Small molecule inhibitors of non-canonical nf-kb pathway and uses thereof

Abstract

The present application relates to small molecule inhibitors of non-canonical NF-kB pathway and uses thereof. In particular, the present application provides the use of an agent capable of inhibiting or preventing the binding of RelB protein to its target DNA or an agent capable of inhibiting the transcription function of RelB protein in the preparation of a medicament for treating or preventing a disease that benefits from the inhibition of non-canonical NF-kB pathway due to the blocked binding of RelB protein to its target DNA.

Description

TECHNICAL FIELD

[0001] The present application relates to small molecule inhibitors of non-canonical NF-kB pathway and uses thereof. BACKGROUND

[0002] Nuclear Factor-kB (NF-kB) is a family of transcription factors involved in many important biological processes, including immune response, inflammatory response, tumorigenesis, cell survival, proliferation, development and differentiation, etc. The mammalian NF-kB family mainly contains five family members: NF-kB1 (p50), NF-kB2 (p52), RelA (p65), RelB and c-Rel. The five proteins all contain a region for DNA binding and dimerization, about 300 amino acids, called REL homology region. At the C-terminal of RelA, RelB and Rel proteins, there is a transcriptionally active region. This region is used to mediate the binding with transcription factors and auxiliary transcription factors such as TATA binding protein (TBP), TFIIB, E1A binding protein 300KD (EP300) and CREB binding protein (CBP). RelB has a leucine zipper structure (LZ) at the N-terminal.

[0003] The activation process of NF-kB pathway mainly includes: classical NF-kB pathway and non-canonical NF-kB pathway. The activation of classical NF-kB pathway depends on the induction of IkB protein degradation, especially IkBa degradation. The dimer composed of RelA and p50, when not activated, NF-kB1 (p105) is constitutively cleaved into p50 and forms a heterodimer with RelA. IkB in the cytoplasm binds to it, thereby inhibiting its nuclear transcription function. Under the stimulation of pathogen and inflammatory factors (TNFa, IL-1b and TLRs), etc., IkB protein kinase (IKK) complex phosphorylates IkB, which is degraded by ubiquitination-dependent method, thereby releasing RelA / p50, and then entering the nucleus to perform the function of transcription factor.

[0004] The non-canonical NF-kB pathway was formally recognized in 2002. The activation of the non-canonical NF-kB pathway depends on the cleavage of NF-kB2 p100. When not activated, NF-kB2 p100 resides in the cytoplasm in complex with RelB. Upon stimulation by extracellular signaling factors, mainly TNF family members (such as RANKL, LTa1b2, CD40L and BAFF, etc.), NF-kB2 p100 is cleaved into p52, removing the inhibitory signal, thus forming the p52 / RelB dimer and entering the nucleus. The activation of the non-canonical NF-kB pathway requires only some specific receptors, mainly TNF receptor superfamily members, including BAFF, CD40, LTbR, RANK, TNFR2 and Fn14, etc. These receptors can all mediate a specific biological function of the non-canonical NF-kB pathway. The common feature of the receptors activating the non-canonical NF-kB pathway is that they can recruit the TRAF3-TRAF2-cIAP1 / 2 complex upon receptor ligand binding and cause its degradation, thus causing NIK aggregation and activation and cleavage of NF-kB2 p100. Figure 1

[0005] Recent studies have shown that the non-canonical NF-kB pathway regulates very important biological processes, such as the development of the lymphatic system, negative selection of T cells, B cell survival and maturation, and bone metabolism, and abnormal activation of this pathway can cause many immune diseases and bone metabolism diseases. In addition, abnormal activation of the non-canonical NF-kB pathway is associated with the occurrence of many tumors.

[0006] ​In cancer, there are three major mechanisms that activate the non-canonical pathway: viral oncogenes, mutations in signaling pathway members, and overexpression of molecules upstream of the signal. In Kaposi's sarcoma and primary effusion lymphoma cells, the human herpesvirus 8 (HHV-8) protein vFLIP-K13 activates the NIK-independent cleavage of p100 to p52 and promotes cell survival and proliferation. The latent membrane protein (LMP)-1 of Epstein-Barr virus (EBV) has also been reported to promote the NIK-dependent cleavage of p100. Consistent with these findings, p52 is highly expressed in EBV-positive Hodgkin's lymphoma, nasopharyngeal carcinoma, and DLBCL. The hepatitis B virus HBx protein also potentiates the p52 signal by increasing the expression of Bcl-3. In hepatocellular carcinoma cell lines, the Bcl-3 / p52 complex activates the Cyclin D1 promoter, promoting cell proliferation and transformation. Chromosomal rearrangements and mutations in the p100 / p52 gene are common in many human hematological malignancies. For example, the C-terminal region of p100 is often deleted, resulting in an activated form of p100 that enters the nucleus and activates NF-kB target genes, leading to cell proliferation and survival. Such mutations are seen in many cancers, such as multiple myeloma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, and B-cell chronic lymphocytic leukemia (CLL). In addition to p52 mutations, mutations in upstream kinases and regulators of the non-canonical NF-kB pathway have also been reported. Mutations in many of the upstream regulators (TRAF2, TRAF3, cIAPl & 2, CD40, BAFFR, BIRC, and NIK) promote the stabilization of NIK and thus activate the non-canonical NF-kB pathway. This mechanism of activation plays an important role in different tumor types, including DLBCL, splenic marginal zone lymphoma, multiple myeloma, Hodgkin's lymphoma, and lung cancer. In the API2-MALT1 fusion oncoprotein in mucosa-associated lymphoid tissue (MALT) lymphoma cell lines, NIK is dissociated, thus activating the non-canonical NF-kB pathway. In addition, in human prostate cancer tumor cells, the activation of STAT3 induces the cleavage of p100 and activates the non-canonical NF-kB pathway.

[0007] The transcriptional targets of the non-canonical NF-kB pathway also vary depending on the cancer cell type. For example, in melanoma cells, the RelB-p52 complex directly regulates the transcription of the EZH2 enhancer, thereby suppressing the initiation of the senescence program. Similarly, in normal human skin fibroblasts and patient-derived CLL cells, the expression of EZH2 is dependent on p52.

[0008] Knockout studies have demonstrated the importance of each molecule in the non-canonical NF-kB pathway in B cell survival and development. For example, BAFF plays an important role in B cell survival. The BAFF-NIK-p52 signaling pathway upregulates the expression of anti-apoptotic genes Bcl-2 and Bcl-xL, which are important for cell survival. The BAFF-NIK-p52 signaling pathway also inhibits the expression of pro-apoptotic genes. During lymph node development, LTβ signals through p52 and p50 to exert biological functions. LTβ activates the expression of adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1). These two molecules are important for the formation and development of lymph nodes in mice. Meanwhile, LTβ also induces the expression of cytokines (CCL20 and CXCL3). These cytokines are important for the formation of lymph nodes and the compartmentalization of B cells and T cells in the lymph nodes. Knocking out TRAF3 in B cells leads to an increase in plasma cells in mice.

[0009] The non-canonical NF-kB pathway plays an important role in some immune cells other than B cells. It is required for the expression of inducible costimulator (ICOS) ligand in B cells. ICOS is important for the development of follicular helper T cells (Tfh cells). Specific knockout of TRAF3 in mouse regulatory T cells (Treg) promotes antigen-induced Tfh cell activation, promotes germinal center formation, and IgG antibody production. TRAF3 -deficient Treg cells exhibit a lack of ICOS expression. In NIK-deficient mice, the number of dendritic epidermal T cells on the skin is significantly reduced. In Th17 cells, the P52-c-Rel heterodimer is involved in the regulation of the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF), IL23R, and CD80. Th17 plays an important role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE) in mice (Yue et al., 2014).

[0010] In DC cells, the activated non-canonical NF-kB pathway induced by CD40L upregulates the expression of indoleamine 2,3 dioxygenase (IDO) and downregulates the expression of proinflammatory factors, thereby increasing the number of Tregs. In addition, knocking out p52 in DCs increases the expression of activation marker molecules CD80 and major histocompatibility complex II (MHC II), which suggests that the activation of p52 negatively regulates the function of DCs in mice. In another study, knocking out NIK in CD11c + Knocking out NIK in DCs does not affect the development of normal lymphoid organs, but leads to the loss of antigen cross-priming. Specific knockout of LTβR in endothelial cells leads to the loss of peripheral lymph node development in mice, demonstrating the role of endothelial cells in lymph node formation.

[0011] In mouse bone marrow-derived macrophages (BMDMs), RelB / p52 can bind to the promoter of the Ifnb gene, preventing the binding of p65 and inhibiting the expression of interferon, which is important for antiviral responses. Bone marrow cell-specific knockout of TRAF2 promotes the expression of proinflammatory cytokines in BMDMs. TRAF3-deficient BMDMs show up-regulation of c-Rel and IRF5 protein expression. In another independent study, bone marrow cell-specific knockout of TRAF3 leads to spontaneous chronic inflammation and tumors in aged (15-22 month) mice.

[0012] The non-canonical NF-kB pathway also plays an important role in different immune diseases. Over-activation of the non-canonical NF-kB pathway can lead to autoimmune diseases and other related pathological states. For example, mice over-expressing p52 develop inflammatory autoimmune disease due to the self-activation of lymphocyte survival, which is caused by the inhibition of the expression of the anti-apoptotic gene Bim1 by p52. In another study, mice constitutively activating p52 develop lung inflammation, accompanied by alveolar damage and fibrosis. In addition, T cells derived from NIK-deficient mice also fail to induce graft-versus-host disease when transplanted into MHC II-mismatched mice.

[0013] In patients with rheumatoid arthritis, activation of NOTCH leads to p52 activation in osteoblasts and synoviocytes. In fact, p52 activation promotes the production of proinflammatory factors and at the same time inhibits osteoblast formation, which leads to chronic inflammation, bone loss, and cartilage destruction, which are all hallmarks of rheumatoid arthritis. In summary, persistent activation of the non-canonical NF-kB pathway signaling is often associated with defects (autoimmunity and tolerance). SUMMARY

[0014] The present application provides a compound of the following formula I:

[0015]

[0016] wherein,

[0017] A1, A2, and A3 are each independently selected from CR1 or N;

[0018] D is selected from CR2, O, S, or NR3;

[0019] E and F are each independently selected from C(R4)2 or NR5;

[0020] L is a linking group;

[0021] Ar is selected from optionally substituted C6-14aryl, optionally substituted C3-C8 carbocyclyl, optionally substituted C3-C10 heterocyclyl, or optionally substituted C5-C10 heteroaryl;

[0022] R1and R2are each independently selected from H, C1-C6alkyl, halogen, C1-C3alkylthio, or OH;

[0023] R3is selected from H or C1-C6alkyl;

[0024] R4and R5are each independently selected from H, C1-C6alkyl, halogen, or hydroxyl, or R4, R5, together with the ring atoms to which they are attached, form a 5-8 membered saturated or unsaturated ring optionally containing 1, 2, or 3 heteroatoms selected from O, S, and N in addition to E and F.

[0025] In certain embodiments, the compound of Formula I has the structure of Formula II:

[0026]

[0027] wherein,

[0028] Ar is selected from optionally substituted C6-14aryl, optionally substituted C3-C8 carbocyclyl, optionally substituted C3-C10 heterocyclyl, or optionally substituted C5-C10 heteroaryl.

[0029] In certain embodiments, the compound of Formula I has the structure of Formula III:

[0030]

[0031] wherein,

[0032] R6is selected from H or C1-C6alkyl;

[0033] Ar is selected from optionally substituted C6-14aryl, optionally substituted C3-C8 carbocyclyl, optionally substituted C3-C10 heterocyclyl, or optionally substituted C5-C10 heteroaryl.

[0034] In certain embodiments, Ar is C6-C14aryl optionally substituted with 1, 2, 3, or 4 substituents selected from hydroxyl, C1-C6alkyl, and halogen.

[0035] In certain embodiments, Ar has at least one hydroxyl substituent. Preferably, the hydroxyl substituent is located at the ortho position.

[0036] The present application also provides a pharmaceutical composition comprising a compound of Formula I, II, or III, or a pharmaceutically acceptable salt thereof, as described herein, and a pharmaceutically acceptable carrier or excipient.

[0037] The present application also provides use of an agent capable of inhibiting the binding of RelB protein to its target DNA in the manufacture of a medicament for treating or preventing a disease that benefits from the inhibition of the non-canonical NF-κΒ pathway by blocking the binding of RelB protein to its target DNA.

[0038] In certain embodiments, the agent is a compound of Formula I, II, and III, or a pharmaceutically acceptable salt thereof, as described herein.

[0039] In certain embodiments, the agent is a nucleic acid molecule selected from the group consisting of a targeting vector, an siRNA, and an expression vector for interfering RNA.

[0040] In certain embodiments, the agent is a specific antibody of RelB.

[0041] The present application also provides a method of treating or preventing a disease that benefits from the inhibition of the non-canonical NF-κΒ pathway, comprising administering to a subject in need thereof a therapeutically effective amount of an agent capable of inhibiting the binding of RelB protein to its target DNA.

[0042] In certain embodiments, the agent is a compound of Formula I, II, or III, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, as described herein.

[0043] The present application also provides an agent capable of inhibiting the binding of RelB protein to its target DNA for use in a method of treatment or prevention of a disease that benefits from the inhibition of the non-canonical NF-κΒ pathway by blocking the binding of RelB protein to its target DNA, comprising a compound of Formula I, II, or III, or a pharmaceutically acceptable salt thereof.

[0044] The present application also provides a genetically engineered cancer cell, wherein the expression of RelB is reduced or completely absent, or the expressed RelB protein has no binding activity or reduced binding activity to its target DNA, as compared to a cancer cell that has not been genetically engineered in the same way.

[0045] The present application also provides a cell culture for drug screening, comprising a cancer cell as described herein. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 : Non-canonical NF-kB pathway therapeutic targets (Vinay Tergaonkar 2016).

[0047] Figure 2: Small molecule drug screening system targeting RelB protein. a, Q-PCR detection of c-Myc RNA expression level in HCT116 cell line; b, Western Blot detection of c-Myc protein expression level in HCT116 cell line; c, Dual luciferase reporter gene detection of c-Myc reporter gene activity. d, MTT experiment detection of HCT116 cell activity at different time points.

[0048] Figure 3 : RelB / p52 / DNA and small molecule drug structure simulation diagram. RelB protein three-dimensional structure diagram; RelB / p52 / DNA and small molecule drug structure simulation (1 and 2 are DNA, 3 is small molecule drug, and the periphery is RelB / p52 protein dimer).

[0049] Figure 4 : Inhibition of c-Myc by small molecule drugs. a, Detection of the effect of small molecule drugs on c-Myc reporter gene in dual luciferase reporter gene experiment; b, Western Blot detection of the effect of small molecule drugs on c-Myc protein level in HCT116 cell line.

[0050] Figure 5 : Detection of cell activity in HCT116 cells. a, Detection of cell activity in HCT116 cells by different concentrations of small molecule drugs; b, summary of figure a; c, IC50 of RS09 and RS47 on HCT116 determined by cell activity detection experiment.

[0051] Figure 6 : Effect of small molecule drugs on the activity of different cell lines. a, Effect of small molecule drugs on the activity of colon cancer cell lines; b, Effect of small molecule drugs on the activity of normal intestinal epithelial cell lines; c, Effect of small molecule drugs on the activity of hepatocarcinoma cell lines; d, Effect of small molecule drugs on the activity of lung cancer cell lines

[0052] Figure 7 : Verification of small molecule drugs that can bind to RelB protein to prevent its binding to target DNA. a, Detection of the effect of small molecule drugs on NF-kB reporter gene in dual luciferase reporter gene experiment; b, Detection of the effect of small molecule drugs on Bcl-3 reporter gene in dual luciferase reporter gene experiment; c, EMSA experiment to verify the effect of small molecule drugs on the binding of RelB protein to the probe; d, Pulldown experiment to verify whether small molecule drugs can bind to RelB molecules; e, Realtime PCR detection of whether small molecule drugs affect the RNA expression level of TNF.

[0053] Figure 8: In the B cell system induced by BAFF stimulation, the inhibitory effect of small molecule drugs on non-canonical NF-kB pathway was detected. a, Realtime PCR detected the RNA expression level of pim2 gene; b, flow cytometry detected the inhibitory effect of small molecule drugs on the survival of human-derived B lymphoma cells; flow cytometry detected the inhibitory effect of small molecule drugs on the survival of mouse primary B cells.

[0054] Figure 9 : Immunofluorescence co-localization of small molecule drugs and RelB protein.

[0055] Figure 10 : Small molecule drugs inhibit the growth of HCT116 cells. a, flow cytometry analysis of the effect of small molecule drugs on HCT116 cell cycle; b, statistical chart of a result; c, flow cytometry analysis of the effect of small molecule drugs on HCT116 cell apoptosis; d, statistical chart of c result; e, effect of small molecule drugs on HCT116 clone formation.

[0056] Figure 11 : Small molecule drugs inhibit the growth of SW620 cells. a, flow cytometry analysis of the effect of small molecule drugs on HCT116 cell cycle; b, statistical chart of a result; c, flow cytometry analysis of the effect of small molecule drugs on HCT116 cell apoptosis; d, statistical chart of c result; e, effect of small molecule drugs on HCT116 clone formation.

[0057] Figure 12 : Small molecule drug RS47 promotes B lymphoma cell apoptosis. a, effect of small molecule drugs on BJAB cell apoptosis; b, statistical chart of a result; c, effect of small molecule drugs on human-derived B lymphoma cell apoptosis; d, effect of small molecule drugs on BMSC cell apoptosis.

[0058] Figure 13 : Small molecule drugs inhibit HCT116 tumor formation in nude mice. a, HCT116 tumor formation in nude mice; b, statistical chart of subcutaneous tumor mass; c, expression level of C-Myc RNA in tumor cells; d, expression level of C-Myc protein in tumor cells. DETAILED DESCRIPTION

[0059] It should be understood that, within the scope of the present application, each of the above technical features of the present application and each of the technical features specifically described below (such as examples) can be combined with each other to form a preferred technical solution.

[0060] The present application discloses small molecule compounds capable of binding to RelB protein, intercalating into the site where RelB protein binds to target DNA, thereby preventing RelB protein from binding to DNA and inhibiting its transcription function, and thus inhibiting the non-canonical NF-κB pathway, and thus being useful for treating or preventing various diseases mediated by the non-canonical NF-κB pathway, including cancer and inflammatory diseases.

[0061] The small molecule compounds of the present application include compounds of the following Formula I or pharmaceutically acceptable salts thereof:

[0062]

[0063] wherein,

[0064] A1, A2 and A3 are each independently selected from CR1 or N;

[0065] D is selected from CR2, O, S or NR3;

[0066] E and F are each independently selected from C(R4)2 or NR5;

[0067] L is a linker;

[0068] Ar is selected from optionally substituted C6-14 aryl, optionally substituted C3-C8 carbocyclyl, optionally substituted C3-C10 heterocyclyl, or optionally substituted C5-C10 heteroaryl;

[0069] R1 and R2 are each independently selected from H, C1-C6 alkyl, halogen, C1-C3 alkylthio, or OH;

[0070] R3 is selected from H or C1-C6 alkyl;

[0071] R4 and R5 are each independently selected from H, C1-C6 alkyl, halogen, or hydroxyl, or R4, R5 together with the ring atom to which they are attached form a 5-8 membered saturated or unsaturated ring optionally containing 1, 2, or 3 heteroatoms selected from O, S, and N in addition to E and F.

[0072] As used herein, "alkyl" refers to straight chain or branched chain C1-C6 alkyl, preferably C1-C4 alkyl, and more preferably C1-C3 alkyl. Typical alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, t-butyl, and the like.

[0073] As used herein, "alkylthio" refers to a thio group substituted with an alkyl group as described herein.

[0074] As used herein, "aryl" refers to a monocyclic, bicyclic, or tricyclic aromatic radical having from 6 to 14 carbon atoms. Preferred aryl groups are C6-10 aryl groups. Typical C6-14 aryl groups include phenyl, naphthyl, phenanthryl, anthryl, indenyl, azulenyl, biphenyl, bisphenylene, and fluorenyl.

[0075] As used herein, "carbocyclyl" includes cycloalkyl and partially saturated carbocyclic groups. Useful cycloalkyl groups are C3-8 cycloalkyl groups. Typical cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. Useful partially saturated carbocyclic groups are cycloalkenyl groups having 3 to 8 ring carbon atoms, which can be cyclopentenyl, cycloheptenyl, and cyclooctenyl.

[0076] As used herein, "halogen" includes fluorine, chlorine, bromine, and iodine.

[0077] As used herein, "haloalkyl" includes C1-C6 alkyl groups, preferably C1-C4 alkyl groups, substituted with one or more fluorine, chlorine, bromine, or iodine atoms. Typical haloalkyl groups include fluoromethyl, difluoromethyl, trifluoromethyl, pentafluoroethyl, 1,1-difluoroethyl, chloromethyl, chlorofluoromethyl, and trichloromethyl.

[0078] As used herein, "heterocyclyl" refers to a saturated or partially saturated 3-7 membered monocyclic ring, or 7-10 membered bicyclic ring system, consisting of carbon atoms and optionally 1-4 heteroatoms selected from O, N, and S. Useful saturated or partially saturated heterocyclic groups include tetrahydrofuranyl, pyranyl, piperidinyl, piperazinyl, pyrrolidinyl, imidazolidinyl, imidazolinyl, dihydroindolyl, iso- dihydroindolyl, quinuclidinyl, morpholinyl, isochromanyl, chromanyl, pyrazolidinyl, and pyrazolinyl.

[0079] As used herein, "heteroaryl" refers to a group containing 5-14 ring atoms, and having 6, 10, or 14 electrons shared in a cyclic array. The ring atoms contained are carbon atoms and optionally 1-3 heteroatoms from oxygen, nitrogen, and sulfur. Useful heteroaryl groups include thienyl (thiophenyl), benzo[d]isothiazol-3-yl, benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furanyl, pyranyl, isobenzo furanyl, chromenyl, xanthenyl, thioxanthyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, including but not limited to 2-pyridyl, 3-pyridyl, and 4-pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, phthalazinyl, naphthylidinyl, quinazolinyl, cinnolinyl, pteridinyl, carbazolyl, beta-carbolinyl, phenanthridinyl, acridinyl, perimidinyl, phenoxazinyl, phenothiazinyl, isoxazolyl, furazanyl, phenoxazinyl, 1,4-dihydroquinoxaline-2,3-dione, 7-aminoiso coumarin, pyrido[1,2-a]pyrimidin-4-one, tetrahydro- five-membered [c]pyrazol-3-yl, pyrazo[1,5-a]pyrimidinyl, benzoisoxazolyl such as 1,2- benzoisoxazol-3-yl, benzoimidazolyl, 2-hydroxyindolyl, thioazolyl, and 2-oxobenzimidazolyl.

[0080] In the present text, for the groups mentioned which can be substituted, the number of substituents, when substituted, can be 1, 2, 3 or 4, and the substituents can be selected from the group consisting of halogen, hydroxy, carboxy, amino, nitro, cyano, Cι-6acylamino, Cι-6acyloxy, Cι-6alkoxy, aryloxy, alkylthio, C6-Cι0aryl, C3-C8cycloalkyl, C2-C6alkenyl, C2-C6alkynyl, saturated and unsaturated heterocyclyl or heteroaryl.

[0081] In formula I of the present application, preferably, at least one of A1, A2and A3is N, preferably at least two of them are N. In certain embodiments, of A1, A2and A3, A1and A3are N, and A2is CR1.

[0082] In preferred embodiments, R1is H. In certain embodiments, R1may be Cι-C6alkyl or Cι-3alkylthio.

[0083] In preferred embodiments, D is O or S.

[0084] In preferred embodiments, E and F are each C(R4)2, and of E and F, one R4of each is H, and the other two R4together with the C to which they are attached form a 5-8 membered saturated or unsaturated ring optionally containing 1, 2 or 3 heteroatoms selected from the group consisting of O, S and N, in addition to the carbons of E and F, preferably a 5-8 membered saturated carbocyclic ring, more preferably a cyclohexane.

[0085] Thus, in certain embodiments, the ring containing A1-A3and D-F in the compound of formula I is a 4b,5,6,7,8,8a-hexahydrobenzo[4,5]-thieno[2,3-d]pyrimidine ring.

[0086] In preferred embodiments of formula I, L is a linking group containing N and C. In certain embodiments, L is -NH-N=C(R7)- or -NH-NH-C(R8)-NH-C(R8)-, wherein R7is H or Cι-C3alkyl; each R8is independently S or O.

[0087] In preferred embodiments of formula I, Ar is preferably C6-Cι4aryl optionally substituted with 1, 2, 3 or 4 substituents selected from the group consisting of hydroxy, halogen and Cι-C3alkyl, more preferably phenyl substituted with 1, 2, 3 or 4 substituents selected from the group consisting of hydroxy, halogen and Cι-C3alkyl.

[0088] Thus, in certain embodiments, the compound of formula I has the following structure of formula II:

[0089]

[0090] wherein

[0091] L is a linking group;

[0092] Ar is selected from optionally substituted C6-14 aryl, optionally substituted C3-C8 carbocyclic, optionally substituted C3-C10 heterocyclic, or optionally substituted C5-C10 heteroaryl.

[0093] In a preferred embodiment of Formula II, L is preferably a linking group containing N and C. In some embodiments, L is -NH-N=C(R7)- or -NH-NH-C(R8)-NH-C(R8)-, wherein R7 is H or a C1-C3 alkyl group; each R7 is independently S and O.

[0094] In a preferred embodiment of Formula II, Ar is preferably a C6-C14 aryl group optionally substituted with 1, 2, 3 or 4 substituents selected from hydroxyl, halogen and C1-C3 alkyl, more preferably a phenyl group substituted with 1, 2, 3 or 4 substituents selected from hydroxyl, halogen and C1-C3 alkyl.

[0095] Therefore, in some embodiments, the compound of formula I has the structure shown in formula III:

[0096]

[0097] In the formula,

[0098] R6 is selected from H or C1-C6 alkyl groups;

[0099] Ar is selected from optionally substituted C6-14 aryl, optionally substituted C3-C8 carbocyclic, optionally substituted C3-C10 heterocyclic, or optionally substituted C5-C10 heteroaryl.

[0100] In a preferred embodiment of Formula III, Ar is preferably a C6-C14 aryl group optionally substituted with 1, 2, 3 or 4 substituents selected from hydroxyl, halogen and C1-C3 alkyl, more preferably a phenyl group substituted with 1, 2, 3 or 4 substituents selected from hydroxyl, halogen and C1-C3 alkyl.

[0101] In some embodiments, the compounds of formula I, II, or III include compounds RS07, RS09, RS10, RS47, and RS51 described herein. In other embodiments, the compounds of formula I, II, and III do not include compounds RS07, RS09, RS10, RS47, and RS51 described herein.

[0102] The compounds of the present invention can be prepared using methods known in the art. In particular, other compounds falling within general formula I herein can be prepared using suitable starting materials and reagents, referring to the methods for preparing compounds RS07, RS09, RS10, RS47 and RS51.

[0103] Some of the compounds of this invention may exist as stereoisomers, including optical isomers. This invention includes all stereoisomers and racemic mixtures of such stereoisomers, as well as individual enantiomers that can be isolated according to methods well known to those skilled in the art.

[0104] Examples of pharmaceutically acceptable salts in this article include inorganic and organic acid salts, such as hydrochloride, hydrobromide, phosphate, sulfate, citrate, lactate, tartrate, maleate, fumarate, mandelate, and oxalate; as well as inorganic and organic base salts formed with bases such as sodium hydroxyl, tris(hydroxymethyl)aminomethane (TRIS, aminobutanetriol), and N-methylglucosamine.

[0105] This document also provides a pharmaceutical composition comprising a compound of formula I, II, or III as described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In this document, "pharmaceutically acceptable carrier" refers to an inactive ingredient, such as a solid, semi-solid, or liquid filler, diluent, coating material, formulation adjuvant, excipient, or carrier, used in combination with a therapeutic agent to form a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to the subject at the dosage and concentration used and is compatible with other components in the formulation. A pharmaceutically acceptable carrier is suitable for the formulation used. For example, if the therapeutic agent is to be administered orally, the carrier may be a gel capsule. If the therapeutic agent is to be administered subcutaneously, ideally, the carrier is non-irritating to the skin and does not cause a reaction at the injection site.

[0106] The pharmaceutical composition can be prepared by mixing the compound described herein with one or more optional pharmaceutically acceptable carriers, having the desired purity. Pharmaceutically acceptable carriers may include buffers (such as phosphates, citrates, and other organic acids); antioxidants (such as ascorbic acid and methionine); preservatives (such as octadecylbenzyldimethylammonium chloride, hexamethyldiammonium chloride, benzalkonium chloride, benzyl chloride, phenol, butanol or benzyl alcohol, alkyl esters of p-hydroxybenzoate such as methylparaben or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); hydrophilic polymers (such as polyvinylpyrrolidone); amino acids (such as glycine, glutamine, asparagine, histidine, arginine, or lysine); monosaccharides; disaccharides; and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as polyethylene glycol (PEG).

[0107] Exemplary pharmaceutical carriers can also include binders such as pregelatinized cornstarch, polyvinylpyrrolidone or hydroxypropyl methylcellulose; fillers such as lactose or other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylate or dicalcium phosphate; lubricants such as magnesium stearate, talc, silicon dioxide, colloidal silicon dioxide, stearic acid, metal stearate, hydrogenated vegetable oil, cornstarch, polyethylene glycol, sodium benzoate, sodium acetate; disintegrants such as starch, sodium starch glycolate; and wetting agents such as sodium lauryl sulfate.

[0108] The pharmaceutical composition can be in any suitable dosage form, including but not limited to dosage forms suitable for various modes of administration, such as dosage forms suitable for oral, intravenous, topical or external administration. The pharmaceutical composition can contain a suitable amount of the compound of the present application, which can be determined by the manufacturer according to the actual situation.

[0109] The present application inhibits the non-canonical NF-κB pathway by inhibiting the binding of RelB protein to its target DNA and inhibiting its transcription function. Based on this, the present application can be implemented using an agent that can inhibit or prevent the binding of RelB protein to its target DNA, or an agent that can inhibit the transcription function of RelB protein. Such agents can be inhibitors of RelB protein expression or activity known in the art, including inhibitors that can reduce RelB protein expression at the genetic level and inhibitors that can inhibit the binding activity of RelB protein to its target DNA at the protein level. The expression of RelB protein can be reduced by administering a suitable nucleic acid molecule. For example, the suitable nucleic acid molecule can be a suitable targeting vector to knock out the RelB gene sequence from the genome, so that the cell of interest does not express RelB protein; or introduce a mutation in the RelB gene sequence, so that the cell of interest expresses an inactive or weakly active RelB protein. Of particular interest are nucleic acid molecules that can introduce a mutation at the known binding site of RelB protein to its target DNA, so that the binding of RelB protein to its target DNA is weak or has no binding activity. The suitable nucleic acid molecule can also be an siRNA that can inhibit RelB expression and an RNA interference vector of RelB gene. Suitable targeting vectors, siRNAs and RNA interference vectors can be constructed using conventional methods in the art. In certain embodiments, the inhibitor of RelB is a protein inhibitor, such as a specific antibody of RelB; or a small molecule compound, such as a compound of formula I, II or III of the present application or a pharmaceutically acceptable salt thereof, particularly including compounds RS51, RS07, RS09, RS10 and RS47.

[0110] It is understood that the non-canonical NF-κΒ pathway, RelB protein, and target DNA thereof described herein are well known in the art. Thus, the diseases that can be treated or prevented herein are diseases mediated by the non-canonical NF-κΒ pathway, especially diseases that benefit from inhibition of the non-canonical NF-κΒ pathway, and more preferably diseases that benefit from inhibition of the non-canonical NF-κΒ pathway by blocking the binding of RelB protein to its target DNA.

[0111] The diseases described herein include any disease caused by over-activation of the non-canonical NF-κΒ pathway, including but not limited to various cancers, autoimmune diseases, inflammatory diseases, and bone-related diseases. In particular, since the agents described herein, especially the compounds of Formula I, II, or III, block the non-canonical NF-κΒ signaling, they can prevent the accumulation of inflammatory cells and tumor stromal cells in the tumor microenvironment by inhibiting the production of chemokines, thereby inhibiting tumor vascular development and formation, and inhibiting tumor growth. Thus, the cancers that can be treated or prevented by the compounds described herein can be solid and hematological tumors, including but not limited to hepatocarcinoma, melanoma, Hodgkin's disease, lymphoma, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, Wilms' tumor, cervical cancer, testicular cancer, soft tissue sarcoma, primary macroglobulinemia, bladder cancer, chronic myelocytic leukemia, primary brain cancer, malignant melanoma, small cell lung cancer, gastric cancer, colon cancer, malignant pancreatic islet tumor, malignant carcinoid cancer, choriocarcinoma, mycosis fungoides, head and neck cancer, osteogenic sarcoma, pancreatic cancer, acute myelogenous leukemia, hairy cell leukemia, rhabdomyosarcoma, Kaposi's sarcoma, urogenital tumor disease, thyroid cancer, esophageal cancer, malignant hypercalcemia, cervical hyperplasia, renal cell carcinoma, endometrial cancer, polycythemia vera, idiopathic thrombocytopenia, adrenal cortex cancer, skin cancer, and prostate cancer. The autoimmune diseases can be systemic lupus erythematosus, multiple sclerosis, and autoimmune hepatitis. The inflammatory diseases can be intestinal inflammation or rheumatoid arthritis. The bone-related diseases can be osteoporosis, etc.

[0112] The present application also provides a method for treating or preventing a disease that benefits from inhibition of the non-canonical NF-κB pathway, comprising administering to a subject in need thereof a therapeutically effective amount of an agent that inhibits or prevents binding of a RelB protein to its target DNA, or an agent that inhibits the transcriptional function of a RelB protein, particularly a compound of Formula I, II, or III, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In practicing the methods of treatment or prevention described herein, a therapeutically or prophylactically effective amount of a compound or a pharmaceutical composition thereof is administered to a subject in need thereof. The subject can be a mammal, particularly a human. The amount administered is an amount effective to ameliorate or eliminate one or more symptoms of the disease. For treatment of a particular disease, the effective amount is an amount sufficient to ameliorate or in some way diminish the symptoms associated with the disease. Such an amount can be administered as a single dose, or can be administered according to a regimen effective to treat the disease. The amount administered can be curative of the disease, but administration is generally to ameliorate the symptoms of the disease. Repeated administration is generally required to achieve the desired amelioration of symptoms.

[0113] While the amount of each component in a pharmaceutical formulation can vary between individuals, those skilled in the art will be able to determine appropriate dosages for each component. In general, the compounds described herein, or a pharmaceutically acceptable salt thereof, are administered orally to mammals in an amount of about 0.0025 to 50 mg / kg body weight per day. Preferably, the compounds are administered orally in an amount of about 0.01 to 10 mg / kg body weight per day. A unit oral dose can include about 0.01 to 50 mg, preferably about 0.1 to 10 mg, of a compound of the present application. The unit dose can be administered once or more than once a day, in one or more tablets or capsules each containing about 0.1 to 50 mg, conveniently about 0.25 to 10 mg, of a compound of the present application or solvate thereof.

[0114] The present application also provides an agent that inhibits or prevents binding of a RelB protein to its target DNA, or an agent that inhibits the transcriptional function of a RelB protein, particularly a compound of Formula I, II, or III, or a pharmaceutically acceptable salt thereof, for use in a method of treatment or prevention for treating or preventing a disease described herein that benefits from inhibition of the non-canonical NF-κB pathway by inhibition of binding of a RelB protein to its target DNA.

[0115] The present application also provides a genetically engineered cancer cell in which the expression of RelB is reduced or in which RelB is not expressed at all, or in which the RelB protein expressed has no or reduced binding activity to its target DNA, as compared to a cancer cell that has not been genetically engineered in the same way. Preferably, the genetically engineered cancer cell is a colon cancer cell.

[0116] The present application therefore also provides a cell culture containing said genetically engineered cancer cells.

[0117] The following examples are illustrative, but not limiting, of the methods and compositions of the present application. Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered in clinical therapy and which are obvious to those skilled in the art are within the spirit and scope of the application.

[0118] I. Experimental Materials

[0119] 1. Experimental Instruments

[0120] The experimental instruments include centrifuge, PCR instrument, spectrophotometer, electrophoresis instrument, etc., all from commercially available products, such as from Beckman Coulter, Eppendorf, Dongsheng Innovation, Thermo, etc.

[0121] 2. Experimental Reagents

[0122]

[0123]

[0124]

[0125] 3. Experimental Animals

[0126] Nude mice: purchased from Shanghai Slake Experimental Animal Co., Ltd., the mice are Babl / c background, T cell immunodeficient mice, SPF level.

[0127] The experimental mice were bred in the SPF level animal house of the Healthy Science Institute of the Shanghai Institute of Life Sciences, Chinese Academy of Sciences.

[0128] 4. Experimental Methods

[0129] 4.1. Construction of Overexpression Plasmid

[0130] a. Find the DNA sequence of the relevant gene on NCBI, find the CDS region, and use the software Primer Premier5 to design amplification primers for cloning the corresponding gene sequence. The 5' end of the upstream primer and the downstream primer carries an enzyme cutting site and a protection base, respectively, and the enzyme cutting site is determined by the enzyme cutting site on the expression vector. The sequence is synthesized by Boshang Company.

[0131] Primer sequence:

[0132] hRelB F: CGgcgatagcATGCTTCGGTCTGGGCCAGCCTC (SEQ ID NO: 1)

[0133] hRelB R: CGacgcgtCTACGTGGCTTCAGGCCCCGGGG (SEQ ID NO: 2)

[0134] b. Prepare a standard primer concentration of 10 μM using the synthesized primers. Perform sequence amplification using a human cDNA library as a template. This experiment utilizes TOYOBO's KOD-Plus high-fidelity PCR enzyme to ensure a high success rate.

[0135] Reaction conditions:

[0136] Reagent Amount (μl) Primer F 1.5 Primer R 1.5 10X Buffer 5 2 mM dNTPs 5 25 mM MgSO4 3 Template 1 KOD Enzyme 1 Double distilled water Make up to 50

[0137] Reaction procedure:

[0138]

[0139]

[0140] c. Use 1% agarose gel electrophoresis to identify whether the target band appears in the PCR test. If positive, perform the following experiments.

[0141] d. PCR product recovery (Transgene)

[0142] a) Take 50 μl of PCR product, add five times the volume of solution BB, mix, and then add to the adsorption column. Let it stand for 1 min to improve the recovery efficiency. Centrifuge at 1000g for 1 min and remove the filtrate.

[0143] b) Then add 650 μl WB, centrifuge at 1000 g for 1 min, and remove the filtrate.

[0144] c) Centrifuge again at 1000g for 1min to remove residual WB from the adsorption column.

[0145] d) Place the adsorption column in a clean centrifuge tube and add 20 μl of ddH2O to the center of the column (preheating to 65°C beforehand can improve the recovery yield). Let stand for 1 min.

[0146] e) Centrifuge at 1000g for 1 min to elute DNA for the next experiment or store at -20 degrees Celsius.

[0147] e. Digestion of the vector and PCR products using a double enzyme digestion system.

[0148] Reagent Amount Vector or PCR product 2 μg or 40 μl endonuclease 1 1 μl endonuclease 2 1 μl 10X Buffer 2 μl ddH2O Make up to 50 μl

[0149] Digest at 4°C overnight or (digest at 37°C for 1 hour).

[0150] f. Agarose gel electrophoresis: cut the gel block containing the target band under UV light.

[0151] g. Gel recovery (Axygen)

[0152] a) Calculate the volume of the gel, 100 mg = 100 μl;

[0153] b) Add 3 volumes of buffer DEA, dissolve the gel at 75°C (red);

[0154] c) Add 0.5 volumes of DEA to DEB, mix well (yellow);

[0155] d) 12000 g 1 min centrifugation, remove the filtrate;

[0156] e) Add 500 μl of buffer Wl, 12000 g 1 min centrifugation, remove the filtrate;

[0157] f) Add 700 μl of buffer W2, 12000 g 30 s centrifugation, remove the filtrate;

[0158] g) Add 700 μl of buffer W2, 12000 g 1 min centrifugation, remove the filtrate;

[0159] h) 12000 g, 1 min centrifugation again to remove residual filtrate;

[0160] i) Change the centrifuge tube, add 25 μl of ddH2O, stand for 1 min;

[0161] j) 12000 g, 1 min centrifugation, collect the supernatant, and determine the concentration.

[0162] h. Use T4 ligase to connect the target fragment and the enzyme-digested vector into recombinant plasmid.

[0163] Connection system:

[0164] Reagent Amount Fragment of interest 6 μl Vector 1 μl T4 Buffer 2 μl T4 Ligase 1 μl

[0165] Connect at room temperature for 1 h.

[0166] i. Transformation

[0167] a) Take the connection product and add it to the competent cells, incubate on ice for 30 min;

[0168] b) 42°C heat shock for 90 s;

[0169] c) Place on ice for 2-3 min, add 800 μl of antibiotic-free LB, incubate at 37°C for 50 min;

[0170] d) Take the bacterial solution, centrifuge at 5000 rpm for 3 min, remove the supernatant to 200 μl, resuspend the bacterial solution;

[0171] e) In a clean bench, coat the LB plate with resistance;

[0172] f) Incubate at 37°C overnight, inverted.

[0173] j. Pick single clones into 4-5 ml LB medium containing resistance, incubate at 37°C, 250 rpm overnight.

[0174] k. Take a small amount of the overnight culture and store in 40% glycerol solution (1:1) and store at -80°C.

[0175] l. Small pipette (Axygen)

[0176] a) Centrifuge the overnight culture at 12000 rpm for 1 min, discard the supernatant;

[0177] b) Add 250 μl of buffer S1 to resuspend the pellet. Do not leave any pellet;

[0178] c) Add 250 μl of buffer S2, gently invert the tube 4-6 times, mix well, and lyse the bacteria until a clear solution is formed. Do not exceed 5 min;

[0179] d) Add 350 μl of buffer S3, gently invert the tube 6-8 times, centrifuge at 12000 g for 10 min;

[0180] e) Take the supernatant from step d) and transfer to a preparation tube (previously placed in a 2 ml centrifuge tube), centrifuge at 12000 g for 1 min, discard the filtrate;

[0181] f) Place the preparation tube back into the centrifuge tube, add 500 μl of buffer W1, centrifuge at 12000 g for 1 min, discard the filtrate;

[0182] g) Place the preparation tube back into the centrifuge tube, add 700 μl of buffer W2, centrifuge at 12000 g for 1 min, discard the filtrate;

[0183] h) Place the preparation tube back into the centrifuge tube, add 700 μl of buffer W2, centrifuge at 12000 g for 1 min, discard the filtrate;

[0184] i) Place the preparation tube back into the 2 ml centrifuge tube, centrifuge at 12000 g for 1 min;

[0185] j) Transfer the preparation tube into a new 1.5 ml centrifuge tube, add 50 μl of ddH2O in the center of the preparation tube, let stand at room temperature for 1 min, centrifuge at 12000 g for 1 min;

[0186] k) Measure the plasmid concentration, and identify the positive band by double enzyme digestion.

[0187] l) Plasmid positive band is sent for sequencing to obtain the target plasmid.

[0188] 4.2, Knockout plasmid construction

[0189] a, This experiment uses Clontech's RNAi Designer tool to design shRNA. First, use RNAi Target Sequence Selector to select the target sequence to be knocked out. Then use shRNA sequence Designer to design the corresponding shRNA sequence according to the selected target sequence. When designing the sequence, restriction enzyme cutting sites should be added at both ends of the shRNA sequence. The designed sequence is sent to Boshang Company for synthesis.

[0190] b, shRNA annealing

[0191] The synthesized shRNA is two complementary single strands, which need to be annealed to form a double-stranded DNA with sticky ends at both ends.

[0192] Annealing reaction system (Biyun Tian)

[0193] Reagent Amount Top strand 10 μl Bottom strand 10 μl 5X Annealing Buffer 10 μl ddH2O Make up to 50 μl

[0194] Annealing reaction program:

[0195] 95℃

[0196] -0.1℃ / 8s; 700 cycles

[0197] 4℃ storage

[0198] c, The following enzyme digestion, ligation, transformation, clone selection, identification, shaking bacteria, and small extraction experiments are the same as above.

[0199] 4.3, Liposome transfection

[0200] a, The cells plated in advance grow to about 70%, and cell transfection can be performed.

[0201] b, Different amounts of transfection reagent Lipo 2000 are selected according to the specifications of the plated cells. Generally, 1 μg of DNA: 2.5 μl of Lipo 2000 is used for transfection. Before transfection, dilute Lipo 2000 with serum-free medium, dilute DNA of the same volume, and mix after standing for 5 min.

[0202] c, The above mixture is allowed to stand for 20 min.

[0203] d, Add the mixture to the cells to be transfected along the wall of the culture plate, and mix the medium gently.

[0204] e. After incubating in an incubator for 4-6 hours, replace the culture medium with fresh culture medium and continue incubation.

[0205] f. Collect samples for experimentation.

[0206] 4.4 RNA Extraction

[0207] a. Remove the culture medium from the culture plate and wash the cells with pre-cooled 1X PBS to remove residual culture medium and discard the supernatant.

[0208] b. Add 1 ml of TRIzol reagent to each sample and thoroughly pipette the cells until they are completely lysed. At this point, the samples can be stored at -80 degrees Celsius.

[0209] c. Add 0.2 ml of chloroform to each sample, votex vigorously for 30 seconds, and incubate at room temperature for 3 minutes.

[0210] d. Centrifuge at 4℃, 12000 rpm, for 15 min.

[0211] e. Take a small amount of the colorless aqueous phase solution from the top layer, approximately 50% of the volume of TRIzol, to avoid taking in impurities that could affect the extraction quality.

[0212] f. Add an equal volume of isopropanol to the extracted colorless upper layer of liquid, mix by inverting the container, and incubate at room temperature for 10 minutes.

[0213] Centrifuge at 4℃, 12000rpm, for 10min, and remove the supernatant.

[0214] i. Add 75% ethanol (prepared with DEPC water) and votex vigorously.

[0215] j. Centrifuge at 4℃, 7500g, for 5 minutes, and discard the supernatant.

[0216] k. Dry the residual ethanol in the tube at room temperature and dissolve it in a certain volume of DEPC water.

[0217] 1. Determine the concentration of RNA.

[0218] 4.5 RNA reverse transcription

[0219] a. Measure the concentration of RNA and calculate the corresponding volume.

[0220] b. Reaction system

[0221] Reagent Amount 5X Master mix 10 μl Total RNA 1 μg in corresponding volume DEPC H2O Make up to 20 μl

[0222] Reaction conditions:

[0223] 37℃ for 15 minutes

[0224] 85°C 5 s

[0225] 4°C Standby

[0226] The reverse-transcribed cDNA diluted four times (adding three volumes of water) can be used for Realtime PCR or frozen at -20°C.

[0227] 4.6. Realtime PCR

[0228] Realtime PCR is performed by ABI 7500 fast, 96-well plate PCR instrument

[0229] Reagent Amount SYBR mix 10 μl Forward primer (F) 0.4 μl Reverse primer (R) 0.4 μl Dye II 0.4 μl Template (cDNA) 4 μl ddH2O 4.8 μl Total volume 20 μl

[0230] Reaction procedure (two-step method):

[0231]

[0232] RT PCR primer:

[0233]

[0234]

[0235] 4.7. Protein extraction

[0236] Protein extraction by loading method:

[0237] a. The sample cells are washed twice with pre-cooled PBS, and the supernatant is removed;

[0238] b. The 5X loading buffer is diluted to 1X in advance;

[0239] c. An appropriate amount of loading lysis solution is added to each sample;

[0240] d. The cells in the culture plate are scraped with a gun head, and the same direction is circled to ensure that all the cells are scraped off;

[0241] e. The gelatinous protein is sucked into a 1.5 ml centrifuge tube, and boiled at 100°C for 10 min;

[0242] Protein extraction by RIPA method:

[0243] a. The sample cells are washed twice with pre-cooled PBS, and the supernatant is removed;

[0244] b. Proteinase inhibitors are added in advance in the RIPA lysis solution, and an appropriate amount of RIPA lysis solution is added to each sample, which is placed on ice for 30 min, and the lysis solution is mixed every 10 min;

[0245] c. Take the lysate into a centrifuge tube, centrifuge at 13000 rpm, 4°C for 15 min;

[0246] d. Take the supernatant as the protein lysate, which can be used to determine the protein concentration by BCA method.

[0247] 4.8. Determine the protein concentration by BCA method

[0248] a. Preparation of standard sample, dilute the BSA (2000 μg / ml) stock solution according to the following table

[0249] Standard Dilution volume (PBS) Stock volume Final concentration (μg / ml) A 0 300 μl stock 2000 B 125 μl 375 μl stock 1500 C 325 μl 325 μl stock 1000 D 175 μl 175 μl standard B 750 E 325 μl 325 μl standard C 500 F 325 μl 325 μl standard E 250 G 325 μl 325 μl standard F 125 H 400 μl 100 μl standard G 25 I 400 μl 0 0

[0250] b. Prepare working reagent: BCA reagent A: BCA reagent B = 50:1;

[0251] c. Add 10 μl of each sample to 200 μl of working reagent, and mix the solution into a detachable 96-well plate;

[0252] d. Incubate at 37°C for 30 min;

[0253] e. Take out and determine the absorbance on the enzyme marker at 562 nm;

[0254] f. Obtain the standard curve using the absorbance value and the standard concentration, and calculate the protein concentration of each sample according to the standard curve;

[0255] g. When performing WB, add protein samples of the same concentration (the sample is diluted with 5X loading buffer).

[0256] 4.9. Western blotting experiment (Western Blot)

[0257] a. Loading: add 20-30 μg of protein sample to each well in the pre-configured 10% SDS-PAGE gel;

[0258] b. Electrophoresis: first concentrate the gel at a constant voltage of 80V for 40 min, and then run at a constant voltage of 120V for 60 min;

[0259] c. Membrane transfer: PVDF membrane needs to be activated with methanol for 2 min first, then put into the membrane transfer solution, wet transfer with sandwich method, arrange the membrane and gel in a certain order, put into the membrane transfer tank, connect the correct electrodes, transfer the membrane under ice bath conditions, 100V, 50 min;

[0260] d. Blocking: after membrane transfer, take out the PVDF membrane and immerse it in blocking solution, block on the shaker for 1 h;

[0261] e. Cut the membrane, cut the PVDF membrane into appropriate size according to the size of the protein band;

[0262] f. Incubate the primary antibody: dilute the antibody according to the appropriate ratio, add it to the corresponding PVDF membrane container, and incubate it overnight at 4 degrees Celsius on a vertical shaker;

[0263] g. Incubate the secondary antibody: after the primary antibody is recovered, wash the membrane in the PBST solution three times for 5 minutes each time, and place it on a horizontal shaker;

[0264] h. Then add the appropriate HRP-labeled secondary antibody, incubate it at room temperature for 1 hour, and place it on a vertical shaker;

[0265] i. Remove the secondary antibody, wash it again in the PBST solution three times for 5 minutes each time, and place it on a horizontal shaker;

[0266] j. Development: use tabletting or bio-luminescence imaging to scan the membrane and obtain the results.

[0267] 4.10 Dual-luciferase reporter gene experiment

[0268] a. According to the transfection method, pre-transfect the cells with the reporter gene plasmid and the internal reference plasmid and the corresponding other plasmids;

[0269] b. Wash the sample cells with PBS twice, add pre-diluted 1X cell lysis solution (included in the kit), and shake vigorously at room temperature for 20 minutes;

[0270] c. Absorb the cell lysis solution, centrifuge at 12,000 rpm for 5 minutes;

[0271] d. Absorb 50 μl of supernatant in a black 96-well enzyme-labeled plate;

[0272] e. During the process of lysing the cells, take out the pre-configured luciferase reagent and dissolve it at room temperature in the dark. The STOP solution needs to be prepared by adding 50X STOP substrate to the STOP buffer;

[0273] f. Use a multifunctional enzyme-labeled instrument, select spontaneous light, quickly add LARII 30 μl to the sample in the enzyme-labeled plate, read at 450 nm, and obtain the activity value of the reporter gene;

[0274] g. After reading, quickly add 30 μl of STOP solution and read again to obtain the internal reference value;

[0275] h. The relative value of the reporter gene is the ratio of the two values.

[0276] 4.11 Cell viability detection experiment

[0277] a. Place an appropriate amount of cells 1000-5000 in a 96-well culture plate, add various treatments, and use 200 μl of culture medium;

[0278] b. At the experimental time point, add WST-1 (Roche) 10 μl to each well, shake gently, continue incubation in incubator for 1-4 h, and observe the color change of the culture medium.

[0279] c. At the appropriate time point, measure the absorbance of the culture medium at OD450 nm using a multifunctional plate reader.

[0280] d. Compare the cell viability according to the different values. The better the cell viability, the greater the absorbance value.

[0281] 4.12. Extraction of nuclear proteins

[0282] Prepare the nuclear protein extraction solution

[0283]

[0284]

[0285] Before using the buffer, add

[0286] Na3VO4 to a final concentration of 1 mM

[0287] β-glycerophosphate to a final concentration of 10 mM

[0288] DTT to a final concentration of 1 mM

[0289] Protease inhibitors

[0290] Nucleoplasm separation:

[0291] a. Digest the cells into single cells and collect them in a 1.5 ml centrifuge tube.

[0292] b. Wash the cells twice with pre-cooled PBS.

[0293] c. After removing the PBS, resuspend the cells with 500 μl of buffer A.

[0294] d. Place on ice for 15 min.

[0295] e. Add 50 μl of 10% Triton (final concentration 1%) and vortex vigorously for 10 s.

[0296] f. Centrifuge at 3000 rpm for 3 min at 4°C.

[0297] g. The supernatant is the cytoplasmic protein.

[0298] h. Add an appropriate amount of buffer A to the precipitate, resuspend and centrifuge once to wash away the cytoplasmic protein, and discard the supernatant.

[0299] i. Add 50 μl of buffer C to the precipitate, blow the cells apart with a pipette, and place on ice for 15 min.

[0300] j. 4°C centrifugation for 3 min at maximum speed;

[0301] k. The supernatant is the nucleoplasmic protein.

[0302] 4.13. EMSA (Electrophoretic Mobility Shift Assay)

[0303] a. Prepare 4% polyacrylamide gel

[0304] TBE buffer (10X) 1ml Distilled water 16.2ml 39:1 Acrylamide / bisacrylamide (40%, w / v) 2ml 80% Glycerol 625 μl 10% Ammonium persulfate 150 μl TEMED 10 μl

[0305] b. Pour the mixture into the gel mold (the mold can be prepared by using conventional protein gel)

[0306] c. EMSA binding reaction

[0307] a) Sample reaction system

[0308] Nuclease free water 5 μl EMSA / Gel-Shift binding buffer (5X) 2 μl Nuclear protein or purified transcription factor 2 μl Labeled probe 1 μl Total volume 10 μl

[0309] b. Add various reagents in the above order, mix well before adding the labeled probe, and place at room temperature (20-25°C) for 10 minutes to eliminate possible non-specific binding of the probe and protein, or to allow the cold probe to react preferentially. Then add the labeled probe, mix well, and place at room temperature (20-25°C) for 20 minutes.

[0310] c) Add 1 μl of EMSA / Gel-Shift loading buffer (colorless, 10X), mix well, and load immediately. Note: Sometimes bromophenol blue can affect protein and DNA binding, so it is recommended to use colorless EMSA / Gel-Shift loading buffer as much as possible. If you feel that you cannot load the sample when using colorless loading buffer, you can add a very small amount of blue loading buffer to the colorless loading buffer until you can observe the blue color.

[0311] d. Electrophoresis:

[0312] a) Use 0.5X TBE as the electrophoresis solution. Pre-electrophorese at a voltage of 10 V / cm for 10 minutes. If there are spare loading wells during pre-electrophoresis, you can add a small amount of diluted 1X EMSA loading buffer (blue) to observe whether the voltage is running normally.

[0313] b) Add the sample mixed with the loading buffer to the loading well. Add 10 μl of diluted 1X EMSA / Gel-Shift loading buffer (blue) to the spare loading well to observe the progress of electrophoresis.

[0314] c) Electrophorese at 10 V / cm. Make sure the temperature of the gel does not exceed 30°C, and if it does, reduce the voltage accordingly. Electrophorese to the lower 1 / 4 of the gel in the blue dye bromophenol blue in the EMSA / Gel-Shift loading buffer, and stop electrophoresis.

[0315] e. Transfer:

[0316] a) Take a piece of nylon membrane that is about the same size or slightly larger than the EMSA gel, cut the corners to make marks, and soak in 0.5X TBE for at least 10 minutes. The nylon membrane can only be handled with forceps throughout, and only the corners that will not touch the sample can be handled.

[0317] b) Take two pieces of filter paper that are about the same size or slightly larger than the nylon membrane, and soak in 0.5X TBE.

[0318] c) Place the soaked nylon membrane on one of the soaked filter papers, making sure there are no air bubbles between the nylon membrane and the filter paper.

[0319] d) Carefully place the EMSA gel on the nylon membrane, making sure there are no air bubbles between the gel and the membrane.

[0320] e) Place the other soaked filter paper on the EMSA gel, making sure there are no air bubbles between the filter paper and the gel.

[0321] f) Using a wet transfer apparatus for Westerns or other similar wet transfer apparatus, transfer the probes, proteins, and complexes of probes and proteins on the EMSA gel to the nylon membrane using 0.5X TBE as the transfer buffer. For an EMSA gel that is about 10 X 8 X 0.1 cm, use the standard Western transfer apparatus from BioRad, and set the transfer at 380 mA (about 100 V) for 30-60 minutes. If the gel is thicker, extend the transfer time accordingly. Keep the transfer buffer cold during the transfer, usually by placing the transfer tank in a 4°C refrigerator or in an ice bath or ice water bath.

[0322] g) After the transfer is complete, carefully remove the nylon membrane, with the sample side up, and place it on a dry piece of filter paper, and gently blot the excess liquid from the bottom surface. Immediately proceed to the next step of cross-linking, and do not allow the membrane to dry out.

[0323] f. Cross-linking:

[0324] a) Use a UV-light cross-linker, and select 254 nm UV light, 120 mJ / cm 2Cross-linking for 45-60 seconds. If there is no UV cross-linking instrument, a portable UV lamp (such as the portable UV detector (EUV002) of Biyun Tian) can be used, and the membrane is irradiated at a distance of about 5-10 cm for 3-10 minutes. A UV lamp in a super-clean workbench can also be used, and the membrane is irradiated at a distance of about 5-10 cm for 3-15 minutes. The optimal cross-linking time can be determined by using a standard product

[0325] b) After cross-linking is completed, the next step of detection can be directly performed; or the membrane can be wrapped with a preservative film and stored at room temperature for 3-5 days, and then the next step of detection is performed. If the detection result shows that the cross-linking effect is poor, and even the band without the probe is very weak, the membrane can be cross-linked again according to the conditions of step A after drying to further improve the cross-linking effect.

[0326] g. Chemiluminescence method for detecting biotin-labeled probes:

[0327] a) Dissolve the blocking solution and the washing solution in a 37-50°C water bath. Note: The blocking solution and the washing solution must be completely dissolved before use. The blocking solution and the washing solution can be used at room temperature to 50°C, but it must be ensured that no precipitate is generated in the two solutions, and special attention should be paid in winter.

[0328] b) Take a suitable container, add 15 ml of the blocking solution, and then put the cross-linked nylon membrane containing the sample. Slowly shake on a side-to-side shaker or a horizontal shaker for 15 minutes.

[0329] c) Take 7.5 μl of streptavidin-HRP conjugate and add it to 15 ml of the blocking solution (1:2000 dilution), and mix well for standby use.

[0330] d) Remove the blocking solution used for the nylon membrane, and add 15 ml of the blocking solution containing the streptavidin-HRP conjugate prepared in the previous step. Slowly shake on a side-to-side shaker or a horizontal shaker for 15 minutes.

[0331] e) Take 25 ml of the washing solution (5X), add 100 ml of heavy distilled water or Milli-Q grade pure water, and mix well to prepare 125 ml of the washing solution.

[0332] f) Transfer the nylon membrane to another container containing 15-20 ml of the washing solution, and rinse for 1 minute.

[0333] g) Remove the washing solution, add 15-20 ml of the washing solution, and slowly wash on a side-to-side shaker or a horizontal shaker for 5 minutes.

[0334] h) Repeat step G three times (a total of four times), and each washing time is about 5 minutes.

[0335] i) Transfer the nylon membrane to another container with 20-25 ml of detection buffer, and shake slowly for 5 minutes on a side-to-side shaker or a horizontal shaker.

[0336] j) Mix 5 ml of BeyoECL Star A solution and 5 ml of BeyoECL Star B solution to prepare the BeyoECL Star working solution. Note: The BeyoECL Star working solution must be prepared fresh.

[0337] k) Take out the nylon membrane and absorb the excess liquid with a paper towel. Immediately place the membrane with the sample side facing up into a clean container on a horizontal table top or on a piece of plastic wrap.

[0338] l) Carefully add a total of 10 ml of the BeyoECL Star working solution prepared in step J to the surface of the nylon membrane, so that the working solution completely covers the nylon membrane. Place at room temperature for 2-3 minutes.

[0339] m) Take out the nylon membrane and absorb the excess liquid with a paper towel. Place the nylon membrane between two pieces of plastic wrap or other suitable transparent film, and secure it in a film cassette (also known as a film holder).

[0340] n) Press the film for 1-5 minutes using X-ray film. You can first press the film for 1 minute, immediately develop and fix, and then adjust the pressing time according to the results; or you can directly press the film for 30 seconds, 1 minute, 3 minutes, 5 minutes, or longer, and then develop and fix together to observe the results.

[0341] 4.14, Pull down experiment

[0342] a, Add biotin and RS47-biotin 1 μM to HCT116 cells in a 10 cm dish, respectively, and treat for 24 h;

[0343] b, Digest the cells, collect the cell suspension, and wash twice with pre-cooled PBS;

[0344] c, Add 1 ml of RIPA (previously add protease inhibitors PMSF and PI) to lyse the cells, and place on ice for 10 min;

[0345] d, Centrifuge the cell lysate at 12000 g for 10 min at 4°C, and keep the supernatant;

[0346] e, Incubate with biotin and RS47-biotin at room temperature for 2 h;

[0347] f, Add 40 μl of streptavidin agarose beads, mix gently, and incubate at room temperature for 1.5 h, with occasional gentle mixing;

[0348] g, Centrifuge at 500 g for 1 min, and discard the supernatant;

[0349] h. Wash 4 times with RIPA lysis buffer;

[0350] i. Add 20 μl loading lysis protein, 100 degrees Celsius boil 10 min;

[0351] j. Centrifuge, the liquid on top can be used for WB experiment.

[0352] 4.15, Flow cytometry analysis of apoptosis

[0353] a. Wash the cell sample twice with PBS;

[0354] b. Resuspend the cells with pre-cooled 1X binding buffer, 80 μl per sample;

[0355] c. Add 5 μl Annexin V antibody to each sample, incubate at 4 degrees Celsius for 30 min in the dark;

[0356] d. Wash the cells once with PBS;

[0357] e. Add pre-configured 7AAD dye solution (5 μl 7AAD per sample, 80 μl binding buffer) before flow cytometry, avoid light for 10 min;

[0358] f. Wash once with PBS, add 300 μl binding buffer per sample, and detect by flow cytometry.

[0359] 4.16, Flow cytometry analysis of cell cycle

[0360] a. Add BrdU to the cell culture medium before collecting the cells, 50 min, final concentration 3.3 nM;

[0361] b. Collect the medium and digested cells into a 15 ml centrifuge tube, 300 g, 3 min;

[0362] c. Wash once with PBS containing 2% FBS;

[0363] d. Resuspend the cells with 1 ml PBS;

[0364] e. Slowly drop 3 ml pre-cooled ethanol drop by drop while vortexing. Incubate at room temperature for 20 min;

[0365] f. Centrifuge and wash once with PBS, resuspend the cell pellet;

[0366] g. Resuspend the cells with 1 ml denaturing solution (2M HCl, prepared fresh every time), mix well, and incubate at room temperature for 20 min;

[0367] h. Centrifuge and wash once with PBS;

[0368] i. Resuspend cells in 1 ml 0.1 M Na2B4O7, pH 8.5 to neutralize residual H + at room temperature for 2 min;

[0369] j. Centrifuge and wash once with PBS (at this point, transfer to 1.5 ml centrifuge tubes);

[0370] k. Add 100 μl PBS + 20 μl BrdU antibody and incubate at room temperature for 20 min in the dark;

[0371] l. Wash once with 1 ml PBS;

[0372] m. Discard supernatant and add 20 μl 7AAD stock solution and incubate for 10 min;

[0373] n. Wash once with 1 ml PBS;

[0374] o. Add 500 μl PBS per sample and run on flow cytometer.

[0375] 4.17. Immunofluorescence staining

[0376] a. Pre-soak slides in 75% ethanol for more than half an hour, then wash the slides with sterile PBS and place the slides in a 12-well culture plate;

[0377] b. Place the appropriate number of cells in the culture plate and incubate for a period of time until the cells have grown to approximately 50% confluency;

[0378] c. Wash the slides twice with PBS;

[0379] d. Fix the cells with 4% formaldehyde solution for 10 min at room temperature;

[0380] e. Wash the slides three times with PBS for 10 min each time;

[0381] f. Place the slides in a ceramic or glass container, not plastic. In a fume hood, add -20°C pre-chilled acetone for 5 min for dehydration fixation;

[0382] g. Remove the slides and wash three times with PBS for 10 min each time;

[0383] h. Place the slides cell-side up on a glass slide and in a humidified chamber with appropriate water;

[0384] i. Place the prepared antibody or nuclear stain on the slide, covering the entire area to be stained. Incubate at room temperature for 30 min in the dark;

[0385] j. Wash the slides three times with PBS for 10 min each time;

[0386] k, suck the liquid on the glass slide, drop the sealing agent to seal the glass slide;

[0387] l, observe the picture under the fluorescence microscope.

[0388] 4.18, clone formation experiment

[0389] a, the exponential growth phase cells are plated at 1000-3000 per six-well plate;

[0390] b, the cells are normally cultured for more than one week until obvious cell clones appear;

[0391] c, pre-cool PBS in the culture dish and wash once;

[0392] d, 4% PFA is fixed at room temperature for 20 minutes;

[0393] e, suck the fixing solution and wash with PBS once;

[0394] f, add crystal violet staining solution and incubate at room temperature for 20 minutes;

[0395] g, suck the crystal violet staining solution and wash with PBS until there is no obvious color in the background;

[0396] h, after natural air drying, scan the picture under the scanner and count the number of clone formation.

[0397] 4.19, tumor cell subcutaneous tumor formation experiment

[0398] a, purchase appropriate number of 4-week-old male nude mice to the animal house one week in advance;

[0399] b, when the tumor cell line grows to the exponential growth phase, the cells are in good condition and are digested into single cell suspension;

[0400] c, count the cells and resuspend the cells in pre-cooled PBS to a cell concentration of 1×10 7 / ml;

[0401] d, the cells are placed on ice in the animal house, and 200l of cell suspension is injected subcutaneously on the back of each nude mouse, i.e. the total number of cells is 2×10 6 ;

[0402] e, during the growth of the tumor, inject the small molecule drug into the peritoneal cavity at a constant volume and quantity

[0403] h, kill the mice after about 3 weeks and measure the volume and mass of the tumor

[0404] i, and select the non-necrotic part of the tumor, fix part with formalin, part with OCT embedding at-80℃, part for collecting protein, and part for collecting RNA.

[0405] 4.20 Statistical Analysis

[0406] Data are expressed as the average of three or more repetitions. The two-tailed Student's T test was used to assess experimental differences between the two groups. Statistical results were considered statistically significant as p < 0.05, *p < 0.05, **p < 0.01, and ***p < 0.001.

[0407] II. Experimental Results

[0408] 1. Establish a small molecule drug screening system targeting RelB protein.

[0409] Abnormal activation of the non-classical NF-κB pathway can lead to many diseases, such as cancer, bone-related diseases, and autoimmune diseases.

[0410] However, no specific target gene regulated by the non-canonical NF-κB pathway has been found in tumor cell lines. The inventors discovered that knocking down RelB expression in the HCT116 colon cancer cell line using shRNA technology significantly reduced c-Myc expression at both the RNA and protein levels. Figure 2 (a, b). This indicates that RelB can regulate c-Myc expression in the HCT116 cell line, and at least knocking down RelB can downregulate c-Myc expression. In the dual luciferase reporter gene assay, knocking down RelB significantly reduced the activity of the c-Myc reporter gene (a, b). Figure 2 (c). This indicates that RelB not only regulates the expression level of c-Myc but also affects its function to some extent. The above experiments demonstrate at different levels that knocking down RelB significantly downregulates c-Myc expression in colon cancer cell lines. As an important oncogene, c-Myc plays a crucial role in tumor development and progression; therefore, c-Myc can be considered a target gene for screening small molecule drugs. Furthermore, in the MTT assay for cell viability, we found that knocking down RelB in the HCT116 colon cancer cell line significantly reduced cell viability at 48 hours compared to the control group. At 72 hours and 96 hours, the difference in cell viability between the experimental group and the control group was even more pronounced. Figure 2 (d). This experiment demonstrates that the non-classical NF-κB pathway can affect the in vitro activity of the colon cancer cell line HCT116.

[0411] In colon cancer cell line HCT116, knockdown of RelB down-regulates the expression of c-Myc at RNA and protein levels, and decreases the activity of c-Myc reporter gene. In addition, knockdown of RelB in HCT116 cells significantly reduces cell viability. Therefore, c-Myc can be used as a target gene downstream of the non-canonical NF-κB pathway to screen small molecule drugs targeting RelB protein.

[0412] 2. Design and screen small molecule drugs targeting RelB protein

[0413] According to the structural model of RelB / p52 protein binding to target DNA Figure 3 , 86 small molecule drugs were initially screened from the small molecule library on the computer, and then according to the preliminary feedback results, 65 small molecule drugs were further screened from the small molecule library on the computer according to the structure of the screened small molecule drugs.

[0414] These small molecules show in the computer simulation that they have certain structures that can bind to RelB protein and be embedded in the binding site of RelB protein and target DNA, thereby preventing RelB protein from binding to DNA and inhibiting its transcription function, thereby inhibiting the non-canonical NF-κB pathway. In particular, certain classes of compounds can form hydrogen bonds with amino acid residues of RelB, including Arg119 and Arg125, or Arg117, Ile205 and Ala127, thereby preventing RelB protein from binding to its target DNA.

[0415] Dual luciferase reporter gene experiments showed that different small molecule drugs had different inhibitory effects on c-Myc reporter gene at a concentration of 10 μM RS09 Figure 4 , a). To further verify whether these small molecules can inhibit the expression of c-Myc, 10 μM of different small molecule drugs were added to HCT116 cell lines for 48 h, and the expression of c-Myc protein in the cells was detected Figure 4 , b). The results showed that small molecule drugs RY06, RS07, RS09, RS47 and RS51 could significantly inhibit the expression of c-Myc protein relative to the control group; while RS10, RS40 and RS44 had little effect on c-Myc at the protein level. The structures of some compounds are as follows:

[0416]

[0417] To further verify whether these small molecule drugs can inhibit the growth of tumor cells (colon cancer cell lines, etc.), cell viability experiments were performed Figure 5, a). The colon cancer cell line HCT116 was treated with different concentrations of small molecule drugs for 48 h, and the cell activity was detected. It was found that at a concentration of 10 μM, RS51, RS07, RS09, RS10 and RS47 could significantly inhibit the activity of HCT116 cells. At a concentration of 2 μM, RS09 and RS47 had a significant inhibitory effect on the activity of HCT116. At a concentration of 1 μM, RS09 and RS47 still had a significant effect on cell activity. Even at 400 nM, they still had a certain effect on cell activity. From the results of the cell activity determination experiment Figure 5 , b), it can be seen that at a low concentration (less than 1 μM), the activity of HCT116 cells in the experimental group treated with RS09 and RS47 has a significant decline.

[0418] In order to more clearly understand the effective concentration of RS09 and RS47 on the inhibition of the activity of colon cancer cell line HCT116, the IC50 of them was determined by the cell activity determination experiment. From the experimental results Figure 5 , c), it can be seen that after treating HCT116 cells for 48 h, the IC50 of RS09 is 0.58 μM, and the IC50 of RS47 is 1.28 μM. Based on the above experiments, we focus on the two small molecule drugs RS09 and RS47.

[0419] The inventors conducted the same experiment on other colon cancer cell lines RKO and 231, and found that RS09 and RS47 also had a significant inhibitory effect on the two cell lines Figure 6 , a). However, the effect on normal colon epithelial cells CRL-1459 was far less obvious than that on colon cancer cell lines Figure 6 , b). The possible reason is that in the normal epithelial cell line, the non-canonical NF-κB pathway is not abnormally activated, while in the colon cancer cell line, the non-canonical NF-κB pathway is abnormally activated, so it is more sensitive to RS09 and RS47.

[0420] The inventors also selected liver cancer cell lines SK-Hcp-1, 7721 and lung cancer cell lines A549, NIH460 for experiments. The results showed that at a concentration of 1 μM which had a significant inhibitory effect on HCT116, neither the liver cancer cell line nor the lung cancer cell line was sensitive to the two small molecule drugs Figure 6 , c, d). This indicates that the inhibition of small molecule drugs on different tumors is selective, which may be related to the activation of the non-canonical NF-κB pathway in tumor cell lines, or there may be some other unknown signal pathways affecting cell activity, which are not inhibited by small molecule drugs.

[0421] 3. RS09 and RS47 target RelB protein to prevent RelB and target DNA binding

[0422] A series of experiments can prove that RS09 and RS47 can prevent RelB / p52 dimer and target DNA binding by binding to RelB, thereby inhibiting cell activity.

[0423] First, in the dual luciferase reporter gene experiment, it was found that overexpression of RelB can significantly increase the activity of the NF-κB reporter gene, and the activity of the NF-κB reporter gene in the experimental group with the addition of RS09 or RS47 is significantly inhibited relative to the control group Figure 7 , a). This shows that RS09 and RS47 can indeed inhibit the activity of the NF-κB pathway activated by RelB.

[0424] In the Bcl-3 luciferase reporter gene experiment, it was found that overexpression of RelB can significantly up-regulate the activity of the reporter gene, and its activity is significantly inhibited in the presence of RS09 or RS47, and the inhibition is more obvious with the increase of the concentration of small molecule drugs Figure 7 , b). This experiment proves to some extent that the small molecule drugs play an inhibitory role by targeting the RelB molecule rather than the RelA molecule.

[0425] In the gel migration experiment (EMSA), we found that the NF-κB probe (DNA) can bind to some proteins (including RelB protein) to form a migration band. In the experimental group with the addition of RelB antibody, a super-migration band will appear due to the binding of the antibody and the RelB protein, indicating that the RelB protein can bind to the NF-κB probe (DNA). In the experimental group with the addition of small molecule drugs in the presence of RelB antibody, we can see that the super-migration band is significantly weakened relative to the control group Figure 7 , c). This experiment shows that RS09 and RS47 can indeed inhibit the binding of RelB protein to its target DNA.

[0426] In the pulldown experiment, we incubated biotin-conjugated RS09 or RS47 with proteins, then pulled down the proteins with beads, and detected which proteins could bind to the small molecule drugs. The experimental results show that in the experimental group with RS09 or RS47, a clear band can be seen, indicating that RS09 and RS47 can indeed bind to RelB protein Figure 7 , d).

[0427] Both luciferase reporter gene assay and EMSA and pulldown assay proved that RS09 and RS47 can bind to RelB protein and prevent it from binding to target DNA to perform inhibitory function. Further, we treated lymphoma cell line BJAB with G28-5 (CD40 agonist) for 0.5 h, which can activate the canonical NF-κΒ pathway but not the non-canonical NF-κΒ pathway. Because the non-canonical pathway activation needs more time than the canonical pathway, we detected TNFα (a target gene downstream of the canonical NF-κΒ pathway) RNA to characterize whether the canonical pathway was activated. The results showed that G28-5 can significantly activate the non-canonical pathway and significantly increase the expression level of TNFα, and the addition of BAY, an inhibitor of the canonical NF-κΒ pathway, can significantly reduce the expression level of TNFα, while RS09 and RS47 have no effect on the expression of TNFα Figure 7 , e). This experiment proved that the small molecule drug has no effect on the canonical pathway and proved that RS09 and RS47 act through RelB but not RelA.

[0428] In B cells, BAFF can activate the non-canonical NF-κΒ pathway but has little effect on the canonical NF-κΒ pathway. Therefore, it can be used as a good experimental system to verify the inhibitory effect of RS09 and RS47 on the non-canonical pathway. In mouse primary B cells, pim2 is a target gene downstream of the non-canonical NF-κΒ pathway, and its RNA expression level will significantly increase after 24 h of BAFF stimulation, while the addition of RS09 or RS47 can significantly inhibit the RNA expression level of the pim2 gene Figure 8 , a). Human-derived B lymphoma cells (PDX) were stimulated with BAFF for 48 h, and RS09 or RS47 was added for the same period of time. Flow cytometry was used to detect the survival of B lymphoma cells. We found that the addition of BAFF significantly increased the survival, while RS09 or RS47 significantly inhibited the survival Figure 8 , b). Human-derived B lymphoma cells can better respond to the state of lymphoma cells in the human body than immortalized B lymphoma cell lines, and are a more optimized experimental system. In mouse primary B cells, we can also observe that the addition of BAFF significantly increases the survival of B cells, while the addition of RS09 or RS47 significantly inhibits the survival of B cells Figure 8 , c).

[0429] Finally, in the immunofluorescence co-localization experiment, we can see that in HCT116 cell line, RS47 and RelB protein exist in the co-localization condition at the cell level. In the resting state of HCT116 cells, they mainly exist in the cytoplasm. This also proves from the side that small molecule drugs can bind to RelB protein Figure 9

[0430] 4. RS47 inhibits the proliferation of colon cancer cell line HCT116 and promotes its apoptosis

[0431] In the BrdU cell incorporation experiment, we added RS47 in the cell culture medium, and finally determined the cell cycle by flow cytometry. The experimental results show that Figure 10 , a, b), compared with the control group, the proportion of cells in S phase of the 1 μM RS47 experimental group decreased significantly from 13.83% to 5.18%. Correspondingly, the cells in G0 / G1 and G2 / M phases increased relatively. It shows that RS47 at a concentration of 1 μM will significantly inhibit the cell cycle and prevent the cells from entering the S phase. For the 2 μM experimental group, we can see that a group of cells appear in the lower left corner of the flow chart, which is a group of dead cells.

[0432] In the apoptosis experiment, we found that at low concentrations (0.5 μM and 1 μM), it basically does not induce cell apoptosis. When the concentration of RS47 increases to 2 μM, obvious cell apoptosis will appear after 72 h of culture Figure 10 , c). From the statistical chart, we can also see that the proportion of living cells at a concentration of 2 μM decreases significantly, and the proportion of early and late apoptosis cells increases significantly Figure 10 , d). Cell cycle experiment and apoptosis experiment show that small molecule drugs at different concentrations have different biological functions. At a relatively low concentration, it mainly regulates HCT116 through affecting the cell cycle, and at a relatively high concentration, it mainly functions through inducing cell apoptosis.

[0433] In the colony formation experiment, even at a concentration of 0.5 μM, the colony formation of HCT116 decreases significantly, whether in the number of colony formation or in the size of the colony Figure 10 , e). At a concentration of 1 μM, almost no colony formation occurs. It shows that under the long-term (7d) action, RS47 can play a good inhibitory effect even at a relatively low concentration (0.5 μM). It shows that the inhibition of tumor cell growth by small molecule drugs is a concentration-dependent and time-dependent process.

[0434] 5. RS47 inhibits the proliferation of colon cancer cell line SW620 and promotes its apoptosis

[0435] ​The inhibitory effect of RS47 was verified in another colon cancer cell line, SW620. Similar to HCT116 cells, in the SW620 cell line, we also found that RS47 could inhibit cells from entering the S phase in a concentration-dependent manner; the proportion of cells entering the S phase decreased with increasing concentration. Figure 11 (a, b). Under high concentration (2 μM) conditions, RS47 can also induce a large number of apoptosis in cells. Figure 11 (c, d). In the colony formation experiment, small molecule drugs also inhibited the colony formation of SW620 cells. Figure 11 (e).

[0436] The above experiments demonstrate that small molecule drugs have an inhibitory effect on various colon cancer cell lines.

[0437] 6. RS47 promotes apoptosis in B-cell lymphoma cells.

[0438] In the B-cell lymphoma cell line BJAB, we found that RS47 significantly induced apoptosis in these cells; with increasing concentration, the number of cells undergoing both early and late apoptosis increased significantly. Figure 12 (a, b). To better simulate the state of lymphoma cells in B-cell lymphoma patients, we selected a more optimized model (human-derived lymphoma cells). RS47 was added to a co-culture system of human-derived myeloma cells and bone marrow stromal cells (BMSCs) to observe whether RS47 had an effect on human-derived myeloma cells. Co-culturing BMSCs and human-derived myeloma cells promoted the survival of human-derived myeloma cells. We found that RS47 promoted apoptosis in human-derived lymphoma cells cultured alone. In the co-culture system, the survival rate of myeloma cells significantly increased. However, after adding RS47, the survival rate of myeloma cells in the experimental group was significantly lower than that in the control group. Furthermore, the decrease in the survival rate of myeloma cells became more pronounced with increasing RS47 concentration. Figure 12 (c). Encouragingly, we found that RS47 had almost no effect on the survival of BMSC cells. Figure 12 (d). That is, in this system, RS47 has a significant pro-apoptotic effect on lymphoma cells, but has almost no effect on stromal cells. This indicates that RS47 has a selective effect on cell apoptosis.

[0439] 7. RS09 inhibits tumorigenesis of HCT116 cells in vivo.

[0440] We conducted a subcutaneous tumor formation experiment in nude mice using the HCT116 cell line. The results showed that the tumor size and mass in the experimental group were significantly smaller than those in the control group. Figure 13 (a, b).

[0441] We selected tumor samples, extracted RNA and protein, and detected c-Myc expression. The results showed that RS09 could inhibit c-Myc expression at both the RNA and protein levels. Figure 13 Figure 13 (c, d). This demonstrates that RS09 can also inhibit tumor cell growth by suppressing the non-canonical NF-κB pathway during tumor formation in mice.

[0442] III. Discussion

[0443] Recent studies have shown that the non-canonical NF-κB pathway also regulates crucial biological processes, such as lymphatic system development, T cell negative selection, B cell survival and maturation, and bone metabolism. Abnormal activation of the non-canonical NF-κB pathway can lead to many immune and bone metabolic diseases. Furthermore, increasing research indicates that the non-canonical NF-κB pathway plays a vital role in the development and progression of an growing number of tumors. In some tumor cell lines, non-canonical NF-κB signaling is persistently activated. This is due, on the one hand, to tumor cells producing cytokines that activate non-canonical NF-κB signaling, such as lymphotoxin (LT), through autocrine secretion; and on the other hand, to the existence of unknown mechanisms within tumor cells (such as mutations in important regulatory genes) that maintain constitutive activation of non-canonical NF-κB signaling. Blocking non-canonical NF-κB signaling can effectively inhibit tumor cell growth and, more importantly, by inhibiting the production of chemokines, prevent the aggregation of inflammatory cells and tumor stromal cells in the tumor microenvironment, thereby inhibiting tumor angiogenesis and formation, and ultimately suppressing tumor growth.

[0444] The inventors have identified a small molecule compound that can bind to the RelB protein, preventing it from binding to target DNA and thus inhibiting its transcriptional function. Research shows that the small molecule drug of this invention does not inhibit classical pathways but can inhibit the activation of non-classical pathways. In in vitro experimental systems, the small molecule drug of this invention can inhibit the cell cycle of colon cancer cell lines HCT116 and SW620 at low concentrations, and induces extensive apoptosis at high concentrations, indicating that the small molecule drug of this invention can regulate cell proliferation and apoptosis. Furthermore, the small molecule drug can also induce apoptosis in B lymphoma cells, while having almost no effect on co-cultured stromal cells. In subcutaneous tumorigenesis experiments in nude mice, this invention found that the small molecule drug of this invention can also inhibit tumorigenesis in the colon cancer cell line HCT116.

[0445] In experiments measuring the effects of small molecule drugs on cell activity using different cell lines, the small molecule drugs of this invention showed significant inhibitory effects on colon cancer cell lines HCT116, RKO, and 231, but no significant inhibitory effect on normal intestinal epithelial cells CRL1459.

[0446] In addition, the present inventors first discovered that knocking down the expression of RelB in a colon cancer cell line can significantly inhibit the expression of the oncogene c-Myc. Based on this, a small molecule drug screening system can be established. SEQUENCE LISTING <110> SHANGHAI INSTITUTE OF NUTRITION AND HEALTH, CHINESE ACADEMY OF SCIENCES SHANGHAI INSTITUTE OF MATERIA MEDICA, CHINESE ACADEMY OF SCIENCES <120> Small molecule inhibitors of non-canonical NF-kB pathway and uses thereof <130> 178300 <160> 14 <170> SIPOSequenceListing 1.0 <210> 1 <211> 33 <212> DNA <213> Artificial Sequence (Artificial Sequence) <400> 1 cggcgatagc atgcttcggt ctgggccagc ctc 33 <210> 2 <211> 31 <212> DNA <213> Artificial Sequence (Artificial Sequence) <400> 2 cgacgcgtct acgtggcttc aggccccggg g 31 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence (Artificial Sequence) <400> 3 ctggaacggt gaaggtgaca 20 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence (Artificial Sequence) <400> 4 aagggacttc ctgtaacaat gca 23 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <400> 5 ttggtgagga tctgcttcca g 21 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <400> 6 tcggcaaatc cgcagctctg at 22 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <400> 7 cacacatcag cacaactacg ca 22 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <400> 8 ttgaccctct tggcagcag 19 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <400> 9 cccagggacc tctctctaat ca 22 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <400> 10 agctgcccct cagcttgag 19 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <400> 11 attgccttca cccaccttc 19 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <400> 12 gccatcaaag taatctcccg 20 <210> 13 <211> 22 <212> DNA <213> Artificial Sequence <400> 13 atctacgagg gctatgctct cc 22 <210> 14 <211> 23 <212> DNA <213> Artificial Sequence <400> 14 ctttgatgtc acgcacgatt tcc 23

Claims

1. Use of an agent capable of inhibiting or preventing the binding of RelB protein to its target DNA or an agent capable of inhibiting the transcriptional function of RelB protein in the manufacture of a medicament for the treatment or prevention of a disease that benefits from the inhibition of the non-canonical NF-kB pathway as a result of the inhibition of the binding of RelB protein to its target DNA, wherein the disease is colon cancer resulting from the over-activation of the non-canonical NF-kB pathway, wherein, The agent is a compound or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, The compound is: or .

Images