric proteins that cause the above-mentioned diseases. Summary of the Invention

[0009] The inventors of this invention have unexpectedly and surprisingly discovered that (S)-hydroxy-acetylpromethazine (a metabolite of acetylpromethazine, a drug commonly used as a sedative / tranquilizer in veterinary medicine) can be used as a monovalent degrader of several proteins in multimeric nuclear pore complexes via the E3 ubiquitin ligase TRIM21. This suggests that TRIM21-mediated TPD can be used as a general strategy for developing TRIM21-based degraders to induce the selective degradation of multimeric proteins, including proteins in biomolecular condensates.

[0010] The inventors have discovered two classes of TRIM21-directed degraders that exhibit high selectivity for novel multimeric substrates. The first class (represented by compounds of formula (I) or (II)) includes the serendipitously discovered (S)-ACE-OH, which acts as a monovalent molecular glue to guide TRIM21 recognition of NUP98, leading to the degradation of a variety of proteins in multimeric nuclear pore complexes. The second class (i.e., bifunctional PROTACs, which contain compounds of formula (I) as ligands recruiting the E3 ubiquitin ligase TRIM21) degrades multimeric protein targets localized to various biomolecular condensates without affecting monomeric proteins in the dilute phase. These two classes demonstrate the potential of TRIM21-directed degraders for differentially modulating proteins in multimeric assemblies such as biomolecular condensates.

[0011] In the first aspect, this document discloses compounds of formula (I) or pharmaceutically acceptable salts thereof, isomers thereof, or deuterated analogs thereof.

[0012] (I)

[0013] in

[0014] X is O, S, or NR. 1 , where R 1 It is hydrogen or alkyl;

[0015] n is 1 to 12;

[0016] R a and R b Each is independently either hydrogen or alkyl;

[0017] R c and R d Each of these groups is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocyclic, or heteroaryl, and each of these groups is unsubstituted or substituted by one or more substituents selected from: halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclic, heteroaryl, cyano, hydroxyl, hydroxyl, alkoxy, -NR m1 C(O)NR n1 R p1 -NR m1 C(O)R n1 -C(O)NR m1 R n1 -NR m1 R n1 -C(O)R m1 or -C(O)OR m1 ;

[0018] Or R c and R d Together with the nitrogen atoms to which they are attached, they form 3 to 12-membered rings containing 0 to 3 additional heteroatoms selected from oxygen, nitrogen, or sulfur, and said rings are unsubstituted or substituted by one or more substituents selected from: halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclic, heteroaryl, cyano, hydroxy, hydroxyalkyl, alkoxy, hydroxyalkoxy, -NR m2 R n2 -C(O)R m2 -C(O)OR m2 -NR m2 C(O)NR n2 R p2 -NR m2 C(O)R n2 or -C(O)NR m2 R n2 ;

[0019] R e It is hydrogen, alkyl, -NR m2R n2 -NR m2 C(O)NR n2 R p2 -NR m2 C(O)R n2 alkyl, aryl, heterocyclic, or heteroaryl groups, each of which is unsubstituted or substituted by one or more substituents selected from: halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclic, heteroaryl, cyano, hydroxy, hydroxyalkyl, alkoxy, hydroxyalkoxy, -NR m3 C(O)NR n3 R p3 -NR m3 C(O)R n3 -C(O)NR m3 R n3 -NR m3 R n3 -C(O)R m3 or -C(O)OR m3 ,

[0020] p is between 0 and 3;

[0021] q is between 0 and 4;

[0022] R f and R g Each of these groups can be independently halogenated, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclic, heteroaryl, cyano, hydroxyl, alkoxy, or -NR. m2 C(O)NR n2 R p2 -NR m2 C(O)R n2 -C(O)NR m2 R n2 -NR m2 R n2 -C(O)R m2 or -C(O)OR m2 ;

[0023] in

[0024] R m1 R n1 R p1 R m2 R n2 R p2 R m3 R n3 and R p3 Each of them is independently hydrogen, alkyl, haloalkyl, alkoxyalkyl-, phenyl, cycloalkyl, heteroaryl, or heterocyclic.

[0025] In the second aspect, this document discloses compounds of formula (II) or pharmaceutically acceptable salts thereof, isomers thereof, or deuterated analogs thereof.

[0026] (II)

[0027] in

[0028] X is O, S, or NR. 1 , where R 1 It is hydrogen or alkyl;

[0029] n is 1 to 12;

[0030] R a and R b Each is independently either hydrogen or alkyl;

[0031] R c and R d Each of these groups is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocyclic, or heteroaryl, and each of these groups is unsubstituted or substituted by one or more substituents selected from: halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclic, heteroaryl, cyano, hydroxyl, hydroxyl, alkoxy, -NR m1 C(O)NR n1 R p1 -NR m1 C(O)R n1 -C(O)NR m1 R n1 -NR m1 R n1 -C(O)R m1 or -C(O)OR m1 ;

[0032] Or R c and R d Together with the nitrogen atoms to which they are attached, they form 3 to 12-membered rings containing 0 to 3 additional heteroatoms selected from oxygen, nitrogen, or sulfur, and said rings are unsubstituted or substituted by one or more substituents selected from: halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclic, heteroaryl, cyano, hydroxy, hydroxyalkyl, alkoxy, hydroxyalkoxy, -NR m2 R n2 -C(O)R m2 -C(O)OR m2 -NR m2 C(O)NR n2 R p2 -NR m2 C(O)Rn2 or -C(O)NR m2 R n2 ;

[0033] R e1 and R e2 Each is independently hydrogen, alkyl, -NR m2 R n2 -NR m2 C(O)NR n2 R p2 -NR m2 C(O)R n2 alkyl, aryl, heterocyclic, or heteroaryl groups, each of which is unsubstituted or substituted by one or more substituents selected from: halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclic, heteroaryl, cyano, hydroxy, hydroxyalkyl, alkoxy, hydroxyalkoxy, -NR m3 C(O)NR n3 R p3 -NR m3 C(O)R n3 -C(O)NR m3 R n3 -NR m3 R n3 -C(O)R m3 or -C(O)OR m3 ;

[0034] p is between 0 and 3;

[0035] q is between 0 and 4;

[0036] R f and R g Each of these groups can be independently halogenated, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclic, heteroaryl, cyano, alkoxy, or -NR. m2 C(O)NR n2 R p2 -NR m2 C(O)R n2 -C(O)NR m2 R n2 -NR m2 R n2 -C(O)R m2 -C(O)OR m2 , cycloalkyl, phenyl, heteroaryl or heterocyclic groups;

[0037] in

[0038] R m1 R n1 R p1 Rm2 R n2 R p2 R m3 R n3 and R p3 Each of them is independently hydrogen, alkyl, haloalkyl, alkoxyalkyl-, phenyl, cycloalkyl, heteroaryl, or heterocyclic.

[0039] In some implementations of the first and second aspects, X is S.

[0040] In some implementations of the first and second aspects, n is 1 to 6, or 2 to 4, or 3.

[0041] In some embodiments of the first and second aspects, R a and R b Each is hydrogen.

[0042] In some embodiments of the first and second aspects, R c and R d Each is either hydrogen or alkyl.

[0043] In some embodiments of the first and second aspects, R c and R d Together with the nitrogen atoms to which they are attached, they form a 5- to 7-membered ring containing 0 to 1 additional heteroatom selected from oxygen, nitrogen, or sulfur, and the ring is unsubstituted or substituted by one or more substituents selected from halogen, alkyl, hydroxy, hydroxyalkyl, alkoxy, or hydroxyalkoxy.

[0044] In some embodiments of the first and second aspects, R c and R d Together with the nitrogen atoms to which they are attached, they form a 6-membered ring containing 0 to 1 additional nitrogen heteroatom, and the ring is substituted by one or more substituents selected from the following: halogen, alkyl, hydroxy, hydroxyalkyl, alkoxy, or hydroxyalkoxy.

[0045] In some embodiments of the first and second aspects, p is 0. In some embodiments of the first and second aspects, q is 0.

[0046] In some implementations of the first aspect, R e It is hydrogen, alkyl, or -NR m2 R n2 , where R m2 and R n2 It is as defined in equation (I).

[0047] In some implementations of the second aspect, R e1 and R e2 Each can be independently hydrogen, alkyl, aryl, or -NR.m2 R n2 , where R m2 and R n2 It is as defined in equation (I). In some implementations of the second aspect, R e1 It is an alkyl group and R e2 It is hydrogen, alkyl, aryl, or -NR m2 R n2 , where R m2 and R n2 It is as defined in equation (I). In some implementations of the second aspect, R e1 and R e2 They are different. In some implementations of the second aspect, with R... e1 and R e2 The attached carbon atom is a chiral center. In some embodiments of the second aspect, with R... e1 and R e2 The attached carbon atom has an (S)- or (R)- configuration. In some embodiments of the second aspect, with R... e1 and R e2 The attached carbon atom has an (S)- configuration.

[0048] In some embodiments of the first and second aspects, X is S; n is 1 to 6, or 2 to 4, or 3; R a and R b Each is hydrogen; p and q are both 0; R c and R d Each is independently an alkyl group; or R c and R d Together with the nitrogen atoms to which they are attached, they form a 5- to 7-membered ring containing 0 to 1 additional heteroatoms selected from oxygen, nitrogen, or sulfur, and said ring is unsubstituted or substituted by one or more substituents selected from: halogen, alkyl, hydroxy, hydroxyalkyl, alkoxy, or hydroxyalkoxy; or R c and R d Together with the nitrogen atoms to which they are attached, they form a 6-membered ring containing 0 to 1 additional nitrogen heteroatom, and said ring is substituted by one or more substituents selected from: halogen, alkyl, hydroxy, hydroxyalkyl, alkoxy, or hydroxyalkoxy; R e It is an alkyl group; or R e1 and R e2 Each is independently hydrogen, alkyl; or R e1 It is hydrogen and R e2 It is hydrogen or alkyl.

[0049] In some embodiments of the first and second aspects, the compound is , ,or .

[0050] In some embodiments of the first and second aspects, the compound is or .

[0051] In a third aspect, this document discloses a method for degrading a target protein in a cell via the TRIM21 E3 ligase ubiquitin-proteasome pathway, the method comprising contacting the target protein with an effective amount of a compound selected from compounds of formula (I), formula (II), or molecular gel compounds or PROTACs carrying compounds of formula (I) or (II) as ligands for recruiting TRIM21 ligases.

[0052] In some implementations of the third aspect, the cell is a cancer cell.

[0053] In some embodiments of the third aspect, the contact is performed in vitro.

[0054] In some embodiments of the third aspect, the contact takes place in vivo.

[0055] In some implementations of the third aspect, the administration of the compound to the subject is further included.

[0056] In some embodiments of the third aspect, the target protein is any protein that forms a multimer, which is defined as multiple copies of a gene-encoded polypeptide that form a complex.

[0057] In some embodiments of the third aspect, the target protein is a multimeric protein that causes human disease.

[0058] In some embodiments of the third aspect, the target protein is a protein aggregate or protein condensate.

[0059] In some embodiments of the third aspect, the target protein is amyloid protein or an amyloid-like aggregate. In some embodiments, the amyloid-like aggregate includes Tau, α-synuclein, and mutant huntingtin.

[0060] In some embodiments of the third aspect, the target protein is NUP98-fusion protein, EWS-FLI1, Tau fibrils, α-synuclein fibrils, mutant huntingtin protein, cGAS, or NLRP3.

[0061] In some embodiments of the third aspect, the degradation is achieved by recruiting the TRIM21 ubiquitin E3 ligase.

[0062] In some embodiments of the third aspect, the compound of formula (I) or the compound of formula (II) induces TRIM21 to be recruited to the vicinity of the NPC to trigger the degradation of nucleoporin.

[0063] In a fourth aspect, this document discloses a compound for degrading target proteins in cells via the TRIM21 E3 ligase ubiquitin-proteasome pathway, said compound being selected from compounds of formula (I), formula (II), or molecular gel compounds or PROTACs carrying said compound (I) or (II) as ligands for recruiting TRIM21 ligases; or the use of the compound in the manufacture of reagents or pharmaceuticals for degrading target proteins in cells via the TRIM21 E3 ligase ubiquitin-proteasome pathway, said compound being selected from compounds of formula (I), formula (II), or molecular gel compounds or PROTACs carrying said compound (I) or (II) as ligands for recruiting TRIM21 ligases.

[0064] In a fifth aspect, this document discloses the use of compounds of formula (I) or formula (II) in PROTAC as ligands for recruiting TRIM21 ligases.

[0065] In a sixth aspect, this document discloses a method for treating or preventing diseases characterized by amyloid or amyloid-like aggregates, the method comprising administering to a subject in need a therapeutically effective amount of a compound of formula (I), a compound of formula (II), or a molecular gel compound or PROTAC carrying a compound of formula (I) or (II) as a ligand for recruiting TRIM21 ligase.

[0066] In some implementations, the disease is Alzheimer's disease, Huntington's disease and Parkinson's disease, type 2 diabetes, autoimmune diseases, or various amyloidosis caused by systemic or localized deposition of amyloid protofibrils in the intracellular and extracellular spaces of tissues and organs. Attached Figure Description

[0067] The following specific embodiments can be best understood in conjunction with the accompanying drawings, which are given by way of example but are not intended to limit the invention to the specific embodiments described.

[0068] Figure 1 The study demonstrated that acepromazine exhibits interferon-enhanced selective anticancer activity:

[0069] - Figure 1A schematic diagram illustrates a competitive cell growth assay in which an equal mixture of WT A549 cells (labeled with H2B-GFP) and IFNGR1-deficient A549 cells (labeled with H2B-mCherry) was treated for four days with IFNγ (20 ng / ml) and a test compound (10 µM), followed by high-content imaging to quantify GFP. + Cell nucleus and mCherry + The ratio of cell nuclei.

[0070] - Figure 1 B shows the 81,845 compounds paired with GFP in the primary screening. + Cell nucleus and mCherry + The effect of the ratio of cell nuclei.

[0071] - Figure 1 C shows the chemical structures of the seed compounds from the primary screening and their representative raw images. Green: H2B-GFP; Red: H2B-mCherry; Scale bar: 50 µm.

[0072] - Figure 1 D shows the effect of the seed compound on GFP. + Cell nucleus and mCherry + The effect of the ratio of cell nuclei. Data represent the means of three independent samples. Statistical significance was determined using one-way ANOVA followed by Dunnett's multiple comparison test.

[0073] - Figure 1 E shows the concentration-response curve of acepromazine on the viability of A549 cells treated with an indicator cytokine (10 ng / ml). Data represent the mean ± sem of three independent samples.

[0074] - Figure 1 F shows crystal violet staining of A549 cells co-cultured with an indicated amount of NK-92MI cells with or without acepromazine (10 µM) for 48 h. Representative results from two independent experiments are shown.

[0075] - Figure 1 G shows the effect of NK-92MI cells on the fitness of WTA549 cells (labeled with H2B-GFP) relative to IFNGR1-deficient A549 cells (labeled with H2B-mCherry) with or without acepromazine (10 µM) treatment for 48 h. GFP was determined by FACS. + Cell nucleus and mCherry + The ratio of cell nuclei. Data represent the means of three independent samples. Student's t-test (two-tailed) was used to determine statistical significance.

[0076] - Figure 1 H shows the concentration-response curves of acepromazine on the viability of indicator cell lines and primary T lymphocytes with and without IFNγ (10 ng / ml). Data represent the mean ± sem of three independent samples.

[0077] Figure 2 This demonstrates that acepromazine exhibits selective anticancer activity that is independent of dopamine receptors and enhances interferon-like activity.

[0078] - Figure 2 A shows the immunoblot of pSTAT1 and STAT1 in A549-Cas9 cells transduced with the indicated sgRNA and treated with IFNγ for 4 h. Representative results from two independent experiments are shown.

[0079] - Figure 2 B illustrates a schematic diagram of the NK92-MI killing assay. An equal mixture of WT A549 cells (labeled with H2B-GFP) and IFNGR1-deficient A549 cells (labeled with H2B-mCherry) was co-cultured with NK-92MI cells for 48 h, followed by FACS to quantify GFP. + Cell nucleus and mCherry + The ratio of cell nuclei.

[0080] - Figure 2 C shows the chemical structure of chlorpromazine.

[0081] - Figure 2 D shows crystal violet staining of A549 (with or without 20 ng / ml IFNγ) treated with different concentrations of acepromazine or chlorpromazine.

[0082] - Figure 2 E shows the expression level of the gene encoding the dopamine receptor in the indicated acepromazine-sensitive cancer cell lines. Normalized transcript expression values, denoted as nTPM, were obtained from the human protein map.

[0083] Figure 3 This demonstrates that the metabolic activation of acepromazine is the basis for its selective anticancer activity.

[0084] - Figure 3 A illustrates the workflow for preparing conditioned medium (CM).

[0085] - Figure 3B shows the effects of ACE, CM, and heated CM (for 3 days) on the activity of DLD-1 (treated with 10 ng / ml IFNγ). Data represent the means of three independent samples. Statistical significance was determined using one-way ANOVA followed by Dunnett's multiple comparison test.

[0086] - Figure 3 C represents the chemical reduction of ACE to ACE-OH.

[0087] - Figure 3 D shows the time-dependent conversion of ACE to ACE-OH in the indicated cell lines, as detected by TLC.

[0088] - Figure 3 E shows the separation of the indicated ACE-OH enantiomers by chiral column chromatography.

[0089] - Figure 3 F shows the concentration-response curves of (S)-ACE-OH and (R)-ACE-OH to the viability of the indicated cell lines with and without IFNγ (10 ng / ml). Data represent the mean ± sem of three independent samples.

[0090] - Figure 3 G shows a heatmap illustrating the expression levels of genes encoding aldehyde-ketone reductase in the indicated cell lines. Download the TPM (transcription per million reads) values ​​from DepMap.

[0091] - Figure 3 H shows the detection of ectopic expression of AKR1C1 / 2 / 3-3xFLAG in DLD-1 cells by anti-FLAG protein blotting and the detection of ACE to ACE-OH conversion by TLC. Representative results from two independent experiments are shown.

[0092] - Figure 3 Figure 1 shows the concentration-response curves of ACE on the viability of DLD-1 cells expressing AKR1C1 / 2 / 3-3xFLAG (treated with 10 ng / ml IFNγ). Data represent the mean ± sem of three independent samples.

[0093] Figure 4 The metabolic activation of acepromazine is shown to be its stereoselective active metabolite (S)-ACE-OH.

[0094] - Figure 4 A shows the mass spectrum of the extracted metabolites from A549 conditioned medium. The main peak corresponds to [M+ H] of ACE-OH. + .

[0095] - Figure 4 B shows the mass spectrum of the extracted metabolites from HCT-116 conditioned medium. The main peak corresponds to [M+ H] in ACE. + .

[0096] - Figure 4 C shows the synthesis of compound 3 from the inactive enantiomer of ACE-OH (compound 1).

[0097] - Figure 4 D shows the three-dimensional structure of compound 3 as determined by X-ray crystallography. Gray: carbon; white: hydrogen; blue: nitrogen; yellow: sulfur; red: oxygen; dark red: bromine.

[0098] - Figure 4 E shows the detection of ectopic expression of AKR1C1 / 2 / 3-3xFLAG in ME-180 cells by anti-FLAG protein blotting. Representative results from two independent experiments are shown.

[0099] - Figure 4 F shows the concentration-response curve of ACE on the viability of ME-180 cells expressing AKR1C1 / 2 / 3-3xFLAG. Data represent the mean ± sem of three independent samples.

[0100] - Figure 4 G presents a heatmap showing the median expression levels of genes encoding AKR1C1 / 2 / 3 in the indicated tumor types and normal tissues. Download the median TPM (transcription per million reads) value from GEPIA 2.

[0101] Figure 5 The anticancer activity of acepromazine mediated by interferon-induced TRIM21 was demonstrated.

[0102] - Figure 5 A scatter plot depicts genes with significantly enriched sgRNAs in A549-Cas9 cells treated with ACE (4–10 µM) and IFNγ (10 ng / ml) compared to IFNγ alone (10 ng / ml) (n = 3 / group) for 3 weeks. P-values ​​were calculated from a negative binomial model using a modified robust sorting aggregation algorithm via MAGeCK.

[0103] - Figure 5 B shows a frequency histogram of all sgRNAs and the sequence of sgRNAs targeting the indicated genes.

[0104] - Figure 5C shows the immunoblot of TRIM21 and β-actin in A549 cells pretreated with the indicated cytokine (10 ng / ml) with or without ACE (10 µM) for 12 h. Representative results from two independent experiments are shown.

[0105] - Figure 5 D shows the immunoblots of TRIM21 and β-actin in A549 cells of the indicated genotype with or without IFNγ (10 ng / ml) treatment for 12 h. Representative results from two independent experiments are shown.

[0106] - Figure 5 E shows the concentration-response curves of ACE for the viability of A549 cells with the indicated genotype, with and without IFNγ (10 ng / ml) treatment. Data represent the mean ± sem of three independent samples.

[0107] - Figure 5 F shows the molecular structure of TRIM21.

[0108] - Figure 5 G shows the crystal structure of the PRYSPRY domain of TRIM21, which is composited with Fc (modified from PDB 2IWG).

[0109] - Figure 5 H shows the immunoblot of TRIM21 and β-actin in A549-TRIM21 knockout (KO) cells rescued with the indicated TRIM21 cDNA. Representative results from two independent experiments are shown.

[0110] - Figure 5 Figure I shows the concentration-response curves of ACE versus the viability of A549-TRIM21 KO cells rescued with the indicated TRIM21 cDNA, with and without IFNγ (10 ng / ml) treatment. Data represent the mean ± sem of three independent samples.

[0111] Figure 6 The study demonstrated the anticancer activity of acepromazine mediated by TRIM21 in multiple cancer cell lines.

[0112] - Figure 6 A- Figure 6 B shows the concentration-response curves of ACE for the viability of SiHa and HuH-7 cells with the indicated genotypes, with and without IFNγ (10 ng / ml) treatment. Data represent the mean ± sem of three independent samples.

[0113] Figure 7This study demonstrates acepromazine-induced TRIM21-dependent degradation of nucleoporin.

[0114] - Figure 7 A- Figure 7 B shows a volcano plot, which depicts the log2 transformation mean fold change (quantified by label-free proteomics) and -log for each protein. 10 The conversion p-value was compared to (A) ACE (10 µM) versus treatment with the medium for 6 h; and (B) ACE (10 µM) versus treatment with ACE and bortezomib (100 nM) for 8 h for A549-TRIM21. D355A The analysis included three independent samples. The p-value was calculated using a Student's t-test (two-tailed).

[0115] - Figure 7 C shows the immunoblotting of the indicated protein in A549 cells pretreated with IFNγ for 8 h using (S)-ACE-OH or (R)-ACE-OH. Representative results from two independent experiments are shown.

[0116] - Figure 7 D shows a schematic cross-section of the NPC (created with Biorender) and the degree of nuclear porin depletion mapped to the cryo-electron tomography structure of the NPC (PDB: 7R5J).

[0117] - Figure 7 E shows a heatmap depicting the time-dependent depletion of nucleoporin in A549 stimulated with IFNγ treated with ACE (10 µM) (quantified by label-free proteomics).

[0118] - Figure 7 Image F shows a transmission electron microscopy image of A549 cells pretreated with IFNγ at the indicated time using ACE (10 µM). Abnormal cell nuclei are marked with red circles. Representative results from three independent experiments are shown. Scale bar: 10 µm.

[0119] - Figure 7 G shows confocal microscopy images of the nuclei (blue, stained with Hoechst 33342), TRIM21-EGFP (green), and NES-mCherry (red) in A549 cells. Cells were pretreated with bortezomib (100 nM) for 2 h, followed by ACE (10 µM) for 4 h, when indicated. Representative results from three independent experiments are shown. Scale bar: 10 µm.

[0120] - Figure 7 H shows a schematic diagram of the TRIM21-TurboID assay.

[0121] - Figure 7 I shows a volcano plot, which depicts the log2 transformation mean fold change (quantified by label-free proteomics) and -log for each protein. 10 The p-value was used to compare the TRIM21-TurboID enriched protein from A549 cells treated with ACE (20 µM) for 4 h with the medium. Three independent samples were included in the analysis. P-values ​​were calculated using a Student's t-test (two-tailed).

[0122] Figure 8 This study demonstrates that acepromazine induces TRIM21-dependent degradation of nucleoporin in multiple cancer cell lines.

[0123] - Figure 8 A shows the immunoblot of the indicated protein in ACE-sensitive or insensitive cell lines treated with the indicated concentration of ACE for 12 h with or without IFNγ (10 ng / ml) pretreatment. Representative results from two independent experiments are shown.

[0124] - Figure 8 B shows the immunoblot of the indicated protein in A549-TRIM21 KO cells treated with ACE (20 μM) for the indicated time.

[0125] - Figure 8 C shows the immunoblotting of the indicated proteins in A549 cells stimulated with IFNγ after 2 h of pretreatment with bortezomib (100 nM), MLN4924 (200 nM), or TAK243 (200 nM) followed by 12 h of ACE treatment (10 µM). Representative results from two independent experiments are shown.

[0126] Figure 9 The results show that NUP98 is the primary target of acepromazine.

[0127] - Figure 9 A illustrates the workflow of a CRISPR suppressor scan (created using Biorender).

[0128] - Figure 9 B shows a violin plot illustrating the log2 transformation fold change in mean abundance for each sgRNA, compared to A549-TRIM21 treated with ACE (dose escalating from 500 nM to 20 µM) for three weeks. D355A The analysis included three independent samples.

[0129] - Figure 9 C shows a scatter plot illustrating A549-TRIM21 treated with ACE for three weeks compared to the medium.D355A Log2 transformation fold change (y-axis) of the mean abundance of each NUP98-targeting sgRNA in cells. sgRNAs are arranged on the x-axis according to the amino acid positions in the NUP98-NUP96 coding sequence, corresponding to the predicted cleavage site positions. Data points represent the average of three independent samples.

[0130] - Figure 9 D illustrates the workflow for isolating ACE-resistant clones from A549-Cas9 cells transduced with sgRNA targeting NUP98 K752.

[0131] - Figure 9 E shows the concentration-response curves of ACE activity against NUP98 mutant clones pretreated with IFNγ (10 ng / ml). Data indicate the mean ± sem for three independent samples.

[0132] - Figure 9 F shows the immunoblotting of the indicated proteins in parental A549 cells and NUP98 mutant clones (R1-R5) stimulated with IFNγ and treated with ACE (10 μM) for 12 h. Representative results from two independent experiments are shown.

[0133] - Figure 9 G shows a volcano plot depicting the log2 transformation mean fold change (quantified by label-free proteomics) and -log for each protein. 10 The p-value was used to compare the TRIM21-TurboID enriched proteins from the NUP98 mutant clone (R1) treated with ACE (20 µM) for 4 h with the medium. Three independent samples were included in the analysis. P-values ​​were calculated using a Student's t-test (two-tailed).

[0134] - Figure 9 H shows a schematic diagram of the PML-GFP degradation determinant assay.

[0135] - Figure 9 I showed ACE against A549-TRIM21 D355A The effect of the indicated PML-GFP-NPC fusion protein levels in cells. Data are mean ± sem for six independent samples.

[0136] - Figure 9 J shows A549-TRIM21 treated with a medium or ACE (10 µM). D355A PML-GFP-NUP98 in cells 结构域2 (Green) Confocal micrograph. Scale bar: 10 µm.

[0137] - Figure 9 K shows NUP98 in the cryo-electron tomography structure of NPC (PDB: 7R5J). APD .

[0138] Figure 10 The deletion in NUP98APD was shown to lead to acepromazine resistance.

[0139] - Figure 10 A shows the cDNA sequence of the NUP98 mutant clone.

[0140] - Figure 10 B shows NUP98 APD The missing segment in the NUP98 mutant clone is highlighted in the structure (PDB: 2Q5Y).

[0141] - Figure 10 C shows a volcano plot, which depicts the log2 transformation mean fold change (quantified by label-free proteomics) and -log for each protein. 10 The p-value was compared between NUP98 mutant clones treated with ACE (10 µM) for 8 h and those treated with the medium (R1). Three independent samples were included in the analysis. P-values ​​were calculated using a Student's t-test (two-tailed).

[0142] - Figure 10 D shows ACE against A549-TRIM21. D355A The effect of the indicated PML-GFP-NUP98 fusion protein level in cells. Data are mean ± sem for six independent samples.

[0143] Figure 11 The crystal structure of the PRYSPRY domain of TRIM21D355A in complex with acepromazine and its metabolites is shown.

[0144] - Figure 11 A shows the relationship between ACE and its metabolites and TRIM21. D355A ITC titration and curve fitting of the PRYSPRY domain.

[0145] - Figure 11 B- Figure 11 D shows TRIM21 complexed with ACE (B), (S)-ACE-OH (C) and (R)-ACE-OH (D). D355A The overall structure of the PRYSPRY domain.

[0146] - Figure 11 E shows TRIM21 D355AThe superposition and binding sites of ACE (magenta), (S)-ACE-OH (cyan) and (R)-ACE-OH (green) within the PRYSPRY domain.

[0147] - Figure 11 F- Figure 11 H shows TRIM21 D355A A close-up view of the binding modes of ACE (F), (S)-ACE-OH (G) and (R)-ACE-OH (H) within the pocket of the PRYSPRY domain.

[0148] - Figure 11 I shows the superimposed ligand conformations of ACE (magenta), (S)-ACE-OH (cyan), and (R)-ACE-OH (green).

[0149] Figure 12 Characterization of the interaction between ACE and its metabolites and TRIM21 PRYSPRY is shown.

[0150] - Figure 12 A shows the relationship between ACE and its metabolites as measured by MST and TRIM21. WT The combination of the PRYSPRY structural domains is shown. The original MST trace and curve fitting are illustrated.

[0151] - Figure 12 B shows such an electron density plot, which illustrates TRIM21 D355A Details of the PRYSPRY domain through the region occupied by the indicated ligand. An omitted diagram of the Fo-Fc of ACE and its metabolites, plotted with 3σ contour lines.

[0152] Figure 13 This demonstrates the selective degradation of multimeric proteins in biomolecular condensates by TRIM21-directed PROTAC.

[0153] - Figure 13 A shows the chemical structure of TrimTAC1.

[0154] - Figure 13 B shows the expression of the indicated reporter protein A549-TRIM21 with or without TrimTAC1 (4 µM) or dBET1 (100 nM) for 4 h. D355A Or a representative image of A549-CRBN cells. Scale bar: 10 µM.

[0155] - Figure 13 C shows TrimTAC1 or dBET1 for A549-TRIM21 D355AConcentration-response curves of normalized intensity of the indicated reporter protein in A549-CRBN cells. Data indicate mean ± sem for three independent samples.

[0156] - Figure 13 D shows the A549-TRIM21 processed with TrimTAC1 (2 µM) at the indicated time. D355A NUP98 in cells FG -mEGFP-BRD4 BD2 The intensity of the sample was determined. Each point represents the mean of three independent samples. Statistical significance was determined using one-way ANOVA followed by Dunnett's multiple comparison test.

[0157] - Figure 13 E shows the indicated treatment of A549-TRIM21 with TrimTAC1 (4 µM), bortezomib (400 nM), and TAK243 (1.5 µM). D355A NUP98 in cells FG -mEGFP-BRD4 BD2 The normalized strength of the data is shown. The data indicate the mean ± sem of six independent samples. Statistical significance was determined using one-way ANOVA followed by Tuki multiple comparison test.

[0158] - Figure 13 F shows the chemical structure of TrimTAC2.

[0159] - Figure 13 G shows a representative image of the indicated FKBP12-mEGFP reporter protein after treatment with the medium or TrimTAC2 (10 μM) for 4 h. Scale bar: 10 µm.

[0160] - Figure 13 H illustrates a schematic representation of the selectivity of TrimTAC for multimeric proteins in biomolecular condensates.

[0161] Figure 14 The design and characterization of TrimTAC are shown.

[0162] - Figure 14 A shows the expression of NUP98 after pretreatment with a medium, bortezomib (400 nM) or TAK-243 (1.5 µM) for 2 h followed by treatment with TrimTAC1 (4 µM) for 4 h. FG -mEGFP-BRD4 BD2 A representative image of A549 cells. Scale bar:

[0163] - Figure 14B shows representative images of A549 cells expressing the indicated reporter protein, with and without TrimTAC2 (10 µM) treatment for 4 h. Scale bar: 10 µM.

[0164] - Figure 14 C shows the expression of NUP98 after pretreatment with a medium, bortezomib (200 nM) or TAK-243 (1 µM) for 2 h followed by treatment with TrimTAC2 (10 µM) for 4 h. FG Representative image of A549 cells with -mEGFP-FKBP12. Scale bar:

[0165] - Figure 14 D shows TrimTAC2 versus A549-TRIM21 D355A Concentration-response curves representing the intensity of the reporter protein in cells. Data indicate the mean ± sem for three independent samples.

[0166] - Figure 14 E shows the indicated treatment of A549-TRIM21 with TrimTAC2 (10 µM), bortezomib (200 nM), and TAK243 (1 µM). D355A NUP98 in cells FG The intensity of -mEGFP-FKBP12. Data indicate the mean ± sem for six independent samples. Statistical significance was determined using one-way ANOVA followed by Tuki multiple comparison test. Detailed Implementation

[0167] definition

[0168] Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0169] As used herein (including the appended claims), unless the context clearly indicates otherwise, singular terms such as “a,” “an,” and “the” include their corresponding plural indicators.

[0170] The term "alkyl" refers to a hydrocarbon group selected from straight-chain and branched saturated hydrocarbon groups containing 1 to 18 carbon atoms (e.g., 1 to 12, further such as 1 to 10, even further such as 1 to 8, or 1 to 6, or 1 to 4). Examples of alkyl groups containing 1 to 6 carbon atoms (i.e., C1-6 alkyl) include, but are not limited to, methyl, ethyl, 1-propyl or n-propyl ("n-Pr"), 2-propyl or isopropyl ("i-Pr"), 1-butyl or n-butyl ("n-Bu"), 2-methyl-1-propyl or isobutyl ("i-Bu"), 1-methylpropyl or sec-butyl ("s-Bu"), 1,1-dimethylethyl or tert-butyl ("t-Bu"). ), 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, and 3,3-dimethyl-2-butyl. The alkyl group may optionally be rich in deuterium, for example, -CD3, -CD2CD3, etc.

[0171] The term "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), and iodine (I).

[0172] The term "haloalkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced by one or more halogen atoms (such as fluorine, chlorine, bromine, and iodine). Examples of haloalkyl groups include haloC1-8 alkyl, haloC1-6 alkyl, or haloC1-4 alkyl, but are not limited to -CF3, -CH2Cl, -CH2CF3, -CCl2, CF3, etc.

[0173] The term "alkyloxy" or "alkoxy" refers to an alkyl group as defined above, which is attached to a portion of the parent molecule by an oxygen atom. Examples of alkoxy groups, such as C1-6 alkyloxy or C1-4 alkyloxy groups, include, but are not limited to, methoxy, ethoxy, isopropoxy, propoxy, n-butoxy, tert-butoxy, pentoxy, and hexoxy.

[0174] The term "alkoxy-alkyl-" refers to an alkyl group as defined above that is further substituted with an alkoxy group as defined above. Examples of alkoxy-alkyl- (e.g., C1-8 alkoxy-C1-8 alkyl- or C1-6 alkoxy-C1-6 alkyl-) include, but are not limited to, methoxymethyl, ethoxymethyl, ethoxyethyl, isopropoxymethyl, or propoxymethyl.

[0175] The term "alkenyl" in this document refers to a hydrocarbon group selected from straight-chain and branched hydrocarbon groups containing at least one C=C double bond and 2 to 18 (e.g., 2 to 8, further such as 2 to 6) carbon atoms. Examples of alkenyl (e.g., C2-6 alkenyl) include, but are not limited to, ethenyl, propenyl, propenyl, 2-methylpropenyl, butenyl, butenyl, butenyl, 3-alkenyl, butenyl, 2-methylbutenyl, hexenyl, hexenyl, 3-alkenyl, hexenyl, 4-alkenyl, and hexenyl, 1,3-dienyl.

[0176] The term "alkynyl" in this document refers to a hydrocarbon group selected from straight-chain and branched hydrocarbon groups, containing at least one C≡C triple bond and 2 to 18 (e.g., 2 to 8, further e.g., 2 to 6) carbon atoms. Examples of alkynyl groups (e.g., C2-6 alkynyl) include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl (propynyl), 1-butynyl, 2-butynyl, and 3-butynyl.

[0177] The term "cycloalkyl" refers to a hydrocarbon group selected from saturated cyclic hydrocarbon groups, including monocyclic and polycyclic (e.g., bicyclic and tricyclic) groups, including fused, bridged, or spirocycloalkyl groups.

[0178] For example, a cycloalkyl group may contain 3 to 12 carbon atoms (e.g., 3 to 10, further, 3 to 8, further, 3 to 6, 3 to 5, or 3 to 4). Even further, for example, the cycloalkyl group may be selected from monocyclic groups containing 3 to 12 carbon atoms (e.g., 3 to 10, further, 3 to 8, 3 to 6). Examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohexyl-1-enyl, 1-cyclohexyl-2-enyl, 1-cyclohexyl-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, and cyclododecyl. Specifically, examples of saturated monocyclic cycloalkyl groups (e.g., C3-8 cycloalkyl groups) include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. In a preferred embodiment, the cycloalkyl group is a monocyclic ring (abbreviated as C3-6 cycloalkyl) containing 3 to 6 carbon atoms, including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Examples of bicyclic cycloalkyl groups include those having 7 to 12 ring atoms arranged in a fused bicyclic arrangement selected from the [4,4], [4,5], [5,5], [5,6], and [6,6] ring systems, or bridged bicyclic groups selected from bicyclic [2.2.1]heptane, bicyclic [2.2.2]octane, and bicyclic [3.2.2]nonane. Further examples of bicyclic cycloalkyl groups include those bicyclic arrangements selected from the [5,6] and [6,6] ring systems.

[0179] The term "aryl" used alone or in combination with other terms refers to a group selected from the following:

[0180] -5 and 6-membered carbon rings, aromatic rings, such as phenyl;

[0181] - Bicyclic ring systems, such as 7- to 12-membered bicyclic ring systems, wherein at least one ring is a carbocyclic and aromatic, such as naphthyl and indanyl; and

[0182] - Tricyclic ring systems, such as 10- to 15-membered tricyclic ring systems, in which at least one ring is a carbocyclic ring and an aromatic ring, such as fluorene.

[0183] The terms "aromatic ring" and "aryl" are used interchangeably throughout this disclosure. In some embodiments, the monocyclic or bicyclic aromatic ring has 5 to 10 cyclic carbon atoms (i.e., C5-10 aryl). Examples of monocyclic or bicyclic aromatic rings include, but are not limited to, phenyl, naphth-1-yl, naphth-2-yl, anthraceneyl, phenanthrene, etc. In some embodiments, the aromatic ring is a naphthyl ring (naphth-1-yl or naphth-2-yl) or a phenyl ring. In some embodiments, the aromatic ring is a phenyl ring.

[0184] The term "heteroaryl" in this article refers to groups selected from the following:

[0185] -5, 6 or 7-membered aromatic monocyclic rings containing at least one heteroatom, such as 1 to 4 heteroatoms, or in some embodiments 1 to 3 heteroatoms, or in some embodiments 1 to 2 heteroatoms, wherein the heteroatoms are selected from nitrogen (N), sulfur (S) and oxygen (O), and the remaining ring atoms are carbon;

[0186] - A 7- to 12-membered bicyclic ring comprising at least one heteroatom, for example, 1 to 4 heteroatoms, or in some embodiments 1 to 3 heteroatoms, or in other embodiments 1 or 2 heteroatoms, wherein the heteroatom is selected from nitrogen, oxygen, or optionally oxidized sulfur (as one or more ring members), the remaining ring atoms are carbon, and wherein at least one ring is aromatic and at least one heteroatom is present in the aromatic ring; and

[0187] -11 to 14-membered tricyclic rings containing at least one heteroatom, such as 1 to 4 heteroatoms, or in some embodiments 1 to 3 heteroatoms, or in other embodiments 1 or 2 heteroatoms, wherein the heteroatom is selected from nitrogen, oxygen or optionally oxidized sulfur (as one or more ring members), the remaining ring atoms are carbon, and wherein at least one ring is aromatic and at least one heteroatom is present in the aromatic ring.

[0188] When the total number of S and O atoms in a heteroaryl group exceeds 1, those heteroatoms are not adjacent to each other. In some embodiments, the total number of S and O atoms in a heteroaryl group is no greater than 2. In some embodiments, the total number of S and O atoms in an aromatic heterocycle is no greater than 1. When a heteroaryl group contains more than one heteroatom ring member, the heteroatoms can be the same or different. The nitrogen atom in one or more rings of a heteroaryl group can be oxidized to form N-oxides.

[0189] The terms "aromatic heterocycle" and "heteroaryl" are used interchangeably throughout this disclosure. In some embodiments, the monocyclic or bicyclic aromatic heterocycle has 5, 6, 7, 8, 9, or 10 cyclic members, wherein 1, 2, 3, or 4 heteroatomic ring members are independently selected from nitrogen (N), sulfur (S), and oxygen (O), and the remaining ring members are carbon. In some embodiments, the monocyclic or bicyclic aromatic heterocycle is a monocyclic or bicyclic ring comprising 1 or 2 heteroatomic ring members independently selected from nitrogen (N), sulfur (S), and oxygen (O). In some embodiments, the monocyclic or bicyclic aromatic heterocycle is a 5- to 6-membered heteroaryl ring, wherein the heteroaryl ring is monocyclic and has 1 or 2 heteroatomic ring members independently selected from nitrogen (N), sulfur (S), and oxygen (O). In some embodiments, the monocyclic or bicyclic aromatic heterocycle is an 8- to 10-membered heteroaryl ring, wherein the heteroaryl ring is bicyclic and has one or two heteroatom ring members independently selected from nitrogen, sulfur, and oxygen.

[0190] Examples of heteroaryl or monocyclic or bicyclic aromatic heterocycles include, but are not limited to (as numbered starting from the connection position specified in priority 1), pyridyl (e.g., 2-pyridyl, 3-pyridyl, or 4-pyridyl), cyclophosphino, pyrazinyl, 2,4-pyrimidinyl, 3,5-pyrimidinyl, 2,4-imidazolyl, imidazopyridyl, isoxazolyl, oxazolyl, thiazolyl, isothiazolyl, thiadiazolyl (e.g., 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, or 1,3,4-thiadiazolyl). Tetraazolyl, thiophene (e.g., thiophene-2-yl, thiophene-3-yl), triazinyl, benzothiophene, furanyl (furyl or furanyl), benzofuranyl, benzimidazolyl, indoleyl, isoindoleyl, indolinyl, oxadiazolyl (e.g., 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl or 1,3,4-oxadiazolyl), phthalazinyl, pyrazinyl, pyridazinyl, pyrroleyl, triazolyl (e.g., 1,2,3-triazolyl, 1,2,4-triazolyl or 1, 3,4-triazolyl), quinolinyl, isoquinolinyl, pyrazolyl, pyrrolopyridyl (e.g., 1H-pyrrolo[2,3-b]pyridin-5-yl), pyrazolopyridyl (e.g., 1H-pyrazolo[3,4-b]pyridin-5-yl), benzoxazolyl (e.g., benzo[d]oxazol-6-yl), pteridinyl, purineyl, 1-oxa-2,3-diazolyl, 1-oxa-2,4-diazolyl, 1-oxa-2,5-diazolyl, 1-oxa-3,4-diazolyl, 1-thio The compounds include 2,3-diazolyl, 1-thia-2,4-diazolyl, 1-thia-2,5-diazolyl, 1-thia-3,4-diazolyl, furazanyl (e.g., furazan-2-yl, furazan-3-yl), benzofurazanyl, benzothiophene, benzothiazolyl, benzooxazolyl, quinazolinyl, quinoxolinyl, naphthidyl, furan-pyridinyl, benzothiazolyl (e.g., benzo[d]thiazolyl-6-yl), indazole (e.g., 1H-indazole-5-yl), and 5,6,7,8-tetrahydroisoquinoline.

[0191] "Heterocyclic group", "heterocyclic" or "heterocyclic" are interchangeable and refer to a non-aromatic heterocyclic group containing one or more heteroatoms selected from nitrogen, oxygen or optional oxidized sulfur as ring members, the remaining ring members being carbon, including monocyclic, fused, bridged and spirocyclic, i.e. monocyclic heterocyclic groups, bridged heterocyclic groups, spirocyclic groups and fused heterocyclic groups.

[0192] The term "monocyclic heterocyclic group" refers to a monocyclic group in which at least one ring member is a heteroatom selected from nitrogen, oxygen, or optionally oxidized sulfur. The heterocycle can be saturated or partially saturated.

[0193] Exemplary monocyclic 4- to 9-membered heterocyclic groups include, but are not limited to (as numbered from the connection position specified as priority 1), pyrrolid-1-yl, pyrrolid-2-yl, pyrrolid-3-yl, imidazolidine-2-yl, imidazolidine-4-yl, pyrrolid-2-yl, pyrrolid-3-yl, piperidin-1-yl, piperidin-2-yl, piperidin-3-yl, piperidin-4-yl, 2,5-piperazinyl, pyranyl, morpholinyl, morpholinoyl, morpholin-2-yl, morpholin-3-yl, ethylene oxide, aziridine-1-yl -yl, aziridin-2-yl, azircyclic octane-1-yl, azircyclic octane-2-yl, azircyclic octane-3-yl, azircyclic octane-4-yl, azircyclic octane-5-yl, cyclothioethane, azircyclic butane-1-yl, azircyclic butane-2-yl, azircyclic butane-3-yl, oxacyclobutane, thiohexacyclobutane, 1,2-dithiohexacyclobutane, 1,3-dithiohexacyclobutane, dihydropyridine, tetrahydropyridyl, thiomorpholinyl, thiazolyl, piperazinyl, high piperazinyl, high Piperidinyl, aziridine-1-yl, aziridine-2-yl, aziridine-3-yl, aziridine-4-yl, oxacycloheptyl, thiocycloheptyl, 1,4-oxathiacycloheptyl, 1,4-dioxacycloheptyl, 1,4-oxathiocycloheptyl, 1,4-oxaazacycloheptyl, 1,4-dithiocycloheptyl, 1,4-thioazacycloheptyl and 1,4-diazacycloheptyl, 1,4-dithiacycloheptyl, 1,4-azithiocyclohexyl, oxacycloheptyl Zollyl, diazazollyl, thiazollyl, dihydrothiophenyl, dihydropyranyl, dihydrofuranyl, tetrahydrofuranyl, tetrahydrothiophenyl, tetrahydropyranyl, tetrahydrothiaranyl, 1-pyrrolinyl, 2-pyrrolinyl, 3-pyrrolinyl, dihydroindolyl, 2H-pyranyl, 4H-pyranyl, 1,4-dioxane, 1,3-dioxolane, pyrazolinyl, pyrazolane, dithiane, dithiopentane, pyrazolane, imidazolinyl, pyrimidinoneyl, or 1,1-dioxo-thiomorpholinyl.

[0194] "Pharmaceutically acceptable salt" means a salt that, to the extent of reliable medical judgment, is suitable for contact with tissues of humans and lower animals without excessive toxicity, irritation, allergic reactions, etc., and is proportionate to a reasonable benefit / risk ratio. Pharmaceutically acceptable salts can be prepared in situ during the final isolation and purification of the compounds disclosed herein, or by reacting a free base functional group with a suitable organic acid or by reacting an acidic group with a suitable base.

[0195] Furthermore, if the compound disclosed herein is obtained as an acid addition salt, the free base can be obtained by alkalizing a solution of the acid salt. Conversely, if the product is a free base, the addition salt (such as a pharmaceutically acceptable addition salt) can be produced by following conventional procedures for preparing acid addition salts from base compounds, by dissolving the free base in a suitable organic solvent and treating said solution with acid. Those skilled in the art will recognize that non-toxic, pharmaceutically acceptable addition salts can be prepared using various synthetic methods without excessive experimentation.

[0196] As defined herein, “pharmaceutically acceptable salts” include salts of at least one compound of formula (I) and salts of stereoisomers of the compound of formula (I), such as enantiomer salts and / or diastereomer salts.

[0197] The term "effective amount" or "therapeutic effective amount" refers to an amount of an active ingredient (such as a compound) that, when administered to a subject for the purposes disclosed herein for degradation or for the treatment of at least one clinical symptom of a disease or disorder, is sufficient to affect such treatment of said disease, disorder, or symptom. "Therapeutic effective amount" can vary depending on: the compound, the disease, disorder, and / or the symptoms of the disease or disorder, the severity of the disease, disorder, and / or the symptoms of the disease or disorder, the age of the subject to be treated, and / or the weight of the subject to be treated. In any given case, the appropriate amount may be apparent to those skilled in the art or may be determined by routine experiments. In some embodiments, "therapeutic effective amount" is the amount of at least one compound disclosed herein and / or at least one stereoisomer thereof and / or at least one pharmaceutically acceptable salt thereof that is effective in "treating" (as defined above) a subject's disease or disorder. In the case of combination therapy, "therapeutic effective amount" refers to the total amount of the combination of substances used to effectively treat the disease, disorder, or symptom.

[0198] Molecular glues can enhance the formation of complexes between E3 ligases and target proteins through binding at protein-protein interfaces, and are generally defined as small molecules that interact with the surfaces of two proteins to induce or enhance the affinity of the two proteins for each other and then induce the degradation of the target protein.

[0199] PROTACs are bifunctional molecules consisting of a recruitment ligand for a ubiquitin ligase (e.g., E3 ubiquitin ligase) linked via a linker and a ligand for targeting endogenous proteins. The function of this bifunctional molecule is to recruit the target protein to the E3 ubiquitin ligase for degradation. In some embodiments, compounds of formula (I) or (II) act as the recruitment ligand for the TRIM21 ubiquitin ligase in the PROTAC compound.

[0200] Target proteins are defined as multimeric proteins that cause disease in humans. Examples include, but are not limited to, NUP98-fusion protein, EWS-FLI1, Tau fibrils, α-synuclein fibrils, mutant huntingtin protein, cGAS, and NLRP3. A multimer refers to multiple copies of a gene-encoded polypeptide that form a complex.

[0201] "Target protein" or "protein" means any protein involved in the metabolism or catabolism of the subject's cells and / or organs, including, in particular, proteins that regulate disease states or conditions to be treated with the relevant compounds disclosed herein. Target proteins may also include proteins derived from microorganisms such as bacteria, viruses, fungi, and protozoa. Typically, target proteins can include, for example, structural proteins; receptors; enzymes; cell surface proteins; and proteins involved in the integration of cellular functions, including those involved in: catalysis, aromatase activity, motility activity, helicase activity, metabolic processes (such as anabolism and catabolism), antioxidant activity, proteolysis, biosynthesis, kinase activity, oxidoreductase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, ligase activity, enzyme regulator activity, signal transduction activity, structural molecule activity, binding activity (to proteins, lipids, or carbohydrates), receptor activity, cell motility, membrane fusion, cell communication, regulation of biological processes, development, cell differentiation, response to stimuli, behavioral proteins, cell adhesion proteins, proteins involved in cell death, proteins involved in transport (including protein transporter activity, nuclear transporter activity, ion transporter activity, channel transporter activity, carrier activity, permease activity, secretory activity, electron transporter activity), pathogenesis, molecular chaperone regulator activity, nucleic acid binding activity, transcription regulator activity, extracellular tissue and biogenetic activity, and translation regulator activity.

[0202] Amyloid proteins are aggregates of proteins characterized by their fibrillary morphology and a β-sheet secondary structure called cross-β. Amyloid-like aggregates refer to a diverse group of protein aggregates that are similar to amyloid proteins but may lack the cross-β structure. Amyloid proteins and amyloid-like aggregates are highly ordered protein structures that form when previously healthy proteins undergo misfolding and lose their normal structure and physiological function. Amyloid proteins or amyloid-like aggregates are associated with human diseases including Alzheimer's disease, Huntington's disease, Parkinson's disease, type 2 diabetes, and many amyloidosis disorders caused by the systemic or localized deposition of amyloid fibrils in the intracellular and extracellular spaces of tissues and organs. Examples of amyloid-like aggregates include, but are not limited to, Tau, α-synuclein, and mutant Huntington's protein.

[0203] Protein multiplication refers to the ability of proteins to interact and thus form complexes with more than one monomer or unit.

[0204] Target proteins can include proteins from eukaryotes and prokaryotes (including humans and other animals, microorganisms, plants and viruses, among many other sources).

[0205] Non-restricted examples of target proteins include B7, B7-1, and B7-2 (which provide a second signal to T cells), TINFRlm, BET bromo domain, TNFR2, NADPH oxidase, BclIBax and other mates in the apoptosis pathway, C5a receptor, HMG-CoA reductase, PDE V phosphodiesterase type, PDE IV phosphodiesterase type 4, PDE I, PDE II, PDE III, squalene cyclase inhibitors, CXCR1, CXCR2, nitric oxide (NO) synthase, cyclooxygenase 1, cyclooxygenase 2, 5HT receptor, dopamine receptor, G protein (Gq), histamine receptor, 5-lipoxygenase, trypsin-like serine protease, thymidylate synthase, purine nucleoside phosphorylase, trypanosome GAPDH, glycogen phosphorylase, carbonic anhydrase, chemokine receptor, JAW STAT, RXR and its analogues, HIV 1 protease, HIV 1. Integrase, influenza ceramidinase, hepatitis B reverse transcriptase, sodium channels, protein P-glycoprotein (and MRP), tyrosine kinase, CD23, CD124, tyrosine kinase p56 lek, CD4, CD5, IL-2 receptor, IL-1 receptor, TNF-αR, ICAM1, Cat+ channels, VCAM, VLA-4 integrin, selectins, CD40 / CD40L, newokmms and receptors, mosme monophosphate dehydrogenase, p38 MAP kinase, RaslRaflMEWERK pathway, interleukin-1 convertase, caspase, HCV, NS3 protease, HCVNS3 RNA helicase, glycine ribonucleotide formyltransferase, rhinovirus 3C protease, herpes simplex virus-1 (HSV-I), protease, cytomegalovirus (CMV) protease, poly(ADP-ribose) polymerase, cyclin-dependent kinase, vascular endothelial growth factor, oxytocin receptor, microsomal transferin inhibitor, bile acid transport inhibitor, 5α-reductase inhibitor, angiotensin I1, glycine receptor, norepinephrine reuptake receptor, endothelin receptor, neuropeptide Y and its receptor, adenosine receptor, adenosine kinase and AMP deaminase, purinergic receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2X1-7), farnesyltransferase, geranylgeranyl transferase, TrkA of NGF α-receptor, β-amyloid protein, tyrosine kinase Flk-IIKDR, porphyrin receptor, integrin receptor, Her-21neu, telomerase inhibitor, tumor-associated protein (TMP), Bcr-Abl tyrosine kinase, cytosolic phospholipase A2, and EGF receptor tyrosine kinase. Other protein targets include, for example, ecdysone 20-monooxygenase, GABA-gated chloride ion channels, acetylcholinesterase, voltage-sensitive sodium channel proteins, calcium release channels, and chloride ion channels.Other target proteins include acetyl-CoA carboxylase, adenylate succinate synthase, protoporphyrinogen oxidase, and enolpyruvate shikimic acid synthase. Exemplary target proteins include, for example, drug resistance proteins and multidrug resistance (MDR) proteins.

[0206] A fusion protein is a protein formed by linking some or all of two or more proteins (such as the target proteins mentioned above).

[0207] A "multimeric protein" is a protein composed of two or more individual polypeptide chains (which combine to form a single protein), such as the target protein mentioned above. The polypeptide chains can be non-covalently or covalently coupled, for example, via disulfide bonds. Multimeric proteins can be homomeric (same polypeptide chain) or heteromeric (different polypeptide chains).

[0208] The “subject” can be a human, a non-human mammal, or a bird. Non-human mammals include, for example, livestock and pets such as sheep, cattle, pigs, dogs, cats, and rodents. Preferably, the subject is a human.

[0209] Example

[0210] In the following examples, Examples 1 to 6 illustrate in detail how ACE can induce TRIM21 to be recruited to the vicinity of NPC to trigger the degradation of nuclear porin. Example 7 further confirms that two TRIM21-directed PROTACs carrying the ACE moiety as a ligand for recruiting TRIM21 ligases selectively degrade multimeric proteins in biomolecular condensates. Examples 8 and 9 prepared two TRIM21-directed PROTACs, namely TrimTAC1 and TrimTAC2. Finally, Example 11 lists the (biological) materials, assays, and methods used in this invention.

[0211] Example 1: Acepromine exhibits interferon-enhanced selective anticancer activity

[0212] Interferon-γ (IFNγ) is a key inflammatory cytokine that coordinates multiple anti-tumor responses in the tumor microenvironment, and the presence of IFNγ can be used to identify small molecules that have enhanced cytotoxicity against cancer cells.

[0213] An in vitro competitive cell growth assay was designed using the lung adenocarcinoma cell line A549: First, IFNGR1 (a subunit encoding the IFNγ receptor) was deleted from A549 cells using CRISPR / Cas9, resulting in a pool of cells that could not sense IFNγ. Figure 2A); Second, histone H2B-GFP and H2B-mCherry fusion proteins were expressed in wild-type (WT) and IFNGR1-deficient A549 cells, respectively; Third, the two engineered cell populations were mixed at a 1:1 ratio and treated with IFNγ and small molecules (81,845 compounds, 10 µM each) from an internal chemical library, and the change in the ratio of green to red cell nuclei was monitored after four days. Figure 1 A).

[0214] Among approximately 80,000 small drug-like molecules (at 10 µM), three compounds (acepromazine (hereinafter referred to as ACE), acetaminophen, and piperazine) were identified as selectively reducing the fitness of WT A549 cells relative to IFNGR1-deficient cells (expressed as the ratio of reduced green nuclei to reduced red nuclei). Figure 1 B to Figure 1 D). These three seed molecules share a common acetyl-substituted phenothiazine core, which is linked to different amine-containing moieties via a propyl chain (D). Figure 1 C). By measuring A549 activity, it was found that both IFNγ and interferon β (IFNβ) enhanced ACE cytotoxicity, but tumor necrosis factor α (TNFα) did not enhance ACE cytotoxicity. Figure 1 E). The natural killer (NK) cell line NK92-MI, which secretes IFNγ upon encountering cancer cells, was used to mimic the source of IFNγ production in the tumor microenvironment. Co-culturing with NK92-MI enhanced the cytotoxicity of ACE against A549 cells. This enhancement was lost in IFNGR1-deficient A549 cells. Figure 1 F- Figure 1 G and Figure 2 B).

[0215] ACE was an antipsychotic drug used in humans during the 1950s, but is currently used only as a sedative in veterinary medicine. The behavioral effects of ACE are primarily attributed to antagonism of postsynaptic dopamine receptors. However, chlorpromazine ( Figure 2 C) As a closely related analogue, it is still used as an antipsychotic in humans, but unlike acepromazine, acetaminophen, and piperacillin, it does not have a carbonyl group on its tricyclic fused ring and has not shown interferon-enhanced anticancer activity. Figure 2 D).

[0216] Similar assays were performed to examine the cytotoxicity of six cancer cell lines (two cervical cancer cell lines, HeLa and SiHa; one hepatocellular carcinoma cell line, HuH-7; one lymphoma cell line, Mino; one colorectal cancer cell line, DLD-1; and one cervical cancer cell line, ME-180), one normal cell line, and activated T lymphocytes from two healthy donors. IFNγ-enhanced cytotoxicity was observed in the two cervical cancer cell lines (HeLa and SiHa) and the hepatocellular carcinoma cell line (HuH-7). In contrast, no IFNγ-enhanced cytotoxicity was observed in the lymphoma cell line (Mino), the colorectal cancer cell line (DLD-1), and the cervical cancer cell line (ME-180). Furthermore, the non-cancerous keratinocyte cell line HaCat and activated T lymphocytes from healthy donors also exhibited resistance to IFNγ-enhanced ACE cytotoxicity. In summary, these data reveal that ACE possesses interferon-enhanced anticancer activity in cancer cell line subsets.

[0217] Furthermore, ACE-sensitive cancer cell lines do not express dopamine receptors ( Figure 2 E). These findings suggest that ACE exerts its anticancer activity independently of dopamine receptors.

[0218] Example 2: Metabolic activation of acepromazine is the basis for its selective anticancer activity.

[0219] The sensitive cell line A549 was treated with ACE, and then the insensitive cell line DLD-1 was treated with conditioned medium, such as... Figure 3 As shown in Figure A. The results showed that untreated ACE had no cytotoxicity against DLD-1, while ACE conditioned with A549 exhibited potent cytotoxicity. Heating did not inactivate the cytotoxic activity in the conditioned medium, indicating that the active ingredient is a small molecule metabolite (…). Figure 3 B). Therefore, total metabolites were extracted from the conditioned media of ACE-sensitive and ACE-insensitive cell lines and separated by thin-layer chromatography (TLC). UV irradiation of TLC revealed a time-dependent conversion of ACE to slower-migrating species in sensitive cell lines (A549 and HeLa), but no such conversion was observed in ACE-insensitive cell lines (DLD-1 and HCT-116). Figure 3 C).

[0220] Since the carbonyl group of ACE is known to be readily reduced to ACE-OH (hydroxyethylpropazine), ACE-OH was prepared in the laboratory and used as a reference. The migration patterns of slower-migrating species matched those of ACE-OH. Figure 3 C). Furthermore, liquid chromatography-mass spectrometry analysis confirmed that the new species differed from ACE by 2 Daltons, corresponding to the addition of two hydrogen atoms (C). Figure 4 A- Figure 4B). These results reveal that sensitive cancer cell lines selectively convert ACE to ACE-OH.

[0221] The conversion of ACE to ACE-OH produces a chiral center. Since enantiomers of chiral drugs can have reducing, no, or even harmful effects, chiral column chromatography was used to separate the racemic mixture of ACE-OH obtained by reducing ACE with LiAlH4 into two enantiomer fractions. Figure 3 D). Crystallization was facilitated by chemical derivatization, followed by X-ray diffraction, and the absolute conformations and optical activities of the two enantiomers were determined to be (S)-(-)-ACE-OH and (R)-(+)-ACE-OH. Figure 4 C- Figure 4 D). A549 and DLD-1 cells were treated with purified ACE-OH enantiomers (i.e., (S)-(-)-ACE-OH and (R)-(+)-ACE-OH). (S)-ACE-OH exhibited enhanced IFNγ toxicity in both cell lines. In contrast, (R)-ACE-OH was inactive in both cell lines. Figure 3 E). In summary, (S)-ACE-OH is the active metabolite of ACE.

[0222] Aldehyde-ketone reductase (AKR) family enzymes catalyze the reduction of ketones to alcohols. Expression levels of genes encoding aldehyde-ketone reductases were compared in ACE-activated versus non-activated cell lines, and high levels of AKR1C1, AKR1C2, and AKR1C3 were found to be associated with ACE activation. Figure 3 F). AKR1C1, AKR1C2, or AKR1C3 were expressed individually in DLD-1 cells, and it was found that they all enabled DLD-1 to convert ACE to ACE-OH (F). Figure 3 G). Furthermore, expression of AKR1C1, AKR1C2, or AKR1C3 makes DLD-1 cells more sensitive to ACE (G). Figure 3 H). Similar results were obtained in another ACE-insensitive cell line, ME-180. Figure 4 E- Figure 4 F). Notably, AKR1C1 / 2 / 3 is overexpressed in cholangiocarcinoma, hepatocellular carcinoma, and squamous cell carcinoma of the lung, but not in normal tissues. Figure 4 G). These observations not only explain the observed anticancer selectivity of ACE, but also suggest that tumor-associated AKR1C1 / 2 / 3 activation of ACE may be a strategy for enriching cytotoxic metabolites in tumors.

[0223] Example 3: Interferon-induced TRIM21-mediated anticancer activity of acepromazine

[0224] This example investigated the mechanism by which interferon enhances the anticancer activity of ACE. A genome-wide CRISPR-Cas9 knockout screening was performed by targeting 19,114 genes with four separate sgRNAs per gene. A549 cells transduced with sgRNA were treated with IFNγ or IFNγ plus a sublethal dose of ACE for three weeks. Genomic DNA was then isolated from surviving cells and next-generation sequencing was performed to measure the abundance of each sgRNA. Using the MAGeCK algorithm, the top six enriched genes were identified, including five involved in IFNγ sensing and signal transduction (IFNGR1, IFNGR2, JAK1, JAK2, and STAT1) and TRIM21 (…). Figure 5 A- Figure 5 B).

[0225] TRIM21 encodes an E3 ubiquitin ligase containing a three-part motif, and its expression is induced by interferon. Western blot analysis of A549 lysates confirmed that TRIM21 is induced by IFNβ or IFNγ, rather than by TNFα. Figure 5 C).

[0226] The following assays in this embodiment confirmed that interferon enhances the anticancer activity of ACE by inducing TRIM21 expression. Two A549 TRIM21 molecules were generated. - / - Cloning. Furthermore, TRIM21 was overexpressed in A549 cells (TRIM21). OE ) to mimic interferon-induced TRIM21 expression ( Figure 5 D). Measured cell viability showed that loss of TRIM21 induced resistance to ACE in A549 cells, while TRIM21... OE This leads to increased sensitivity to ACE, exceeding the effect of IFNγ. Figure 5 E). Similar results were obtained in two other ACE-sensitive cell lines (SiHa and HuH-7). Figure 6 A- Figure 6 B). These results confirm that interferon-induced TRIM21 mediates the anticancer activity of ACE.

[0227] TRIM21 is an innate immune sensor that recognizes cytosolic antibody-coated viruses to trigger an antiviral response. The C-terminal PRYSPRY domain of TRIM21 has a hydrophobic pocket in the crystallizable fragment (Fc) region that binds antibodies. Figure 5 F). Select three residues in this pocket that are important for Fc binding and make alanine substitution mutants (D355A, W381A, and W383A) in TRIM21. - / - Cellular expression ( Figure 5G). The W381A and W383A mutants did not restore ACE sensitivity. In contrast, the D355A mutant showed increased ACE sensitivity compared to WTTRM21. Figure 5 H). The identification of both loss-of-activity and gain-of-activity mutations indicates that TRIM21 PRYSPRY This pocket in the protein is involved in protein-compound or protein-protein interactions that mediate ACE activity.

[0228] Example 4: Acetaminophen-induced TRIM21-dependent degradation of nucleoporin

[0229] This example confirms that ACE functions by guiding TRIM21 to degrade some proteins required for cell viability, because TRIM21 has an N-terminal RING domain with E3 ubiquitin ligase activity.

[0230] Each of the following nucleoporins (GLE1, NUP35, SMPD4, NUP155, NUP214, AAAS, NUP88, and NUP210) was found to be degraded by at least 50% during ACE treatment at time points of 4, 6, 8, and 12 hours. Specifically, label-free quantitative proteomics was used to compare A549 TRIM21 (D355A) treated with the medium or ACE. OE Cellular proteome ( Figure 7 A). In the list above, several nuclear porins (NUP35, NUP155, SMPD4, and GLE1) were found to be significantly depleted after ACE treatment.

[0231] This phenomenon was also observed in ACE-sensitive cell lines, but not in ACE-insensitive or TRIM21-deficient cell lines. Figure 8 A- Figure 8 B).

[0232] Furthermore, inhibition of the proteasome (via bortezomib treatment) or E1 ubiquitin activator (via TAK243 treatment) prevented ACE-induced depletion of these proteins. Figure 7 B and Figure 8 C). The following section confirms that ACE-OH does indeed induce nucleoporin degradation in a stereoselective manner, because ACE is an inactive prodrug, and only its metabolite (S)-ACE-OH possesses anticancer activity. Indeed, (S)-ACE-OH, rather than (R)-ACE-OH, triggers the degradation of NUP35, NUP155, SMPD4, and GLE1 in IFNγ-stimulated A549 cells. Figure 7 C).

[0233] The nuclear pore complex (NPC) consists of approximately 1000 building blocks, which are assembled from copies of approximately 30 proteins and organized into three stacked loops (inner loop, cytoplasmic loop, and nuclear loop) that are laterally attached to cytoplasmic filaments and nuclear baskets. Figure 7 D). By monitoring the abundance of each nuclear porin in ACE-treated A549 cells over time, it was found that the inner loop of NPCs was rapidly depleted (as short as four hours after ACE treatment), followed by the depletion of subunits from the cytoplasmic loop. Subunits specific to the nuclear loop and nuclear basket were not depleted within a 12-hour window. Figure 7 D- Figure 7 E). Consistent with the degradation of the NPC subunit, high-resolution imaging using transmission electron microscopy revealed nucleus distortion and fragmentation in ACE-treated A549 cells. Figure 7 F). Furthermore, in A549 cells expressing GFP-labeled TRIM21 and NES (nuclear output signal)-labeled mCherry, ACE treatment resulted in the entry of both proteins into the nucleus, consistent with impaired NPC function. Bortezomib treatment rescued ACE-dependent mislocalization of TRIM21-mEGFP and NES-mCherry. Figure 7 G). In summary, these results indicate that ACE guides the recruitment of TRIM21 from the cytosol to attack the inner loop of NPCs.

[0234] The model of ACE-induced TRIM21 recruitment to NPCs was confirmed by expression of TRIM21 fused with the biotin ligase TurboID in A549 cells. TurboID converts biotin into the reactive intermediate biotin-AMP to covalently label proteins near TRIM21. Figure 7 H). Cells were treated with biotin alone or with biotin plus ACE, followed by streptavidin beads to capture biotinylated proteins. Label-free quantitative proteomics revealed that the ACE-treated group was enriched in NUP35, NUP54, NUP58, and GLE1 (H). Figure 7 I). Notably, NUP35 and GLE1 were also the proteins most depleted after ACE treatment. In summary, these results indicate that ACE induces TRIM21 to be recruited to the vicinity of the NPC to trigger the degradation of nucleoporins.

[0235] Example 5: NUP98 is the main target site of acepromazine.

[0236] The following assays confirmed that nucleoporin degradation was attributed to ACE cytotoxicity, as NPCs are a key pathway connecting the nucleoplasm and cytoplasm. Mutations in NPCs that could induce cellular resistance to ACE were identified. A recently described CRISPR repressor scanning approach was used to design a full-coverage sgRNA library targeting the entire coding sequences of 27 nucleoporins (excluding the nuclear loop-specific subunits), enabling the in situ generation of a large number of diverse nucleoporin variants via Cas9 nuclease. Figure 9 A). A549-Cas9 cells transduced with a full-coverage sgRNA library were then cultured for three weeks in the absence or presence of ACE. Next-generation sequencing revealed enrichment of sgRNAs targeting amino acids E721, K750, K752, and F777 in NUP98, forming hotspots in the ACE-treated group. Figure 9 B).

[0237] NUP98 is a nucleoporin synthesized from the NUP98-NUP96 precursor, which utilizes its self-proteolytic domain (NUP98). APD It undergoes self-protein hydrolysis. The identified hotspots associated with ACE resistance fall on NUP98. APD Inside( Figure 9 C). The underlying mechanisms of drug resistance were further elucidated by transducing A549-Cas9 cells with the most enriched sgRNA (targeting K752), and five resistant clones were selected after ACE treatment. Figure 9 D- Figure 9 E). Sequencing of the NUP98 cDNA at the sgRNA cleavage site revealed short exon skipping, leading to NUP98 APD Missing middle section ( Figure 10 A- Figure 10 B). Western blot analysis of these drug-resistant clones revealed the disappearance of NUP98 and the presence of proteins greater than 180 kDa, indicating impaired NUP98-NUP96 self-proteolytic processing. Figure 9 F). In one of the drug-resistant clones, ACE does not induce TRIM21 to degrade nucleoporin near the NPC (F). Figure 9 G and Figure 10 C).

[0238] Further investigation was conducted into how impaired NUP98-NUP96 processing evades TRIM21 recognition to generate ACE resistance. The complex organization of NPCs presented challenges for detailed structure-function analysis. Degradation determinants were designed using the promyelocytic leukemia (PML) nucleosome, a subnuclear structure formed via phase separation. PML-GFP expression resulted in bright nuclear foci that were readily visualized using high-content imaging. Thirteen proteins were identified within NPCs near NUP98, and PML-GFP fusions corresponding to 32 fragments of these 13 proteins (including NUP35, NUP88, NUP93, NUP96, NUP98, NUP155, NUP160, NUP188, NUP205, NUP214, AAAS, GLE1, and SPMD4) were generated. Figure 9 H). High-content imaging of PML-GFP-NUP98 结构域2 It stands out as the most sensitive to ACE-mediated degradation. Figure 9 H and Figure 9 I). Through truncation analysis, the minimum degradation determinant was narrowed down to NUP98. APD The amino acid positions in the middle are 733-880 ( Figure 9 I and Figure 10 D). Therefore, the convergence of CRISPR repressor screening and PML-GFP-degradation determinant assays to the same APD domain in NUP98 indicates that NUP98 is a major target of acepromazine.

[0239] Example 6: Crystal structure of TRIM21PRYSPRY complexed with acepromazine and its metabolites

[0240] The PRYSPRY domain of the WT and D355A mutant TRIM21 was purified to further explore the mechanism of action of ACE and its metabolites at the molecular level. Isothermal titration calorimetry (ITC) revealed the relationship between ACE (dissociation constant / Kd: 5.66 µM), (R)-ACE-OH (Kd: 9.11 µM), and (S)-ACE-OH (Kd: 17.9 µM) and TRIM21. PRYSPRY (D355A) has moderate binding affinity. Figure 11 A). In the case of titration compounds up to 50 µM (ACE), using WT TRIM21 PRYSPRY No binding signal was detected. Micro-thermophoresis (MST) was then used to supplement the ITC results, and ACE (Kd: 44.6 µM) and (S)-ACE-OH (Kd: 237.3 µM) were observed to bind with WT TRIM21. PRYSPRY weak binding ( Figure 12A). (R)-ACE-OH and WTTRIM21 were not detected by MST. PRYSPRY These biophysical measurements reveal the binding of ACE and its metabolites with TRIM21. PRYSPRY The weak binding of (S)-ACE-OH alone cannot explain why (S)-ACE-OH is active while ACE and (R)-ACE-OH are inactive.

[0241] Then, TRIM21 complexes with ACE, (S)-ACE-OH, and (R)-ACE-OH were determined at resolutions of 1.6 Å, 1.89 Å, and 1.74 Å, respectively. PRYSPRY The eutectic structure of (D355A) Figure 11 B- Figure 11 E and Figure 12 B). These structures reveal that the tricyclic phenothiazine rings of these ligands occupy shallow hydrophobic pockets formed by Y328, M330, A335, L371, W383, and F450. The oxygen atom in the acetyl or hydroxyl group of the ligand acts as a hydrogen bond acceptor, interacting with the backbone of L371 (B). Figure 11 F- Figure 11 H). Notably, the aliphatic chains of the ligands exhibit flexibility, leading to different orientations of the amine groups. In the cases of ACE and (R)-ACE-OH, the amine groups extend outside the pocket, while in the case of (S)-ACE-OH, they form polar interactions with the side chains of E389 and Q395 (H). Figure 11 I). Since (S)-ACE-OH is an active metabolite with degradative activity, these structural observations suggest that the side chain conformation of these ligands is a key determinant of degradative activity.

[0242] Example 7: TRIM21-directed PROTAC selectively degrades multimeric proteins in biomolecular condensates

[0243] TRIM21 in combination with ACE PRYSPRY The eutectic structure of (D355A) reveals aliphatic chains as potential exit vectors in PROTAC design. TRIM21-based PROTACs (TrimTAC1) were prepared by linking ACE and JQ1 (a high-affinity binder for BRD4) with three-carbon aliphatic chains. Figure 13 (A and Example 8). dBET1 (CAS: 1799711-21-9, purchased from TargetMol) (which is a CRBN-based PROTAC targeting BRD4) was used as a positive control.

[0244] By stably expressing the second Bromo domain of BRD4 (BRD4) in A549 cells BD2mEGFP-tagged variants of ) were used with TRIM21 (D355A) or CRBN to provide cell models for evaluating the PROTAC activity of TrimTAC1. dBET1 potency (half-maximal degradation concentration / DC) 50 31 nM) Degradation of mEGFP-BRD4 BD2 TrimTAC1, at concentrations up to 100 µM, degrades mEGFP-BRD4. BD2 The aspect is inactive ( Figure 13 B- Figure 13 C).

[0245] TRIM21 has already been utilized in the Trim-Away technique, which uses antibodies to guide TRIM21 in degrading intracellular proteins. Recent follow-up mechanistic studies have revealed that target-induced clustering triggers intermolecular dimerization of the RING domain of TRIM21 to activate its ubiquitination activity. Furthermore, adjacent NUP98... APD The distances between them are 28.2 nm and 23.7 nm in the expanding and contracting NPCs, respectively, which is compatible with TRIM21 clustering. Figure 9 K). The following assays confirm that TRIM21-directed PROTAC can drive the selective degradation of target proteins present in multimeric assemblies that allow the dimerization of the TRIM21 RING domain.

[0246] The N-terminal FG (phenylalanine-glycine) repeat domain of NUP98 is related to mEGFP-BRD4. BD2 Reporter protein fusion. The resulting fusion protein (NUP98) FG -mEGFP-BRD4 BD2 In A549 cells, nuclear condensates are formed, which are efficiently and rapidly degraded by both TrimTAC1 and dBET1 when TRIM21 (D355A) or CRBN is expressed. Figure 13 B- Figure 13 D). Pretreatment of cells with inhibitors targeting the proteasome (bortezomib) or E1 ubiquitin activator enzyme (TAK243) can prevent TrimTAC1-induced NUP98. FG -mEGFP-BRD4 BD2 Nuclear condensate degradation ( Figure 13 E and Figure 14 A).

[0247] To avoid over-reliance on JQ1, a second TRIM21-based PROTAC was synthesized, namely TrimTAC2 (based on SLF, the synthetic ligand of FKBP12). Figure 13(F and Example 9). TrimTAC2 effectively degraded NUP98 without affecting soluble mEGFP-FKBP12. FG -mEGFP-FKBP12 condensates ( Figure 14 B- Figure 14 E).

[0248] mEGFP-FKBP12 was fused with three other aggregate-forming proteins (PML (PML-NB), Coilin (Cajal bodies), and NPM1 (nucleolus)) to further test the general applicability of TrimTAC to degrade proteins present in biomolecular aggregates. mEGFP-FKBP12 was also fused with kinases CDK4 and BTK as examples of non-aggregating proteins. In A549 cells expressing TRIM21 (D355A), TrimTAC2 degraded aggregate-forming fusion proteins (PML, Coilin, and NPM1), but not non-aggregating fusion proteins (CDK4 and BTK). Figure 13 In summary, these results indicate that TRIM21-directed PROTAC possesses the unique ability to degrade proteins in biomolecular condensates while retaining proteins in the dilute phase.

[0249] Example 8: Preparation of TrimTAC1

[0250]

[0251] 0. Preparation of Compound 8

[0252]

[0253] 0.1 Synthesis of Compound 7

[0254] 1-(10H-dibenzo[2,1-b:1',2'-e][1,4]thiazin-2-yl)ethyl-1-one (5.61 g, 23.2 mmol) and 2-[(3-bromopropyl)oxy]tetrahydropyran (6.73 g, 30.1 mmol) were added to a solution of sodium hydride (1.11 g, 27.8 mmol) in DMF (80 mL) at 0°C. The mixture was kept at this temperature for 5 h. The reaction mixture was poured into ice water (100 mL) and extracted with EtOAc (100 mL x 2). The combined organic layers were washed three times with brine (100 mL), dried over Na2SO4, and filtered. The filtrate was concentrated to obtain the residue. The crude product was then dissolved in MeOH (100 mL). TsOH, H2O (2.2 g, 11.6 mmol) was added, and the mixture was stirred at 25°C for another 3 h. The solvent was removed and the residue was purified by silica gel chromatography (EtOAc / petroleum ether = 1 / 2) to give 7 (3.43 g, 11.41 mmol, 48.9%) as a white solid. MS (ESI): m / z = 300.2.

[0255] 0.2 Synthesis of Compound 8

[0256] At 0ºC, 4-methylbenzenesulfonyl chloride (1.38 g, 7.22 mmol) was added to a solution of 7 (1.83 g, 6.01 mmol), 4-(dimethylamino)pyridine (0.15 g, 1.2 mmol), and DCM (50 mL), followed by triethylamine (1.25 mL, 9.02 mmol). The mixture was warmed to 25ºC and stirred for 15 h. After 7 was consumed, the solution was poured into ice water (25 mL) and extracted with DCM (50 mL x 2). The combined organic layers were washed with citric acid (50 mL) and brine (50 mL), dried over Na2SO4, and filtered. The filtrate was concentrated to give the residue, which was purified by silica gel chromatography (EtOAc / petroleum ether = 1 / 5) to give 8 (2.33 g, 5.08 mmol, 88.7%) as a yellow oil. MS (ESI): m / z = 454.2.

[0257] 1. Synthesis of Compound 10

[0258] (3-(piperazin-1-yl)propyl)carbamate tert-butyl ester (316 mg, 1.3 mmol) was added to a solution of 8 (453 mg, 1 mmol) in 20 mL of ACN at 25ºC, followed by the addition of potassium carbonate (276.05 mg, 1.997 mmol). The mixture was stirred at 85ºC for 15 h. The reaction mixture was filtered. The filtrate was concentrated to give a residue, which was purified by rapid column chromatography (MeOH / DCM = 1 / 20) to give 10 (491 mg, 0.49 mmol, 48.9%) as a white solid. MS (ESI): m / z = 525.2.

[0259] 2. Synthesis of Compound 11

[0260] Hydrogen chloride (2 mL, 8.0 mmol) was added to a solution of 10 (277 mg, 0.49 mmol) in DCM (3 mL) at 0ºC. The mixture was stirred at 25ºC for 2 h. After 10 was consumed, the solvent was poured off and the residue was freeze-dried to give 11 (crude) as a white solid. MS (ESI): m / z = 425.2.

[0261] 3. Synthesis of TrimTAC1

[0262] At 0ºC, 11 (crude product, 0.49 mmol), JQ-1 (196 mg, 0.49 mmol, IUPAC name:

[0263] (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazaphen-6-yl)tert-butyl acetate, CAS: 1268524-70-4, purchased from Macklin) and DIPEA (0.161 mL, 0.973 mmol) were added to a solution of DMAc (5 mL) with HATU (322 mg, 0.85 mmol). The mixture was warmed to 25ºC and stirred for 1 h. After 11 was consumed, the reaction mixture was poured into ice water (25 mL) and extracted with EtOAc (20 mL x 3). The combined organic layers were washed three times with brine (10 mL), dried over Na₂SO₄ and concentrated. The crude product was purified by silica gel chromatography (DCM / MeOH = 1 / 13) to give TrimTAC1 as a white solid (153 mg, 0.19 mmol, 38.7% in two steps). MS (ESI): m / z = 807.2. 1H NMR (400 MHz, DMSO) δ 7.56 (d, J = 8.0Hz, 1H), 7.49 (d, J = 8.5 Hz, 2H), 7.45 - 7.40 (m, 3H), 7.35 - 7.14 (m, 3H),7.13 - 6.93 (m, 2H), 4.50 (t, J = 7.2 Hz, 1H), 4.08-3.94 (m, 2H), 3.65-3.57(m, 2H), 3.27 - 3.20 (m, 2H), 3.15-3.09 (m, 3H), 2.69 - 2.65 (m, 2H), 2.62 -2.55 (m, 8H), 2.41 (s, 3H), 2.37 - 2.31 (m, 2H), 1.79-1.73 (m, 2H), 1.62 (s,3H), 1.26 - 1.24 (m, 4H).

[0264] Example 9: Preparation of TrimTAC2

[0265]

[0266] 1. Synthesis of Compound 13

[0267] To a solution of 8 (500 mg, 1.1 mmol) and K₂CO₃ (305 mg, 2.2 mmol) in ACN (20 mL), tert-butyl (2-(2-(2-(piperazin-1-yl)ethoxy)ethoxy)ethyl)carbamate (524 mg, 1.65 mmol) was added. The mixture was stirred at 25ºC. The mixture was stirred at 85ºC for 15 h. The reaction mixture was filtered. The filtrate was concentrated to give a residue, which was purified by rapid column chromatography (MeOH / DCM = 1 / 20) to give 13 (484 mg, 0.81 mmol, 73.1%) as a white solid. MS (ESI): m / z = 599.2, [M+H] + .

[0268] 2. Synthesis of Compound 14

[0269] Hydrogen chloride (3 mL, 12 mmol, 4 M 1,4-dioxane) was added to a solution of 13 (484 mg, 0.81 mmol) in DCM (5 mL) at 0ºC. The mixture was stirred at 25ºC for 2 h. After 13 was consumed, the solvent was poured off and the residue was dissolved in water (5 mL). The pH of the mixture was adjusted to 8 with 2 M aqueous NaHCO3. The solvent was then poured off and the residue was freeze-dried to give 14 (229 mg, 0.46 mmol, 56.9%) as a white solid. MS (ESI): m / z = 499.2, [M+H] + .

[0270] 3. Synthesis of Compound 15

[0271] Glutaric anhydride (52 mg, 0.46 mmol) and DIPEA (177 mg, 1.37 mmol) were added to a solution of 14 (229 mg, 0.46 mmol) in DCM (5 mL). The mixture was stirred at 25ºC for 1 h. Water (50 mL) was then added and the reaction mixture was stirred for another 30 min. The reaction mixture was concentrated. The residue was purified by preparative HPLC to give 15 (159.4 mg, 0.26 mmol, 57.7%) as a colorless oil. MS (ESI): m / z = 613.2, [M+H]+.

[0272] 4. Synthesis of TrimTAC2

[0273] 15 (104 mg, 0.17 mmol), COMU (145 mg, 0.34 mmol, chemical name (1-cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylaminomorpholine carbium hexafluorophosphate and CAS: 1075198-30-9) and N-methylmorpholine (34 mg, 0.34 mmol) were dissolved in DMF (2 mL) and incubated for 1 min. SLF (96 mg, 0.18 mmol, chemical name 1-(3,3-dimethyl-1,2-dioxopentyl)-(2S)-2-piperidinecarboxylic acid, (1R)-1-(3-aminophenyl)-3-(3,4-dimethoxyphenyl)propyl ester and CAS: 195513-96-3) and the catalyst 4-dimethylaminopyridine were added to DMF (1 mL), and the reaction was stirred at 25ºC for 2 h. The reaction mixture was diluted to 1 mL with water / acetonitrile / formic acid (50 / 50 / 0.1) and purified by preparative HPLC to obtain TrimTAC2 as a white solid (61 mg, 0.055 mmol, 33.5%). MS (ESI): m / z = 1120.2, [M+H] + . 1 H NMR (400 MHz, cdcl3) δ 9.03 (d, J =15.4 Hz, 1H), 7.69 - 7.57 (m, 2H), 7.50 - 7.40 (m, 2H), 7.25 - 7.14 (m, 4H),7.11 (d, J = 7.4 Hz, 1H), 7.01 - 6.91 (m, 2H), 6.87 (d, J = 8.0 Hz, 1H), 6.79- 6.74 (m, 1H), 6.70 - 6.65 (m, 2H), 5.80 - 5.67 (m, 1H), 4.05 - 3.93 (m,3H), 3.85 (s, 3H), 3.84 (s, 3H), 3.66 - 3.52 (m, 9H), 3.46 - 3.40 (m, 2H), 3.36 - 3.29 (m, 1H), 3.23 - 3.17(m, 1H), 3.07 - 2.70 (m, 10H), 2.68 - 2.45(m, 9H), 2.43 - 2.32 (m, 3H), 2.27 - 2.16 (m, 1H), 2.11 - 1.99 (m, 5H), 1.78 - 1.60 (m, 4H), 1.28 - 1.16 (m, 8H), 0.88 (t, J = 7.5 Hz, 3H).

[0274] Example 10: Chiral synthesis of compound 5 and determination of the absolute configuration of ACE-OH

[0275]

[0276] Compound 1 (7 mg, 21.3 μmol, 1.0 equivalent) in a flame-dried screw-cap culture tube was azeotropically reacted with toluene three times. CH₂Cl₂ (0.21 mL), triethylamine (5.8 μL, 42.7 μmol, 2.0 equivalent), p-bromobenzoyl chloride (9.4 mg, 42.7 μmol, 2.0 equivalent), and DMAP (one catalytic tablet) were added sequentially to the tube at 0ºC. The resulting mixture was then stirred at room temperature under an argon atmosphere for 1 h, followed by quenching with saturated aqueous NH₄Cl. The reaction mixture was extracted with dichloromethane (10 mL * 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Purification by preparative TLC (methanol:dichloromethane, 1:20) yielded compound 2 (9.2 mg, 79%) as a white solid. 1 H NMR (500MHz, CDCl3): δ 7.89 (d, J = 8.0 Hz, 2H), 7.58 (d, J = 8.0 Hz, 2H), 7.23 -7.13 (m, 3H), 7.08 - 6.88 (m, 4H), 6.00 (q, J = 6.9 Hz, 1H), 4.10 (s, br,2H), 3.09 (t, J = 7.0 Hz, 2H), 2.63 (s, 6H), 2.38 (s, br, 2H), 1.65 (d, J =6.0 Hz, 3H). 13 C NMR (126 MHz, CD3OD): δ 166.38, 146.66, 146.62, 142.70,133.03, 132.27, 130.76, 129.15, 128.59, 128.39, 128.32, 126.72, 126.63,123.82, 121.24, 117.08, 115.01, 74.68, 57.73, 46.02, 45.19, 25.37, 22.51. [α]23.0 D = -72.28° (c = 0.184, MeOH).

[0277]

[0278] Compound 3 (7 mg, 13.7 μmol, 1.0 equivalent) was dissolved in ethyl acetate (10 mL) and the solution was washed with saturated aqueous K₂CO₃ (5 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give an oily free tertiary amine. The free tertiary amine in a flame-dried screw-cap culture tube was azeotropically reacted with toluene three times. CH₂Cl₂ (0.10 mL) and iodomethane (0.2 mL) were added sequentially to the tube at room temperature. The resulting mixture was then stirred at room temperature under an argon atmosphere for 30 min and concentrated under reduced pressure to give a crude compound 4 as a white solid. Compound 5 (4.2 mg, 54%) was purified by preparative reversed-phase HPLC (10%–95% CH₃CN (0.1% HCOOH) in water (containing 0.1% HCOOH)) to give a white solid compound 5. 1 H NMR (500 MHz, CD3OD): δ 8.44 (s, 1H), 7.94 (d, J =8.0 Hz, 2H), 7.68 (d, J = 8.5 Hz, 2H), 7.25 (t, J = 7.5 Hz, 1H), 7.19 (t, J =7.5 Hz, 2H), 7.12 (s. br, 2H), 7.07 (d, J = 8.0 Hz, 1H), 7.00 (t, J = 7.5 Hz,1H), 6.03 (q, J = 6.5 Hz, 1H), 4.18 - 4.04 (m, 2H), 3.43 (t, br, J = 8.0 Hz,2H), 2.98 (s, 9H), 2.25 (s, br, 2H), 1.67 (d, J = 6.5 Hz, 3H). 13 C NMR(126 MHz, CD3OD): δ 168.90, 166.55, 146.28, 146.01, 143.08, 133.09, 132.23, 130.67,129.27, 128.91, 128.79, 128.63, 127.38, 127.20, 124.43, 121.60, 117.34,115.87, 74.96, 65.60, 53.55, 53.52, 53.49, 44.77, 22.41, 21.61. [α]27.2 D = -91.67° (c = 0.084, MeOH).

[0279] Note: Crude compound 4 was used directly to obtain crystals. For characterization, crude compound 4 was purified by preparative reversed-phase HPLC to obtain compound 5.

[0280] Data on compound 4, X-ray crystallographic data are shown in Figure 4 D.

[0281] Table 1. Crystallographic and Refined Data of Compound 3

[0282]

[0283] Table 2.3 Atomic coordinates (*10) 4 ) and equivalent isotropic displacement parameters (Å) 2 *10 3 U(eq) is defined as orthogonalized U. ij One-third of the tensor trace.

[0284]

[0285] Based on the crystal and refined data of compound 3, the configuration of compound 3 was determined, and therefore the active metabolite of ACE was determined to be (S)-(-)-ACE-OH.

[0286] Example 11: (Bio)materials, assays, and methods used in this invention

[0287] Unless otherwise stated herein, materials including chemical materials, biological materials, and reagents are commercially available or can be prepared by those skilled in the art using known methods.

[0288] Unless otherwise specified herein, the determinations and methods (including the conditions under which those determinations or methods are performed) are known to those skilled in the art.

[0289] A. Cell Culture

[0290] Human cell lines A549, HCT-116, HeLa, DLD-1, and HEK293T were gifts from the laboratory of Dr. Deepak Nijhawan at the University of Texas Southwestern Medical Center. NK-92MI was a gift from Dr. Feng Shao at the National Institute of Biological Sciences, Beijing. Mino was obtained from ATCC. SiHa, HuH-7, ME-180, and HaCaT were obtained from the Cell Resource Center, Peking Union Medical College (Beijing, China). All cell lines were confirmed to be mycoplasma-free by PCR. Cells were cultured using standard adherent cell culture methods in a tissue culture incubator at 37ºC and 5% CO2. A549 and DLD-1 were grown in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 2 mM L-glutamine. HCT-116, HEK293T, HeLa, SiHa, HuH-7, ME-180, and HaCaT cells were grown in DMEM medium containing 10% FBS and 2 mM L-glutamine. NK-92MI cells were grown in α-limit essential medium (MEM) supplemented with 0.2 mM inositol, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid, 10 mM HEPEs, non-essential amino acids (NEAA), and 20% FBS.

[0291] B. Chemicals

[0292] Acephalosporin (CAS: 3598-37-6), chlorpromazine (CAS: 69-09-0), bortezomib (CAS: 179324-69-7), MLN4924 (CAS: 905579-51-3), TAK243 (CAS: 1450833-55-2), and biotin (CAS: 58-85-5) were purchased from TargetMol (Topscience, Shanghai, China). All these chemicals were prepared as 10 mM stock solutions in DMSO (Solarbio LifeScience, Beijing, China) (CAS: 67-68-5) and further diluted to the desired concentrations in DMSO.

[0293] C. Construction of reporter cell lines

[0294] Control sgRNA-H2B-GFP and IFNGR1 sgRNA-H2B-mcherry were cloned into the pLenti-U6-Puro backbone by replacing their multiple cloning sites with sgRNA fused with H2B-GFP or H2B-mcherry. The resulting plasmids were packaged into lentiviruses for transduction into A549 cells. Stable cell lines were obtained by selecting transduced cells with 2 µg / ml puromycin.

[0295] D. High-throughput small molecule screening

[0296] Initial screening was performed using a compound library of 81,845 small molecules containing FDA-approved drugs (Life Chemicals, Niagara-on-the-Lake, Canada). The screening procedure was as follows: A549 control sgRNA-H2B-GFP cells and A549 IFNGR1 sgRNA-H2B-mcherry cells were mixed. Three thousand mixed cells in 50 μL of medium were plated in each well of a 384-well clear, flat-bottomed, white polystyrene TC-treated microplate (Corning, Corning, USA) and allowed to adhere to the plate overnight. Compounds from the screening library were added to the cells at a final concentration of 10 μM using a Biomek FXP automated workstation. Seventy-two hours later, images were collected using the PerkinElmer Opera LX high-content screening system according to the vendor's instructions. The number of green and red cells was measured using Columbus online analysis software.

[0297] E. NK lethality determination

[0298] 10,000 A549 cells were seeded in 6-well plates. The original culture medium was removed the next day. NK-92MI cells were added to the target cells at various effector / target cell (E / T) ratios. DMSO or 10 μM acepromazine was added to the cells simultaneously. After 48 h, the cells were stained with crystal violet (Beyotime, Shanghai, China, C0121) at room temperature for 20 min, followed by destaining with water. Images were captured using a VILBER FX7 imager.

[0299] To perform a quantitative NK cell killing assay using flow cytometry, equal volumes of green and red WT A549 and IFNGR1 KO A549 cells were mixed and treated with acepromazine and different amounts of NK cells. After 48 h, the number of green and red cells was measured by flow cytometry.

[0300] F. Cell viability assay

[0301] Three thousand A549 cells were seeded in 100 μL of culture medium into each well of a 96-well clear, flat-bottomed white polystyrene TC-treated microplate (Corning, Corning, USA). Cells were then administered with serially diluted compounds using a D300e digital dispenser (Tecan, Mannedorf, Switzerland). After 72 h, cell viability was measured using the CellTiter-Glo luminescence cell viability assay kit (Promega, Madison, USA) according to the manufacturer's instructions. Luminescence was recorded using an EnVision multimodal plate reader (PerkinElmer, Waltham, USA). IC50 was determined using GraphPad Prism with baseline correction (normalized relative to DMSO control), asymmetric (four-parameter) equations, and least-squares fitting.

[0302] G. LC-MS

[0303] Working standard solutions of 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, and 500 nM were prepared using stock standard solutions of 1 mM acepromazine and acepromazine-OH vials by serial dilution in methanol / water (1:1). Biological samples were resuspended in 1 mL methanol / water (1:1). Standard solutions and biological samples were transferred to 250 μL end-capsule vials for analysis. Standards and samples were analyzed using an Agilent 1290 infinity UHPLC system coupled to an Agilent 6495 triple quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA) with ESI sources. Separation was performed using a Zorbax Eclipse Plus C18 column (2.1 mm x 50 mm, 1.8 µm; Agilent Technologies, Santa Clara, CA). The injection volume was 2 µL. The mobile phase consisted of 0.1% formic acid (A) in water and 0.1% formic acid (B) in acetonitrile. The following gradients were applied: 0–1 min, 5% B; 1–4 min, 5%–95% B; 4–5 min, 95% B; 5–5.01 min, 95%–5% B; 5.01–7 min, 5% B. The flow rate was 0.3 mL / min, and the column temperature was 35ºC. The mass spectrometer was operated in both multiple reaction monitoring (MRM) and positive ESI modes. For the analysis of acepromazine and acepromazine-OH, MRM ion pairs were monitored at m / z 327.2→86.1 (CE 19 V) and 329.2→328.7 (CE 3 V), respectively. The residence time was 200 ms. The absolute concentrations of acepromazine and acepromazine-OH were calculated using Agilent Mass Hunter Quantitative Analysis B.07.00 software.

[0304] H. Whole-genome CRISPR screening

[0305] In total, 2.6 * 10⁶ A549-cas9 cells were infected with a Brunello CRISPR sgRNA library at approximately 0.3 multiples of infection (MOI) for 100* coverage. Twenty-four hours post-infection, cells were reseeded and selected for 3 days with 2 µg / ml puromycin. Cells were then divided into four groups: untreated, IFNγ, ACE, and a combination of IFNγ and ACE. Concentrations were increased in a gradient to ensure substantially uniform cell numbers. After 4 weeks of culture, cells were harvested as previously described, and genomic DNA was extracted. DNA fragments containing sgRNA sequences were amplified from the isolated genomic DNA using two rounds of PCR with NEBNext Ultra II Q5 premix (NEB, Ipswich, USA). For the first round of PCR, twenty 25 µl PCR reactions (each containing 2.6 mg of genomic DNA template) were performed using a thermal cycler program with forward primer NGS-Lib-KO-Fwd-1 (5'-CCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNNNNGCTTTATATCTTGTGGAAAGGACGAAACACC 3') and reverse primer NGS-Lib-KO-Rev-0 (5'-CAGACGTGTGCTCTTCCGATCTCCGACTCGGTGCCAC TTTTTCAA-3'). The thermal cycler program consisted of an initial denaturation at 98ºC for 5 min, followed by 19 cycles (98ºC denaturation for 10 s, 69ºC annealing for 30 s, and 65ºC extension for 45 s) and a final extension at 65ºC for 5 min. The products from the first round of PCR were collected and purified using a DNA purification and concentration kit (Zymo, Irvine, USA) and diluted to 2 ng / mL. For the second round of PCR, eight 25 µl PCR reactions (each containing 2 ng of purified first-round PCR product) were performed on each sample using the indexed forward primer NEBNext i5 (5'-AATGATACGGCGACCACCGAGATCTACAC-8-nucleotide index-ACACGACGCTCTTCCGATCT-3') and the indexed reverse primer NEBNext i7 (5'-CAAGCAGAAGACGGCATACGAGAT-8-nucleotide index-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3'). The second round of PCR used the same cycling conditions as the first round, except that 10 cycles were used. The products of the second round of PCR were then subjected to electrophoresis on a 2% agarose gel.The expected approximately 300 bp amplicons were excised from the gel and sequenced using an Illumina HiSeq PE150 (Berry Genomics, Beijing, China). The raw sequencing data have been saved to [data]. As previously mentioned, the CRISPR screening data were analyzed using MAGeCK (v0.5.9.2).

[0306] I. Protein blotting

[0307] Cells were washed with DPBS to remove residual culture medium and then lysed in SDS lysis buffer (20 mM HEPES, 2 mM MgCl2, 10 mM NaCl, 1% SDS, pH 8.0) containing 0.5 units / µl of totipotent nuclease and an EDTA-free protease inhibitor mixture (Roche, Basel, Switzerland). Protein lysates were centrifuged (12000 g, 10 min, 4ºC), and the concentration of the supernatant was determined using a BCA protein assay. Proteins from samples, ranging from 5 µg to 30 µg, were separated on 4%–20% gradient SDS-PAGE gels and transferred to 0.5 μm nitrocellulose membranes. The membranes were blocked in 5% skim milk PBST solution (0.1% v / v Tween-20) for 30 min, then incubated overnight with primary antibody at 4ºC and 1 h with secondary antibody at room temperature. The primary antibodies used are as follows: rabbit polyclonal anti-Stat1 (1:5,000, Cell Signaling Technology, 9172), rabbit monoclonal anti-phosphorylated Stat1 (1:5,000, Cell Signaling Technology, 7649), mouse monoclonal anti-Flag-HRP (1:10,000, Sigma-Aldrich, A8592), mouse monoclonal anti-β-actin-HRP (1:10,000, Huaxingbio, HX18271), rabbit monoclonal anti-TRIM21 (1:5000, Abcam, AB207728), rabbit polyclonal anti-GLE1 (1:5000, ABclonal, A13207), anti-SMPD4 (1:5000, ABclonal, A15473), and anti-NUP35 (1:5000, ABclonal, A15473). Anti-NUP98 (1:5000, ABclonal, A12762) and anti-NUP98 (1:5000, ABclonal, A0530) were used. The secondary antibody used was goat polyclonal anti-rabbit IgG (1:10,000, Cell Signaling Technology, 7074). HRP enzyme activity was detected using M5 HiPer ECL Western blotting substrate (Mei5bio, Beijing, China). Western blot images were acquired using a VILBER FUSION FX7 imager.

[0308] J. Quantitative mass spectrometry

[0309] A549 cells were treated with 10 μM acepromazine or DMSO in triplicate for 6 h, followed by quantitative proteomics analysis (Deep kinase, Beijing, China). In short, cell pellets were lysed using lysis buffer (8 M urea, 50 mM Tris-HCl, 1% Triton X-100, pH = 7.4) containing protease and phosphatase inhibitors (catalog number 539134, Merck; catalog number 524625, Merck). Protein concentrations were determined using the Pierce BCA protein assay kit (catalog number 23225, ThermoScientific). Cell lysates were reduced with 10 mM dithiothreitol (DTT), alkylated with 40 mM iodoacetamide (IAM), and then quenched with 5 mM dithiothreitol (DTT). Alkylated samples were purified using the SP3 method. All LC-MS / MS experiments were performed on an Orbitrap Exploris 480 (Thermo Scientific) equipped with a UltiMate 3000_UPLC system. MS analyses were performed using an Orbitrap Exploris 480 mass spectrometer (Thermo Scientific). A project-specific DIA spectral library was first generated using DIA-MS2pep. The protein sequences used for database searching were a reviewed human proteome (uniport_UP000005640, 82685 entries). Run-specific FDRs for peptide and protein level identification were estimated using Percolator. The spectral library was further generated and submitted to the DIA-NN software for protein quantification. Precursor and protein FDRs were set to 1%.

[0310] K. TurboID

[0311] A549 cells pretreated overnight with 10 ng / ml IFNγ were treated with 100 nM bortezomib for 2 h, followed by treatment with 20 μM ACE or DMSO for 4 h. Cells were then treated with 50 μM biotin for 2 h. Cells were scraped off and washed with PBS. The cell pellet was lysed in 180 μL RIPA lysis buffer (50 mM Tris pH 8, 150 mM NaCl, 1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 1* protease inhibitor mixture (Sigma-Aldrich), and 1 mM PMSF) and incubated overnight at 4ºC with 60 μL streptavidin beads. The beads were washed sequentially with 1 M KCl, 0.1 M Na2CO3, RIPA buffer, and TBS. The beads were then eluted with 25 μl of elution buffer I (50 mM Tris-HCl pH 7.5, 2 M urea, 5 ng / μl sequencing-grade modified trypsin (Promega), 1 mM DTT) and 50 μl of elution buffer II (50 mM Tris-HCl pH 7.5, 2 M urea, 5 mM iodoacetamide). The eluent was digested overnight at 32ºC on a metal bath at 400 rpm. The eluent was terminated with 1 μl of trifluoroacetic acid and dried by vacuum centrifugation, followed by proteomics analysis.

[0312] L. Nucleoporin sgRNA Cloning and CRISPR Repressor Screening

[0313] The nucleoporin sgRNA full-coverage library contains 9740 sgRNAs, covering all sgRNAs with NGG PAM and cleavage sites within the 27 nucleoporin (table) coding sequences. The nucleoporin full-coverage library was synthesized by Genewiz (Suzhou, Jiangsu, China). Oligonucleotides containing sgRNA sequences were cloned in a mixed manner into lenti-guide-puro (#52963; Addgene, Watertown, Massachusetts, USA). Then, 1 * 10 7 A549-Cas9 cells were infected with a full-coverage library at approximately 0.3 multiples of infection (MOI), achieving a coverage of 1000*. After selection for 3 days using 2 µg / ml puromycin, cells were divided into two groups: DMSO and ACE, with concentrations increasing in a gradient. After 3 weeks of culture, cells were harvested using the above method, the genome was extracted, sgRNA-containing DNA fragments were cloned, next-generation sequencing was performed, and the data were analyzed.

[0314] Isolation of the M. acepromazine-resistant A549-sgNup98-752 clone

[0315] Parental A549 cells were infected with a lentivirus containing sgNup98-752. Stable cell lines were obtained by selecting transduced cells with 2 mg / ml puromycin. One million cells were plated on 10 cm plates and treated with 10 μM acepromazine for three consecutive weeks, changing the medium every 3–4 days. Acetpromazine-resistant clones were isolated and amplified, and their sensitivity to acepromazine was tested using cell viability assays.

[0316] N. Protein expression and purification

[0317] Human TRIM21 PRYSPRY (residues 287-465) was cloned into the pET28a vector with an N-terminal 6xhis tag. The D355A mutation was introduced into TRIM21 PRYSPRY via overlap extension PCR. Bacterial protein expression was performed in *E. coli* BL21-CodonPlus(DE3)-RIL cells. Cells were grown in 2xTY medium (supplemented with 0.5% glucose, 2 mM MgSO4, and kanamycin) at 37ºC and induced with 1 mM isopropyl-bD-thiogalactopyranoside (IPTG) at an OD600 of approximately 0.6–1. Cells were lysed by sonication in binding buffer A (50 mM Tris-HCl pH 8.0, 1 M NaCl, 2 mM TECP) supplemented with 10 mM imidazole and 1x cOmplete mini EDTA-free protease inhibitor mixture (Roche, Basel, Switzerland). The lysate was clarified by centrifugation at 15,000 g for 1 h at 4ºC. The recombinant protein was purified using Ni NTA beads (Smart Lifesciences, Changzhou, China, SA004010) and washed with buffer B (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 1 mM TECP, 30 mM imidazole). The protein was eluted with buffer C (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 1 mM TECP, 30 mM imidazole). The flow-through containing TRIM21 PRYSPRY was concentrated by ultrafiltration, loaded onto a Superdex 75 Increase 10 / 300 GL column (Cytiva, Marlborough, MA, USA), and fractionated in 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5 mM TECP. All purified protein was concentrated to 20 mg / mL for crystallization.

[0318] O. Isothermal titration calorimetry (ITC)

[0319] ITC was performed using Microcal PEAQ-ITC (Malvern Panalytical, Malvern, Worcestershire, UK). The tank contained the protein solution, and the syringe contained the compound solution. Experiments were conducted at 25ºC. The titration consisted of 19 injections (an initial injection of 0.4 μl, followed by 18 injections of 2 μl each). The stirring speed in the reaction tank was 1000 rpm. Data were analyzed using Microcal PEAQ analysis software with nonlinear least squares regression. Baseline drift was corrected during data analysis.

[0320] P. Crystallization and X-ray data collection

[0321] Protein-ACE / S-OH-ACE / R-OH-ACE cocrystals were obtained by mixing the compound with purified TRIM21 PRYSPRY (D355A) at a molar ratio of 2:1. All crystals were grown in hanging drops by vapor diffusion at 20ºC. The Hampton Crystal Index was used. TM Initial crystallization screening was performed. The optimal eutectic was grown at 20ºC over a pore solution containing 0.1 M Tris (pH 7.0) and 3.5 M sodium formate. Large seed crystals were used to improve crystal quality. X-ray diffraction data were collected at the BL02U1 beamline (Trim21-ACE complex and Trim21-R-OH-ACE complex) and BL17UM beamline (Trim21-S-OH-ACE complex) of the Shanghai Synchrotron Radiation Facility. 1, 2 Use XDS, POINTLESS, and AIMLESS programs to process data. 3-5 .

[0322] Q. Structural Determination and Refinement

[0323] The previously reported TRIM21-IgG-Fc complex structure (PDB ID: 2IWG) was used as the search model. 7 By using a program phaser 6 Molecular substitution was performed to resolve the crystal structure of TRIM21 complexed with the ligand. This was achieved using phenix.refine. 9 Structural refinement combined with the use of Coot 8 To assist in further manual model building.

[0324] R. TRIM21 MST Measurement between PRYSPRY and Compounds

[0325] The purified TRIM21 PRYSPRY domain was labeled using the RED-NHS 2nd generation protein labeling kit (MO-L011, NanoTemper Technologies GmbH, Munich, Germany). During measurements, the labeled protein was diluted to 50 nM in PBST buffer (137 mM NaCl, 2.5 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4, 0.05% Tween-20). ACE, (S)-ACEOH, and (R)-ACEOH were prepared by 16 consecutive dilutions in PBST buffer, with a maximum concentration of 5 mM. The mixture of protein and compounds was loaded into a standard-treated capillary (MO-K022, NanoTemper Technologies GmbH, Munich, Germany). Measurements were performed at 25ºC using 80% MST power on a Monolith NT.115 (NanoTemper Technologies GmbH, Munich, Germany) with a blue / red filter. All experiments were repeated three times for each measurement. Data analysis was performed using NanoTemper® affinity analysis software.

[0326] S. Confocal Image Acquisition and Processing

[0327] Transfected cells were cultured on glass plates (NEST, 801002) and stained with Hoechst (1 ug / ml, Absin, abs813337) for 5 minutes, followed by imaging. All confocal images were captured using a Nikon A1 sim confocal microscope and then processed using IMARIS software.

[0328] Opera quantification of T. TrimTAC-induced aggregate degradation

[0329] 10,000 A549-Nup98N-mEGFP-BD2-IRES-mcherry cells or other aggregate-associated cells were plated in 384-well clear, flat-bottomed white polystyrene TC-treated microplates (Corning, Corning, USA) and allowed to adhere overnight. Cells were then administered serially diluted with a compound containing TrimTAC. Four hours later, images were collected using the PerkinElmer Opera LX high-content screening system, following the supplier's instructions. Total EGFP and mcherry intensities were measured using Columbus online analysis software.

[0330] U. NPC degradation assay

[0331] All NPC proteins were separated into multiple domains, and an A549-PML-mEGFP-NPC domain cell line was constructed. Cells were seeded at 10,000 cells / well in 384-well clear, flat-bottomed white polystyrene TC-treated microplates (Corning, Corning, USA) and allowed to adhere overnight. Cells were then treated with or without 10 μM acepromazine for 8 hours. Images were collected using the PerkinElmer Opera LX high-content screening system according to the vendor's instructions. Total EGFP intensity was measured using Columbus online analysis software.

[0332] V. Statistical Analysis

[0333] Statistical analysis was performed using GraphPad Prism 8.0. Student's t-test was used to evaluate statistically significant differences between two sample groups. When comparing more than two independent groups, analysis of variance (ANOVA) was used to evaluate statistical significance. Multiple comparison tests were performed when the ANOVA was significant. All tests were two-tailed, and p < 0.05 was considered statistically significant.

[0334] It should be understood that if any prior art disclosure is mentioned herein, such mention does not constitute an admission that the disclosure is part of the general knowledge of the art in any country.

[0335] Although the foregoing invention has been described in considerable detail by way of illustration and example for the purpose of clarity, it will be apparent to those skilled in the art that certain minor changes and modifications may be made. Therefore, the description and embodiments should not be construed as limiting the scope of the invention.

Claims

1. A method for degrading a target protein in a cell via the TRIM21 E3 ligase ubiquitin-proteasome pathway, the method comprising contacting the target protein with an effective amount of a compound selected from: compounds of formula (I) serving as ligands for recruiting TRIM21 ligases, compounds of formula (II), or molecular gel compounds or PROTACs carrying compounds of formula (I) or (II). The compound of formula (I) is a compound having formula (I) or a pharmaceutically acceptable salt thereof, its isomers or its deuterated analogues. (I) in X is O, S, or NR. 1 , where R 1 It is hydrogen or alkyl; n is 1 to 12; R a and R b Each is independently either hydrogen or alkyl; R c and R d Each of these groups is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocyclic, or heteroaryl, and each of these groups is unsubstituted or substituted by one or more substituents selected from: halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclic, heteroaryl, cyano, hydroxyl, hydroxyl, alkoxy, -NR m1 C(O)NR n1 R p1 -NR m1 C(O)R n1 -C(O)NR m1 R n1 -NR m1 R n1 -C(O)R m1 or -C(O)OR m1 ; Or R c and R d Together with the nitrogen atoms to which they are attached, they form 3 to 12-membered rings containing 0 to 3 additional heteroatoms selected from oxygen, nitrogen, or sulfur, and said rings are unsubstituted or substituted by one or more substituents selected from: halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclic, heteroaryl, cyano, hydroxy, hydroxyalkyl, alkoxy, hydroxyalkoxy, -NR m2 R n2 -C(O)R m2 -C(O)OR m2 -NR m2 C(O)NR n2 R p2 -NR m2 C(O)R n2 or -C(O)NR m2 R n2 ; R e It is hydrogen, alkyl, -NR m2 R n2 -NR m2 C(O)NR n2 R p2 -NR m2 C(O)R n2 alkyl, aryl, or heterocyclic groups, each of which is unsubstituted or substituted by one or more substituents selected from: halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclic, heterocyclic, cyano, hydroxy, hydroxyalkyl, alkoxy, hydroxyalkoxy, -NR m3 C(O)NR n3 R p3 -NR m3 C(O)R n3 -C(O)NR m3 R n3 -NR m3 R n3 -C(O)R m3 or -C(O)OR m3 ; p is between 0 and 3; q is between 0 and 4; R f and R g Each of these groups can be independently halogenated, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclic, heteroaryl, cyano, hydroxyl, alkoxy, or -NR. m2 C(O)NR n2 R p2 -NR m2 C(O)R n2 -C(O)NR m2 R n2 -NR m2 R n2 -C(O)R m2 or -C(O)OR m2 ; in R m1 R n1 R p1 R m2 R n2 R p2 R m3 R n3 and R p3 Each is independently hydrogen, alkyl, haloalkyl, alkoxyalkyl-, phenyl, cycloalkyl, heteroaryl, or heterocyclic; or The compound of formula (II) is a compound having formula (II) or a pharmaceutically acceptable salt thereof, its isomers or its deuterated analogues. (II) in X is O, S, or NR. 1 , where R 1 It is hydrogen or alkyl; n is 1 to 12; R a and R b Each is independently either hydrogen or alkyl; R c and R d Each of these groups is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocyclic, or heteroaryl, and each of these groups is unsubstituted or substituted by one or more substituents selected from: halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclic, heteroaryl, cyano, hydroxyl, hydroxyl, alkoxy, -NR m1 C(O)NR n1 R p1 -NR m1 C(O)R n1 -C(O)NR m1 R n1 -NR m1 R n1 -C(O)R m1 or -C(O)OR m1 ; Or R c and R d Together with the nitrogen atoms to which they are attached, they form 3 to 12-membered rings containing 0 to 3 additional heteroatoms selected from oxygen, nitrogen, or sulfur, and said rings are unsubstituted or substituted by one or more substituents selected from: halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclic, heteroaryl, cyano, hydroxy, hydroxyalkyl, alkoxy, hydroxyalkoxy, -NR m2 R n2 -C(O)R m2 -C(O)OR m2 -NR m2 C(O)NR n2 R p2 -NR m2 C(O)R n2 or -C(O)NR m2 R n2 ; R e1 and R e2 Each is independently hydrogen, alkyl, -NR m2 R n2 -NR m2 C(O)NR n2 R p2 -C(O)NR m2 R n2 alkyl, aryl, heterocyclic, or heteroaryl, wherein each of the alkyl, cycloalkyl, aryl, heterocyclic, or heteroaryl groups is unsubstituted or substituted by one or more substituents selected from: halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclic, heteroaryl, cyano, hydroxy, hydroxyalkyl, alkoxy, hydroxyalkoxy, -NR m3 C(O)NR n3 R p3 -NR m3 C(O)R n3 -C(O)NR m3 R n3 -NR m3 R n3 -C(O)R m3 or -C(O)OR m3 ; p is between 0 and 3; q is between 0 and 4; R f and R g Each of these groups can be independently halogenated, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclic, heteroaryl, cyano, hydroxyl, alkoxy, or -NR. m2 C(O)NR n2 R p2 -NR m2 C(O)R n2 -C(O)NR m2 R n2 -NR m2 R n2 -C(O)R m2 -C(O)OR m2 , cycloalkyl, phenyl, heteroaryl or heterocyclic groups; in R m1 R n1 R p1 R m2 R n2 R p2 R m3 R n3 and R p3 Each of them is independently hydrogen, alkyl, haloalkyl, alkoxyalkyl-, phenyl, cycloalkyl, heteroaryl, or heterocyclic.

2. The method according to claim 1, wherein X is S.

3. The method according to claim 1 or 2, wherein n is 1 to 6, or 2 to 4, or 3.

4. The method according to any one of claims 1 to 3, wherein R a and R b Each is hydrogen.

5. The method according to any one of claims 1 to 4, wherein R c and R d Each is either hydrogen or alkyl.

6. The method according to any one of claims 1 to 5, wherein R c and R d Together with the nitrogen atoms to which they are attached, they form a 5- to 7-membered ring containing 0 to 1 additional heteroatom selected from oxygen, nitrogen, or sulfur, and the ring is unsubstituted or substituted by one or more substituents selected from halogen, alkyl, hydroxy, hydroxyalkyl, alkoxy, or hydroxyalkoxy.

7. The method according to any one of claims 1 to 6, wherein R c and R d Together with the nitrogen atoms to which they are attached, they form a 6-membered ring containing 0 to 1 additional nitrogen heteroatom, and the ring is substituted by one or more substituents selected from the following: halogen, alkyl, hydroxy, hydroxyalkyl, alkoxy, or hydroxyalkoxy.

8. The method according to any one of claims 1 to 7, wherein p is 0.

9. The method according to any one of claims 1 to 8, wherein q is 0.

10. The method according to any one of claims 1 to 9, wherein R e It is hydrogen, alkyl, or -NR m2 R n2 , where R m2 and R n2 It is as defined in equation (I).

11. The method according to any one of claims 1 to 10, wherein R e1 and R e2 Each can be independently hydrogen, alkyl, aryl, or -NR. m2 R n2 , where R m2 and R n2 It is as defined in equation (I).

12. The method according to any one of claims 1 to 10, wherein R e1 It is an alkyl group and R e2 It is hydrogen, alkyl, aryl, or -NR m2 R n2 , where R m2 and R n2 It is as defined in equation (I).

13. The method according to any one of claims 1 to 12, wherein R e1 and R e2 They are different.

14. The method according to any one of claims 1 to 13, wherein with R e1 and R e2 The attached carbon atom is a chiral center.

15. The method according to any one of claims 1 to 14, wherein with R e1 and R e2 The attached carbon atoms are in (S)- or (R)- configuration.

16. The method according to any one of claims 1 to 15, wherein with R e1 and R e2 The attached carbon atom has an (S)- configuration.

17. The method according to any one of claims 1 to 16, wherein X is S; n is 1 to 6, or 2 to 4, or 3; R a and R b Each is hydrogen; p and q are both 0; R c and R d Each is independently an alkyl group; or R c and R d Together with the nitrogen atoms to which they are attached, they form a 5- to 7-membered ring containing 0 to 1 additional heteroatoms selected from oxygen, nitrogen, or sulfur, and said ring is unsubstituted or substituted by one or more substituents selected from: halogen, alkyl, hydroxy, hydroxyalkyl, alkoxy, or hydroxyalkoxy; or R c and R d Together with the nitrogen atoms to which they are attached, they form a 6-membered ring containing 0 to 1 additional nitrogen heteroatom, and said ring is substituted by one or more substituents selected from: halogen, alkyl, hydroxy, hydroxyalkyl, alkoxy, or hydroxyalkoxy; R e It is an alkyl group; or R e1 and R e2 Each is independently hydrogen, alkyl; or R e1 It is hydrogen and R e2 It is hydrogen or alkyl.

18. The method according to any one of claims 1 to 17, wherein the compound is , ,or .

19. The method according to any one of claims 1 to 17, wherein the compound is or .

20. The method according to any one of claims 1 to 19, wherein the contact is performed in vitro.

21. The method according to any one of claims 1 to 20, wherein the contact is performed in vivo.

22. The method according to any one of claims 1 to 21, wherein the method further comprises administering the compound to a subject.

23. The method according to any one of claims 1 to 22, wherein the target protein is any protein that forms a multimer, the multimer being defined as multiple copies of a gene-encoded polypeptide that form a complex.

24. The method according to any one of claims 1 to 23, wherein the target protein is a multimeric protein that causes human disease.

25. The method according to any one of claims 1 to 24, wherein the target protein is a protein aggregate or protein condensate.

26. The method according to any one of claims 1 to 24, wherein the target protein is amyloid protein or an amyloid protein-like aggregate.

27. The method according to any one of claims 1 to 24, wherein the amyloid-like protein aggregates comprise Tau, α-synuclein, and mutant huntingtin.

28. The method according to any one of claims 1 to 24, wherein the target protein is NUP98-fusion protein, EWS-FLI1, Tau fibrils, α-synuclein fibrils, mutant huntingtin protein, cGAS, or NLRP3.

29. The method according to any one of claims 1 to 28, wherein the degradation is achieved by recruiting TRIM21 ubiquitin E3 ligase.

30. The method according to any one of claims 1 to 29, wherein the compound of formula (I) or the compound of formula (II) induces TRIM21 to be recruited to the vicinity of the NPC to trigger the degradation of nucleoporin.

31. A method for treating or preventing a disease characterized by amyloid protein or amyloid-like aggregates, the method comprising administering to a subject in need a therapeutically effective amount of a compound of formula (I), a compound of formula (II), or a molecular gel compound or PROTAC carrying a compound of formula (I) or (II) as a ligand for recruiting TRIM21 ligase.

32. The method of claim 31, wherein the disease is Alzheimer's disease, Huntington's disease and Parkinson's disease, type 2 diabetes, autoimmune disease, or amyloidosis caused by systemic or local deposition of amyloid fibrils in the intracellular and extracellular spaces of tissues and organs.

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