Scaffold protein capable of regulating intracellular signal transduction and method for regulating intracellular signal transduction using same

Abstract

The present invention relates to a method for regulating intracellular signal transduction using a scaffold protein, and a scaffold protein capable of regulating intracellular signal transduction. The present invention relates to a method for regulating intracellular signal transduction using a scaffold protein, and a scaffold protein capable of regulating intracellular signal transduction. The scaffold protein according to the present invention selectively binds to a plurality of target kinases to regulate the interaction among the proteins, thereby exhibiting the effect of reconstructing or converting an originally activated intracellular signal transduction pathway into another signal transduction pathway. Specifically, the scaffold protein of the present invention may regulate the flow of signal transduction by mediating or blocking binding between a specific kinase and a downstream signal transduction protein, and thus may effectively control the growth or death of cells by inhibiting cell proliferation signals or selectively activating apoptosis signals.

Description

Scaffold protein capable of regulating intracellular signal transduction and method for regulating intracellular signal transduction using the same

[0001] The present invention relates to a scaffold protein capable of regulating intracellular signal transduction and a method for regulating intracellular signal transduction using the same. More specifically, the invention relates to a scaffold protein that specifically binds to a kinase involved in intracellular signal transduction, a method for rewiring intracellular signal transduction using the same, and a composition for preventing or treating cancer comprising the scaffold protein.

[0002]

[0003] Generally, cells respond to external stimuli, such as growth factors, cytokines, and stress stimuli, by recognizing signals through cell membrane receptors and transmitting them into the cell through a sequential activation process of various signaling proteins, including kinases. This intracellular signaling process plays a crucial role in precisely regulating various physiological functions, such as cell growth, differentiation, proliferation, survival, and apoptosis.

[0004] However, in cases where abnormalities occur in intracellular signaling systems, such as in cancer cells, specific signaling pathways become abnormally overactivated, leading to the continuous transmission of signals for cell survival and proliferation. Consequently, the suppression of apoptosis signals can induce abnormal cell proliferation. For this reason, various studies have been conducted to regulate intracellular signaling pathways for the treatment of diseases such as cancer, and in particular, approaches to inhibit the activity of specific kinases or block signal transmission have been attempted.

[0005] In this regard, Korean Registered Patent Publication No. 10-1710896 discloses a technology in which a specific protein participates in the MAPK signaling pathway to regulate apoptosis signaling, and proposes that apoptosis can be inhibited or regulated through interactions between intracellular signaling proteins. Additionally, Korean Registered Patent Publication No. 10-2261588 discloses a technology for improving immune responses or disease states by regulating apoptosis-related signaling using a specific biomaterial.

[0006] However, the aforementioned prior art focuses only on inhibiting or regulating the activity of specific signaling pathways or signaling factors, and does not disclose technology for switching proliferation signaling already activated within a cell to another signaling pathway or intentionally rewiring the signaling pathway itself.

[0007] Under these circumstances, the inventors discovered that by using a scaffold protein capable of selectively linking multiple kinases involved in intracellular signal transduction, excessively activated proliferation signaling within cancer cells can be rewired into apoptosis signaling pathways, and based on the observation that this can induce selective death of cancer cells, they completed the present invention.

[0008]

[0009] The object of the present invention is to provide a scaffold protein capable of regulating intracellular signal transduction.

[0010] Another objective of the present invention is to provide a method for regulating intracellular signal transduction using a scaffold protein capable of regulating intracellular signal transduction.

[0011] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer comprising the scaffold protein.

[0012]

[0013] To achieve the above-described objective, the present invention provides a scaffold protein comprising a first domain that specifically binds to a kinase involved in cell proliferation signaling, and a second domain that specifically binds to a kinase involved in apoptosis signaling.

[0014] In the present invention, the first domain specifically binds to BRAF, and the second domain can specifically bind to one or more of MKK4 and MKK7 kinases.

[0015] In the present invention, the scaffold protein may further include a third domain that specifically binds to JNK.

[0016] In the present invention, the first domain may be composed of the amino acid sequence of SEQ ID NO. 1 or SEQ ID NO. 2.

[0017] In the present invention, the second domain may be composed of the amino acid sequence of SEQ ID NO. 3, SEQ ID NO. 4, or SEQ ID NO. 5.

[0018] In the present invention, the third domain may be composed of the amino acid sequence of SEQ ID NO. 6 or SEQ ID NO. 7.

[0019] In the present invention, the first domain and the second domain can be connected by a linker.

[0020] In the present invention, the scaffold protein may be composed of the amino acid sequence of SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, or SEQ ID NO. 22.

[0021]

[0022] In addition, the present invention provides a method for regulating signal transduction within mammalian cells other than humans using the scaffold protein.

[0023] In the present invention, the method may include the step of expressing the scaffold protein within a cell.

[0024] In the present invention, regulating signal transduction within the cell may involve converting a cell proliferation signal into a cell death signal.

[0025] In the present invention, the cell may be a cancer cell.

[0026]

[0027] The present invention also provides a pharmaceutical composition for the prevention or treatment of cancer, comprising the scaffold protein.

[0028] In the present invention, the cancer disease may be one or more selected from the group consisting of colorectal cancer, liver cancer, cervical cancer, lung cancer, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, breast cancer, ovarian cancer, uterine cancer, pancreatic cancer, stomach cancer, skin cancer, bladder cancer, thyroid cancer, brain cancer, and prostate cancer.

[0029]

[0030] The present invention also provides a method for preventing or treating cancer, comprising the step of administering a composition containing the scaffold protein.

[0031]

[0032] The present invention relates to a scaffold protein capable of regulating intracellular signal transduction and a method for regulating intracellular signal transduction using the same. The scaffold protein according to the present invention selectively binds to a plurality of target kinases and regulates the interaction between these proteins, thereby reconfiguring or switching the originally activated intracellular signal transduction pathway to another signal transduction pathway. Specifically, the scaffold protein of the present invention can regulate the flow of signal transduction by mediating or blocking the binding between a specific kinase and a downstream signal transduction protein. Through this, it can effectively control cell proliferation or death by inhibiting cell proliferation signals or selectively activating apoptosis signals.

[0033] Furthermore, since the scaffold protein of the present invention acts by regulating protein-protein interactions without directly inhibiting the active site, it exhibits excellent signal modulation effects even in small amounts while minimizing side effects caused by non-specific inhibition. Therefore, a composition containing the scaffold protein of the present invention as an active ingredient can be usefully utilized as a pharmaceutical composition for effectively preventing or treating diseases such as cancer caused by abnormal intracellular signal transduction.

[0034]

[0035] FIGS. 1a to 1c each show the results of the mini-protein-BRAF model, mini-protein-MKK4 model, and mini-protein-JNK1 model selected through docking simulation according to one embodiment of the present invention.

[0036] Figure 2a shows the results of measuring the binding strength of a mini protein to BRAF according to one embodiment of the present invention.

[0037] Figures 2b and 2c show the results of measuring the binding strength of a mini protein to MKK4 according to one embodiment of the present invention.

[0038] FIG. 2d shows the results of measuring the binding strength of a mini protein to JNK1 according to one embodiment of the present invention.

[0039] Figure 3a shows the Western blot results of a scaffold protein prepared according to one embodiment of the present invention.

[0040] FIG. 3b shows a schematic diagram of a scaffold protein prepared according to one embodiment of the present invention.

[0041] Figure 4a shows the results of MKK4 expression according to one embodiment of the present invention and the results of co-expressing MKK4 and a mini protein.

[0042] Figure 4b shows the result of normalizing the degree of MKK4 phosphorylation according to one embodiment of the present invention.

[0043] FIG. 4c is a schematic diagram of the process of phosphorylating BRAF by MKK4 and a mini-protein complex according to one embodiment of the present invention.

[0044] Figure 5 shows the JNK1 phosphorylation results of a scaffold protein according to one embodiment of the present invention.

[0045] Figure 6 shows the results of measuring the survival rate of cancer cells when a scaffold protein according to one embodiment of the present invention is expressed.

[0046] FIGS. 7a to 7d show the co-immunoprecipitation results regarding whether a scaffold protein prepared according to one embodiment of the present invention interacts with a target kinase.

[0047] Figure 8 shows the results of confirming whether a scaffold protein prepared according to one embodiment of the present invention increases the expression and phosphorylation levels of c-Jun.

[0048] Figures 9a and 9b respectively show the results of a scaffold protein prepared according to one embodiment of the present invention inducing apoptosis in HepG2 liver cancer cells and SW480 colon cancer cells.

[0049]

[0050] Specific embodiments of the present invention will be described in more detail below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled expert in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0051] In the specification and claims of the present invention, expressions such as "comprising," "having," and their variations mean that the described features or components are present, and unless otherwise limited, do not exclude the possibility of adding one or more other features or components.

[0052] Singular expressions used in the specification and claims of the present invention shall be interpreted to include plural expressions unless the context clearly specifies them to be singular, and plural expressions shall also be interpreted to include singular expressions unless the context clearly specifies them to be plural.

[0053] Numerical ranges expressed in the specification and claims of the present invention include lower limits, upper limits, and all values ​​between them unless specifically defined otherwise, and should be interpreted as including all possible numerical ranges obtained by combining lower limits and upper limits.

[0054] Expressions indicating degree, such as "approximately" used in the specification of the present invention, should be interpreted to include the tolerance of the corresponding numerical value.

[0055]

[0056] The present invention relates to a method for regulating intracellular signal transduction using a scaffold protein. Specifically, the present invention relates to a method for regulating intracellular signal transduction using a scaffold protein that specifically binds to a kinase involved in intracellular signal transduction.

[0057] Generally, kinases involved in intracellular signal transduction act as key regulatory factors that transmit signals by phosphorylating specific amino acid residues of substrate proteins in response to external stimuli or intracellular signals. These phosphorylation reactions induced by kinases induce the activation or inactivation of signal transduction proteins and enable the stepwise amplification and transmission of signals through sequential phosphorylation reactions. Such kinase-mediated signaling processes play an essential role in precisely regulating various cellular physiological phenomena, such as cell growth, differentiation, proliferation, survival, migration, and apoptosis. In particular, the interactions and activation sequences between different kinases determine the selectivity and specificity of particular signal transduction pathways, and changes in these regulatory mechanisms can alter the functional state of the cell.

[0058] Based on these points, the present invention provides a method for rewiring existing signaling pathways to other signaling pathways by selectively regulating the interactions between kinases involved in intracellular signal transduction using scaffold proteins. Through this, the present invention inhibits signals that induce cell proliferation or survival and selectively activates signals that induce apoptosis, thereby providing a novel therapeutic strategy and perspective distinct from existing signal inhibition methods for various diseases, including cancer, in which excessive cell proliferation is induced due to the abnormal activation of intracellular signal transduction.

[0059] In the present invention, the regulation of intracellular signal transduction using a scaffold protein can be performed not only on cells cultured in vitro, but also on cells present in the body, and can be performed on mammalian cells or mammalian cells excluding humans.

[0060] The method for regulating intracellular signal transduction according to the present invention may include the step of expressing the scaffold protein within the cell.

[0061] Specifically, a nucleic acid containing a gene encoding the scaffold protein can be inserted into an expression vector and then introduced into a cell via a transfection or transduction method. The expression vector may be a plasmid vector, a viral vector, or an equivalent expression system, and can be appropriately selected depending on the cell type and purpose. The nucleic acid introduced into the cell is expressed as a scaffold protein by the cell's transcription and translation mechanisms, and can perform a function of regulating intracellular signal transduction.

[0062] In the present invention, the cell in which signal transduction regulation occurs through a scaffold protein may be a cancer cell.

[0063] Specifically, the cancer may be one or more selected from the group consisting of colorectal cancer, liver cancer, cervical cancer, lung cancer, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, breast cancer, ovarian cancer, uterine cancer, pancreatic cancer, stomach cancer, skin cancer, bladder cancer, thyroid cancer, brain cancer, and prostate cancer.

[0064] In the present invention, by using a scaffold protein capable of rewiring an activated proliferation signaling pathway within cancer cells into a signaling pathway that induces apoptosis, selective death of cancer cells can be induced, thereby achieving a preventive or therapeutic effect against cancer.

[0065] In this regard, various signaling pathways regulating cell survival and apoptosis operate in a complex manner within cancer cells, and numerous kinases are involved as key regulators in these pathways. Kinases activated by growth factor receptors transmit signals for cell survival and proliferation through successive phosphorylation reactions, whereas kinases responding to stress stimuli or damage signals mediate signaling that induces apoptosis. While the balance between these proliferation and apoptosis signals is precisely regulated in normal cells, in cancer cells, proliferation signals often become dominant due to the overactivation or dysregulation of specific kinases. Consequently, apoptosis signals are not effectively transmitted, creating a signaling environment in which the abnormal survival and proliferation of cancer cells persist.

[0066] In the present invention, by using a scaffold protein capable of binding to both a specific kinase involved in proliferation signals within cancer cells and a specific kinase involved in death signals, the kinase involved in proliferation signals and the kinase involved in death signals are physically brought into close proximity, thereby enabling the proliferation signal transmitted within the cancer cell to be rewired into a death signal.

[0067] In this regard, the intracellular signal transduction method using the scaffold of the present invention may be a method for inducing apoptosis signals within cancer cells.

[0068] The scaffold protein of the present invention may include a first domain and a second domain capable of binding to a kinase involved in intracellular signal transduction.

[0069] The first domain above can specifically bind to a kinase involved in cell proliferation signaling, and specifically, it may be a domain that binds to BRAF.

[0070] BRAF (B-Raf proto-oncogene, serine / threonine kinase) is a key kinase in the MAPK (Mitogen-Activated Protein Kinase) signaling pathway within cells. When activated by growth factor stimulation, it sequentially phosphorylates MEK (Mitogen-Activated Protein Kinase) and ERK (Extracellular Signal-Regulated Kinase), thereby transmitting intracellular proliferation signals. This BRAF-MEK-ERK signaling axis plays a role in maintaining cell survival and proliferation by promoting cell cycle progression and the expression of anti-apoptotic proteins. In particular, if BRAF undergoes mutation or becomes overactivated, the corresponding proliferation signals can be continuously activated regardless of external stimuli. As a result, apoptosis signals are suppressed, and abnormal cell proliferation is induced, leading to the development of diseases such as cancer.

[0071] Meanwhile, the second domain can specifically bind to a kinase involved in cell death signaling, and specifically, the second domain may be a domain capable of binding to MKK (Mitogen-Activated Protein Kinase Kinase).

[0072] More specifically, the above MKK may be one or more of MKK4 and MKK7.

[0073] MKK4 and MKK7 are upstream kinases activated in response to stress stimuli, playing a role in activating downstream JNK family proteins by phosphorylating them. Activated JNK1, JNK2, and JNK3 induce the expression of genes associated with apoptosis by phosphorylating various substrate proteins, including nuclear transcription factors. This MKK4 / MKK7-JNK signaling axis is known as a representative pathway mediating apoptotic signals under conditions such as DNA damage, oxidative stress, or cytotoxic stimuli. Therefore, the signaling pathway leading from MKK4, MKK7, and JNK1 through JNK3 functions as a critical mechanism for regulating apoptotic signals that determines cell survival.

[0074] In the present invention, by using a scaffold protein comprising a first domain that binds to BRAF and a second domain that binds to MKK, cell proliferation signals induced or transmitted from BRAF can be selectively rewired into a cell death signaling pathway mediated by MKK.

[0075] In addition, the scaffold protein of the present invention may further include a third domain capable of specifically binding to a kinase involved in cell death signaling.

[0076] Specifically, the third domain may be a domain capable of binding to JNK (c-Jun N-terminal Kinase), and the JNK may be one or more selected from the group consisting of JNK1, JNK2, and JNK3.

[0077] The scaffold protein of the present invention further includes a domain that binds to JNK, thereby effectively capturing and positioning JNK activated by MKK, which allows for clearer and more selective regulation of the flow of intracellular signal transduction. Through this, cell proliferation signals induced by BRAF can be more effectively converted into a death signaling pathway leading to MKK and JNK.

[0078] In one embodiment of the present invention, domains capable of binding to BRAF, MKK, and JNK, respectively, were derived through protein structure-based design and binding prediction, and a scaffold protein containing said domains was constructed. Furthermore, as a result of introducing the thus constructed scaffold protein into a cell, it was confirmed that MKK and JNK were selectively phosphorylated, thereby confirming that intracellular signal transduction can be regulated in the direction of death signals by the scaffold protein.

[0079] In the present invention, the first domain may be composed of the amino acid sequence of SEQ ID NO. 1 or SEQ ID NO. 2 below, the second domain may be composed of the amino acid sequence of SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5 below, and the third domain may be composed of SEQ ID NO. 6 or SEQ ID NO. 7 below.

[0080]

[0081] [Sequence No. 1]

[0082] LVTLEFQGVYQEGLKLQCIQGQGDQMCSQMVGVALMVWILSIQLPTSTNWWMFQICQNARLITVVKSKF

[0083]

[0084] [Sequence No. 2]

[0085] TKHDSLALTQIIEKIMSRVTTNMFALHFGKMHLNSCGRQGTGMIRKDGWQGAGIGRDAYRLRQKHAIR

[0086]

[0087] [Sequence No. 3]

[0088] SRRTCVKSANAWQWMSIERYCISYNGQNIQRNCESINGGLDSKEPLLSERQMMGQQMGKYNIAVNSERV

[0089]

[0090] [Sequence No. 4]

[0091] NWSRWFYTSAAHSMFVISCLQWVMEGHMGTHKDTCANLIFAIGMEPWVAAICIKQRKQNTYEHDKIITS

[0092]

[0093] [Sequence No. 5]

[0094] QVFGNIFWSWAYSNGAYMGKADWRLARGRIRGMMEAGHICGRINPSGSYWSNKILLLFQNFRDSYGEDY

[0095]

[0096] [Sequence No. 6]

[0097] ENMVQTHKKFQNNMKRRYIMDLTRVCKCHEATYMWKIWDRDSAEIQALRNEIAYIIVLQDCMLDQNKQT

[0098]

[0099] [Sequence No. 7]

[0100] AGVIYAIWTADEDQLWFFITNNQMHSQLYKNDEKVMSCRWCKIQSLTEDKTILSTSSAQEGHTREGDRR

[0101]

[0102] In addition, the nucleotide sequences encoding the amino acids of the above sequence numbers are as follows, and the amino acid sequences of sequence numbers 1 to 7 are each encoded by the nucleotide sequences of sequence numbers 8 to 14.

[0103]

[0104] [Sequence No. 8]

[0105] ctggttaccctggaatttcagggcgtttaccaggaaggtctgaaactgcagtgcattcagggtcaaggcgatcagatgtgctctcagatggttggcgttgcactgatggtctggattctgagcattcagctgccgacctctaccaactggtggatgttccagatttgccagaacgctcgtctgattaccgtggtcaaaagcaaattt

[0106]

[0107] [서열번호 9]

[0108] accaaacacgatagcctggcactgacccagattattgagaaaatcatgagccgcgtcaccaccaacatgatgtttgcgctgcacttcggcaaaatgcacctgaatagctgcggtcgtcaaggcaccggcatgattcgcaaagacggttggcaaggcgcaggtattggtcgcgacgcatatcgtctgcgtcaaaaacacgcaattcgc

[0109]

[0110] [서열번호 10]

[0111] agtcgtcgtacctgcgttaaaagcgcaaacgcttggcagtggatgagcattgaacgttactgcatcagctacaacggtcagaacatccagcgtaactgcgaaagcattaacggcggtctggattccaaagaaccgctgctgtctgaacgtcaaatgatgggccagcagatgggcaaatacaacattgcggttaatagcgaacgcgtt

[0112]

[0113] [서열번호 11]

[0114] aactggagccgttggttttatacctctgccgcccatagcatgttcgttatttcctgcctgcagtgggttatggagggtcacatgggcacccataaagatacctgcgcgaacctgatcttcgccattggtatggaaccgtgggttgcagctatctgcatcaaacagcgcaaacagaacacctacgagcacgacaaaatcatcacctct

[0115]

[0116] [서열번호 12]

[0117] caggttttcggcaacatcttttggagctgggcgtatagtaacggcgcgtatatgggcaaagcagattggcgtttagcacgcggtcgtattcgcggtatgatggaagcaggccatatttgcggtcgtattaatccgagcggtagctattggagcaacaaaatcctgctgctgttccagaactttcgcgatagttacggcgaagattac

[0118]

[0119] [서열번호 13]

[0120] gagaacatggtccagacccacaaaaaattccagaacaacatgaaacgccgctacatcatggatctgacccgcgtttgtaaatgccacgaagccacctacatgtggaaaatctgggatcgcgatagcgcagaaattcaggcactgcgtaacgaaatcgcgtacatcatcgtcctgcaggattgcatgctggatcagaacaaacaaacc

[0121]

[0122] [서열번호 14]

[0123] gcaggtgttattacgcgatttggaccgcagacgaagatcagctgtggttcttcatcaccaacaaccagatgcacagccagctgtacaaaaacgacgagaaag tcatgtcctgtcgttggtgcaaaatccagagcctgaccgaggacaaaaccattctgagcaccagtagcgcacaagaaggtcatacccgcgaaggcgatcgtcgc

[0124]

[0125] Meanwhile, in the scaffold protein of the present invention, the first domain and the second domain, and the second domain and the third domain can be connected by a linker.

[0126] Specifically, the linker may be a GS linker, and the first domain and the second domain may be connected to the first linker, and the second domain and the third domain may be connected to the second linker.

[0127] The first linker above may be composed of the amino acid sequence of SEQ ID NO. 15 below, and the second linker above may be composed of the amino acid sequence of SEQ ID NO. 16 below.

[0128]

[0129] [Sequence No. 15]

[0130] GGSGSGG

[0131]

[0132] [Sequence No. 16]

[0133] GSGSGSG

[0134]

[0135] In addition, the nucleotide sequences encoding the amino acid sequences of the above linker are as follows, and the amino acid sequences of SEQ ID NOs 15 and 16 are each encoded by the nucleotide sequences of SEQ ID NOs 17 to 18.

[0136]

[0137] [Sequence No. 17]

[0138] ggcggttctggttcgggcgga

[0139]

[0140] [Sequence No. 18]

[0141] ggaagtggtagcggcagcggg

[0142]

[0143] The scaffold protein used for regulating intracellular signal transduction according to the present invention may be composed of the first domain, the first linker, the second domain, the second linker, and the third domain, and may have a structure of the first domain-first linker-second domain-second linker-third domain.

[0144] Specifically, the scaffold protein of the present invention may be composed of the amino acid sequence of SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, or SEQ ID NO. 22 below.

[0145]

[0146] [Sequence No. 19]

[0147] TKHDSLALTQIIEKIMSRVTTNMMFALHFGKMHLNSCGRQGTGMIRKDGWQGAGIGRDAYRLRQKHAIRGGSGSGGSRRTCVKSANAWQWMSIERYCISYNGQNIQRNCE SINGGLDSKEPLLSERQMMGQQMGKYNIAVNSERVGSGSGSGENMVQTHKKFQNNMKRRYIMDLTRVCKCHEATYMWKIWDRDSAEIQALRNEIAYIIVLQDCMLDQNKQT

[0148]

[0149] [Sequence No. 20]

[0150] TKHDSLALTQIIEKIMSRVTTNMMFALHFGKMHLNSCGRQGTGMIRKDGWQGAGIGRDAYRLRQKHAIRGGSGSGGQVFGNIFWSWAYSNGAYMGKADWRLARGRIRGMMEAGHICGRINPSGSYWSNKILLLFQNFRDSYGEDYGSGSGSGENMVQTHKKFQNNMKRRYIMDLTRVCKCHEATYMWKIWDRDSAEIQALRNEIAYIIVLQDCMLDQNKQT

[0151]

[0152] [서열번호 21]

[0153] LVTLEFQGVYQEGLKLQCIQGQGDQMCSQMVGVALMVWILSIQLPTSTNWWMFQICQNARLITVVKSKFGGSGSGGNWSRWFYTSAAHSMFVISCLQWVMEGHMGTHKDTCANLIFAIGMEPWVAAICIKQRKQNTYEHDKIITSGSGSGSGAGVIYAIWTADEDQLWFFITNNQMHSQLYKNDEKVMSCRWCKIQSLTEDKTILSTSSAQEGHTREGDRR

[0154]

[0155] [서열번호 22]

[0156] LVTLEFQGVYQEGLKLQCIQGQGDQMCSQMVGVALMVWILSIQLPTSTNWWMFQICQNARLITVVKSKFGGSGSGGQVFGNIFWSWAYSNGAYMGKADWRLARGRIRGMMEAGHICGRINPSGSYWSNKILLLFQNFRDSYGEDYGSGSGSGAGVIYAIWTADEDQLWFFITNNQMHSQLYKNDEKVMSCRWCKIQSLTEDKTILSTSSAQEGHTREGDRR

[0157]

[0158] In addition, the nucleotide sequences encoding the amino acid sequences of the scaffold protein are as follows, and the amino acid sequences of SEQ ID NOs 19 to 22 are each encoded by the nucleotide sequences of SEQ ID NOs 23 to 26.

[0159]

[0160] [Sequence No. 23]

[0161] accaaacacgatagcctggcactgacccagattattgagaaaatcatgagccgcgtcaccaccaacatgatgttttgcgctgcacttcggcaaaatgcacctgaatagctgcggtcgtcaaggcaccggcatgattcgcaaagacggttggcaaggcgcaggtatt ggtcgcgacgcatatcgtctgcgtcaaaaacacgcaattcgcggcggttctggttcgggcggaagtcgtcgtacctgcgttaaaagcgcaaacgcttggcagtggatgagcattgaacgttactgcatcagctacaacggtcagaacatccagcgtaactgcgaaa gcattaacggcggtctggattccaaagaaccgctgctgtctgaacgtcaaatgatgggccagcagatgggcaaatacaacattgcggttaatagcgaacgcgttggaagtggtagcggcagcggggagaacatggtccagacccacaaaaaattccagaacaacat gaaacgccgctacatcatggatctgacccgcgtttgtaaatgccacgaagccacctacatgtggaaatctgggatcgcgatagcgcagaaattcaggcactgcgtaacgaaatcgcgtacatcatcgtcctgcaggattgcatgctggatcagaacaaacaaacc

[0162]

[0163] [Sequence No. 24]

[0164] accaaacacgatagcctggcactgacccagattattgagaaaatcatgagccgcgtcaccaccaacatgatgtttgcgctgcacttcggcaaaatgcacctgaatagctgcggtcgtcaaggcaccggcatgattcgcaaagacggttggcaaggcgcaggtattggtcgcgacgcatatcgtctgcgtcaaaaacacgcaattcgcggcggttctggttcgggcggacaggttttcggcaacatcttttggagctgggcgtatagtaacggcgcgtatatgggcaaagcagattggcgtttagcacgcggtcgtattcgcggtatgatggaagcaggccatatttgcggtcgtattaatccgagcggtagctattggagcaacaaaatcctgctgctgttccagaactttcgcgatagttacggcgaagattacggaagtggtagcggcagcggggagaacatggtccagacccacaaaaaattccagaacaacatgaaacgccgctacatcatggatctgacccgcgtttgtaaatgccacgaagccacctacatgtggaaaatctgggatcgcgatagcgcagaaattcaggcactgcgtaacgaaatcgcgtacatcatcgtcctgcaggattgcatgctggatcagaacaaacaaacc

[0165]

[0166] [서열번호 25]

[0167] ctggttaccctggaatttcagggcgtttaccaggaaggtctgaaactgcagtgcattcagggtcaaggcgatcagatgtgctctcagatggttggcgttgcactgatggtctggattctgagcattcagctgccgacctctaccaactggtggatgttccagatttgccagaacgctcgtctgattaccgtggtcaaaagcaaatttggcggttctggttcgggcggaaactggagccgttggttttatacctctgccgcccatagcatgttcgttatttcctgcctgcagtgggttatggagggtcacatgggcacccataaagatacctgcgcgaacctgatcttcgccattggtatggaaccgtgggttgcagctatctgcatcaaacagcgcaaacagaacacctacgagcacgacaaaatcatcacctctggaagtggtagcggcagcggggcaggtgttatttacgcgatttggaccgcagacgaagatcagctgtggttcttcatcaccaacaaccagatgcacagccagctgtacaaaaacgacgagaaagtcatgtcctgtcgttggtgcaaaatccagagcctgaccgaggacaaaaccattctgagcaccagtagcgcacaagaaggtcatacccgcgaaggcgatcgtcgc

[0168]

[0169] [서열번호 26]

[0170] ctggttaccctggaatttcagggcgtttaccaggaaggtctgaaactgcagtgcattcagggtcaaggcgatcagatgtgctctcagatggttggcgttgcactgatggtctggattctgagcattcagctgccgacctctaccaactggtggatgttccagatt tgccagaacgctcgtctgattaccgtggtcaaaagcaaatttggcggttctggttcgggcggacaggttttcggcaacatcttttggagctgggcgtatagtaacggcgcgtatatgggcaaagcagattggcgtttagcacgcggtcgtattcgcggtatgatgg aagcaggccatatttgcggtcgtattaatccgagcggtagctattggagcaacaaaatcctgctgctgttccagaactttcgcgatagttacggcgaagattacggaagtggtagcggcagcggggcaggtgttatttacgcgatttggaccgcagacgaagatca gctgtggttcttcatcaccaacaaccagatgcacagccagctgtacaaaaacgacgagaaagtcatgtcctgtcgttggtgcaaaatccagagcctgaccgaggacaaaaccattctgagcaccagtagcgcacaagaaggtcatacccgcgaaggcgatcgtcgc

[0171]

[0172] Thus, the present invention can regulate intracellular signal transduction by converting intracellular proliferation signals into death signals using a scaffold protein comprising a domain capable of binding to both a kinase involved in intracellular proliferation signal transduction and a kinase involved in intracellular death signal transduction.

[0173] In one embodiment of the present invention, it was confirmed that when a scaffold protein was expressed in cancer cells, the survival rate of the cancer cells was significantly reduced as the signaling pathway involved in cell proliferation signals was inhibited and the signaling pathway involved in apoptosis signals was selectively activated.

[0174]

[0175] The present invention also provides a scaffold protein capable of regulating intracellular signal transduction.

[0176] As previously explained, the scaffold protein of the present invention comprises a first domain that specifically binds to BRAF, which is involved in cell proliferation signaling, and a second domain that specifically binds to MKK, which is involved in cell death signaling, thereby being able to regulate intracellular signaling, and specifically, to rewire intracellular proliferation signals into death signals.

[0177] In addition, the scaffold protein of the present invention may further include a third domain that specifically binds to JNK, which is involved in cell death signaling.

[0178] The first domain of the scaffold protein may be composed of the amino acid sequence of SEQ ID NO. 1 or SEQ ID NO. 2, the second domain may be composed of the amino acid sequence of SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5, and the third domain may be composed of SEQ ID NO. 6 or SEQ ID NO. 7.

[0179] In the scaffold protein of the present invention, the first domain and the second domain, and the second domain and the third domain may be connected by a linker, and the linker may be a GS linker, and the first domain and the second domain may be connected by a first linker, and the second domain and the third domain may be connected by a second linker.

[0180] The first linker above may be composed of the amino acid sequence of SEQ ID NO. 15, and the second linker above may be composed of the amino acid sequence of SEQ ID NO. 16.

[0181] In addition, the scaffold protein may be composed of the amino acid sequence of SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, or SEQ ID NO. 22.

[0182] Meanwhile, the same content as previously explained can be applied to the characteristics of the scaffold protein, such as its structure, composition, and amino acid sequence.

[0183]

[0184] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer, comprising the scaffold protein.

[0185] The above cancer disease may be one or more selected from the group consisting of colorectal cancer, liver cancer, cervical cancer, lung cancer, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, breast cancer, ovarian cancer, uterine cancer, pancreatic cancer, stomach cancer, skin cancer, bladder cancer, thyroid cancer, brain cancer, and prostate cancer.

[0186] The above pharmaceutical composition may further comprise a suitable pharmaceutically acceptable carrier, excipient, or diluent according to conventional methods. The pharmaceutically acceptable carrier is one that is commonly used in formulations and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0187] The pharmaceutical composition of the present invention may additionally include lubricants, humectants, sweeteners, flavorings, emulsifiers, suspending agents, preservatives, etc., in addition to the above components. Regarding suitable pharmaceutically acceptable carriers and formulations, each component may be preferably formulated using the method disclosed in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0188] The pharmaceutical composition of the present invention can be administered orally or parenterally, and parenteral administration includes intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc.

[0189] Oral formulations include, for example, tablets, pills, hard and soft capsules, liquids, suspensions, emulsifiers, syrups, granules, etc., and these formulations may additionally contain diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine) and lubricants (e.g., silica, talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycol) in addition to the active ingredient. Furthermore, the tablet may contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, and in some cases may contain disintegrants or boiling mixtures such as starch, agar, alginic acid or its sodium salt and / or absorbents, coloring agents, flavoring agents and sweeteners. The above formulation can be prepared by conventional mixing, granulation, or coating methods.

[0190] In addition, a representative formulation for parenteral administration is an injectable preparation, and solvents for the injectable preparation may include water, Ringer's solution, isotonic physiological saline, or suspension. The sterile fixation oil of the injectable preparation may be used as a solvent or suspension medium, and any non-irritating fixation oil, including mono- and di-glycerides, may be used for this purpose. Furthermore, the injectable preparation may use fatty acids such as oleic acid.

[0191] The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including the type and severity of the patient's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects by taking all of the above-mentioned factors into account, and this can be easily determined by a person skilled in the art.

[0192] Specifically, the effective amount of the composition according to the present invention may vary depending on the patient's age, gender, and weight, and generally, 0.001 to 150 mg, preferably 0.01 to 100 mg per kg of body weight, may be administered daily or every other day, or divided into 1 to 3 doses per day. However, since the amount may be increased or decreased depending on the route of administration, gender, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

[0193] The present invention also relates to a method for preventing or treating cancer, wherein the method may include the step of administering a composition comprising a scaffold protein.

[0194] In the present invention, the subject to administration may be an individual, and the individual may be an animal, typically a mammal.

[0195]

[0196] Examples

[0197]

[0198] The present invention will be explained in more detail through the following examples. However, these examples represent some experimental methods and configurations to illustrate the invention, and the scope of the invention is not limited to these examples.

[0199]

[0200] Preparation Example 1: Preparation of a signal-regulating scaffold protein

[0201]

[0202] 1-1. Preparation of BRAF, MKK4, and JNK1 Receptor Models

[0203]

[0204] 3D atomic coordinates of BRAF for the de novo design of a mini-protein binding to BRAF were prepared from the X-ray crystal structure of BRAF complexed with MEK1 (PDB entry: 4MNE). The construction of an all-atom model for the V600E mutant BRAF was initiated by utilizing the structure of the kinase domain (residues 449-722) of wild-type BRAF and inserting residues 465-468, which were missing from the original X-ray structure.

[0205] Next, 15 soluble mutations (I543A, I544S, I551K, Q562R, L588N, K630S, Y673S, A688R, L706S, Q709R, S713E, L716E, S720E, P722S, and K723G) were introduced, and a receptor model containing a total of 16 mutations (V600E mutation BRAF15mut) was completed by substituting valine (Val), residue 600, with glutamate (Glu).

[0206] The 3D atomic coordinates of the V600E mutant BRAF15mut were optimized using homology modeling with the latest version of the MODELLER program. In the final step, the structure was optimized using a conjugate gradient algorithm and molecular dynamics simulations to minimize spatial constraint intrusion.

[0207] In addition, for the design of a mini-protein that binds to MKK4, the X-ray structure of the human MKK4 activity domain (PDB entry: 3ALN) was used without further modification, and for the design of a mini-protein that binds to JNK1, the X-ray structure of the human JNK activity domain (PDB entry: 2NO3) was used without further modification.

[0208]

[0209] 1-2. Screening of Basic Scaffolds for the Preparation of Mini-proteins Binding to BRAF, MKK4, and JNK1

[0210]

[0211] To manufacture mini-proteins (i.e., domains) targeting the kinases BRAF, MKK4, and JNK1, a module protein was designed that binds to an allosteric site located structurally opposite to the active site for each kinase without affecting the phosphorylation activity of each kinase.

[0212] First, for the de novo design of a mini-protein that inhibits BRAF activity, the partial structure of the endogenous ligand protein 14-3-3 was determined as a basic scaffold from the X-ray crystal structure of BRAF (PDB entry: 4MNE) complexed with MEK1, and a segment (residues 164-232) containing three α-helices that play a key role in binding to BRAF by encircling the C-terminal tail was isolated from the 14-3-3 protein.

[0213] Subsequently, by explicitly adding hydrogen atoms to all heavy atoms, an all-atom model with accurate geometry and complete atomic-level details was constructed and selected as the basic scaffold for BRAF-binding miniproteins.

[0214] In addition, the design of the mini-protein that binds to MKK4 and JNK1 was carried out in the same way as BRAF.

[0215]

[0216] 1-3. Mini-protein docking simulation

[0217]

[0218] Based on the receptor model prepared in Preparation Example 1-1 and the basic scaffold protein determined in Preparation Example 1-2, a docking simulation was performed using a multi-scale Monte Carlo-based algorithm implemented in the RosettaDock program.

[0219] During the simulation, the mini-proteins were systematically translated and rotated relative to the fixed structures of each target protein, thereby extensively exploring various potential binding orientations. The docking protocol focused on identifying the most probable binding modes by optimizing the side chain positions of the mini-protein ligands, a process performed by combining rotamer packing and explicit gradient-based minimization of rigid-body degrees of freedom. Through this process, the side chain orientation and overall scaffold position were fine-tuned to maximize interaction with the target surface.

[0220] Protein-protein complexes generated were evaluated and ranked using a complex binding energy function that includes several key energy factors, such as van der Waals interactions, electrostatic interactions (with reduced weighting applied), implicit solvation energy based on a Gaussian model, direction-dependent hydrogen bonding, and statistical preference for side chain rotamer arrangement.

[0221] Among the 1,000 different binding configurations generated during the simulation process for each target, the structure with the lowest calculated binding energy was selected as the final mini-protein-target complex model.

[0222] The results of the mini-protein-BRAF model, mini-protein-MKK4 model, and mini-protein-JNK1 model selected as above are shown in Figs. 1a, 1b, and 1c, respectively.

[0223]

[0224] 1-4. De novo design of miniproteins targeting BRAF, MKK4, and JNK1

[0225]

[0226] Starting from the most likely binding configuration, we mutated all 69 amino acid residues of the mini-protein scaffold to find a mini-protein that could bind strongly and specifically to a target protein.

[0227] The design of mini-proteins was performed using the RosettaRemodel blueprint protocol, enabling residue-level custom design under the constraint that the original three-helix bundle topology derived from the scaffold must be maintained.

[0228] Prior to design optimization, each target protein structure was placed on a spatial hashing grid to detect and exclude adverse van der Waals collisions at protein-protein interfaces. In the core design process, the FastDesign protocol was applied, a method that integrates rotamer optimization and gradient-based energy minimization to purify both the backbone and side chain structures of mini-protein candidates. In particular, for residues located at binding interfaces, iterative adjustments were made to enhance shape complementarity and interaction specificity with the target protein.

[0229] Each design outcome was evaluated using the Rosetta energy score function, simultaneously assessing the structural quality of the predicted 3D folded structure and the interaction strength at the binding interface. Out of a total of 1,000 mini-protein designs, top candidates with excellent energy profiles and predicted binding characteristics were selected for subsequent analysis.

[0230]

[0231] 1-5. Selection of Mini-protein Candidates Targeting BRAF, MKK4, and JNK1 Using Hydration Free Energy

[0232]

[0233] To evaluate the aqueous solubility of each mini-protein inhibitor candidate, the hydration free energy (△G) was calculated using a solvent-contact model. sol ) calculated.

[0234] Assuming that the total hydration free energy of a miniprotein can be approximated as the sum of individual atomic contributions, △G for the miniprotein sol The value was calculated using the following Equation 1.

[0235]

[0236] [Equation 1]

[0237]

[0238]

[0239] In the above Equation 1, S i represents the atomic hydration energy for atom i, and V j represents the fragmental volume of atom j, and O i max represents the maximum accessible volume for atom i. The Gaussian envelope function is the interatomic distance r ij It was used to model the spatial decay of solvent accessibility as a function of , and the parameter σ was set to 3.5 Å.

[0240] Atomic parameter S i , O i max and V j It was derived from previously optimized values ​​using a standard genetic algorithm based on experimental hydration free energy data for dipeptides.

[0241] △G calculated thereafter solThe values ​​were used to evaluate the solubility potential of each candidate, and mini-proteins with more favorable (i.e., lower) hydration free energy were given priority; finally, the candidates selected for experimental verification were chosen based on these calculated hydration energy values.

[0242] Finally, 50 mini-proteins targeting BRAF, 200 mini-proteins targeting MKK4, and 50 mini-proteins targeting JNK1 were selected as candidates.

[0243]

[0244] 1-6. Expression and Purification of Mini-protein Candidates

[0245]

[0246] The mini-protein candidates selected in Preparation Examples 1-5 above were synthesized in an E. coli expression vector pMAL-c5X containing an MBP tag.

[0247] E. coli BL21(DE3) possessing a tag-binding module was cultured in LB medium containing ampicillin. Cells were grown at 37°C until an OD600 of 0.5 was reached, and then protein expression was induced with 0.5 mM IPTG at 18°C ​​for 18 hours. Subsequently, cells were collected by centrifugation.

[0248] For purification, the bacterial pellet was suspended in cold purification buffer [20 mM HEPES pH 7.4, 300 mM NaCl, 1 mM PMSF, 5 mM β-mercaptoethanol], sonicated over ice, and then centrifuged at 17,000 xg for 50 minutes at 4 ℃. The supernatant was applied to glutathione cephalosporose resin (Cytiva) or amylose resin (NEB) while gently stirring at 4 ℃ for 4 hours. Proteins bound to the resin were washed three times with washing buffer [20 mM HEPES pH 7.4, 300 mM NaCl, 5 mM β-mercaptoethanol] and then eluted with GST elution buffer [20 mM HEPES pH 7.4, 300 mM NaCl, 10 mM reduced L-glutathione (GSH)] or MBP elution buffer [20 mM HEPES pH 7.4, 200 mM NaCl, 1 mM EDTA, 10 mM maltose]. The concentration of the purified protein was measured using the Bradford protein quantification method with BSA as the standard.

[0249]

[0250] 1-7. Selection of miniproteins targeting BRAF, MKK4, and JNK1

[0251]

[0252] 1-7-1. BRAF, MKK4, and JNK1 Expression and Purification

[0253]

[0254] Human-derived BRAF domains (residues 444-733; UniProt ID: P15056, including V600E mutation), MKK4 (residues 37-399; UniProt ID: P45985), and JNK1 (UniProt ID: P45983-3) were cloned into the E. coli expression vector pGEX4T-1 containing a GST tag and the mammalian expression vector pcDNA3.1(+) containing a 6x myc tag, respectively, and expression and purification were performed based on the method of Preparation Examples 1-6.

[0255]

[0256] 1-7-2. Measurement of Mini-protein Binding Affinity to Target Kinases

[0257]

[0258] The binding affinity of the mini proteins prepared in Preparation Examples 1-6 above for each target kinase was measured by performing an ELISA (Enzyme-linked immunosorbent assay).

[0259] Each biotinylated target kinase was added to a streptavidin-coated plate (ThermoFisher Scientific, #15500) and reacted at room temperature (RT) for 1 hour. The plate was washed with TBS-T buffer [50 mM Tris, pH 7.5, 137 mM NaCl, 0.05% Tween-20] and then blocked with SuperBlock blocking buffer (ThermoFisher Scientific). Subsequently, purified MBP-tag binding module (100 nM) was added and reacted at room temperature for 2 hours.

[0260] After removing the reaction solution and thoroughly washing and blocking the plate, the anti-MBP primary antibody was added. After 1 hour of reaction, the HRP-conjugated secondary antibody was added to the washed plate and reacted for 1 hour. Subsequently, the plate was washed, and 100 μL of TMB solution was added to induce an enzyme-substrate reaction with HRP for 5 minutes, followed by the addition of 100 μL of reaction stop solution. The amount of residual bound module protein was measured by absorbance at 450 nm using a Varioskan LUX plate reader (ThermoFisher Scientific).

[0261] The results for BRAF measured in this way are shown in Fig. 2a, the results for MKK4 in Figs. 2b and 2c, and the results for JNK1 in Fig. 2d.

[0262] In addition, as a result of measuring binding affinity, candidates 36, 42, 44, and 47 among the mini-protein candidates targeting BRAF showed high binding affinity, candidates 70, 126, 154, and 198 among the mini-protein candidates targeting MKK4 showed high binding affinity, and candidates 38 and 147 among the mini-protein candidates targeting JNK1 showed high binding affinity.

[0263] Based on this, BRAF-targeting mini-protein candidates 42 and 44, MKK4-targeting mini-protein candidates 70, 154, and 198, and JNK1-targeting mini-protein candidates 38 and 147 were selected as mini-proteins to manufacture the final scaffold protein.

[0264] The amino acid sequences of the mini-protein candidates selected in this way are shown in Table 1 below.

[0265]

[0266] Target Kinase Mini Protein Number Sequence Number Amino Acid Sequence BRAF421LVTLEFQGVYQEGLKLQCIQGQGDQMCSQMVGVALMVWILSIQLPTSTNWWMFQICQNARLITVVKSKF442TKHDSLALTQIIEKIMSRVTTNMMFALHFGKMHLNSCGRQGTGMIRKDGWQGAGIGRDAYRLRQKHAIRMKK4703SRRTCVKSANAWQWMSIERYCISYNGQNIQRNCESINGGLDSKEPLLSERQMMGQQMGKYNIAVNSERV1544NWSRWFYTSAAHSMFVISCLQWVMEGHMGT HKDTCANLIFAIGMEPWVAAICIKQRKQNTYEHDKIITS1985QVFGNIFWSWAYSNGAYMGKADWRLARGRIRGMMEAGHICGRINPSGSYWSNKILLLFQNFRDSYGEDYJNK1386ENMVQTHKKFQ NNMKRRYIMDLTRVCKCHEATYMWKIWDRDSAEIQALRNEIAYIIVLQDCMLDQNKQT1477AGVIYAIWTADEDQLWFFITNNQMHSQLYKNDEKVMSCRWCKIQSLTEDKTILSTSSAQEGHTREGDRR

[0267]

[0268] 1-8. Preparation of Scaffold Proteins

[0269]

[0270] A scaffold protein for cell signal rearrangement was prepared based on the mini proteins selected in Preparation Example 1-7-2 above.

[0271] A plasmid expressing an artificially designed Flag-tag scaffold was inserted into the mammalian expression vector p3xFlag-CMV-10, transfected into HEK293 cells using Lipofectamine3000, and then cultured for 24 and 48 hours. Subsequently, the cell lysates were analyzed by Western blot using an anti-Flag antibody, and the results are shown in Figure 3a.

[0272] Each mini-protein was connected via GS linkers (linkers 1 and 2), and a schematic diagram of the prepared scaffold is shown in FIG. 3b. In addition, the amino acid sequences of the linkers and the prepared scaffold proteins are shown below.

[0273]

[0274] [Sequence No. 19] Amino acid sequence of scaffold protein #1

[0275] TKHDSLALTQIIEKIMSRVTTNMMFALHFGKMHLNSCGRQGTGMIRKDGWQGAGIGRDAYRLRQKHAIRGGSGSGGSRRTCVKSANAWQWMSIERYCISYNGQNIQRNCE SINGGLDSKEPLLSERQMMGQQMGKYNIAVNSERVGSGSGSGENMVQTHKKFQNNMKRRYIMDLTRVCKCHEATYMWKIWDRDSAEIQALRNEIAYIIVLQDCMLDQNKQT

[0276]

[0277] [Sequence No. 20] Amino acid sequence of scaffold protein #2

[0278] TKHDSLALTQIIEKIMSRVTTNMMFALHFGKMHLNSCGRQGTGMIRKDGWQGAGIGRDAYRLRQKHAIRGGSGSGGQVFGNIFWSWAYSNGAYMGKADWRLARGRIRGMM EAGHICGRINPSGSYWSNKILLLFQNFRDSYGEDYGSGSGSGENMVQTHKKFQNNMKRRYIMDLTRVCKCHEATYMWKIWDRDSAEIQALRNEIAYIIVLQDCMLDQNKQT

[0279]

[0280] [Sequence No. 21] Amino acid sequence of scaffold protein #3

[0281] LVTLEFQGVYQEGLKLQCIQGQGDQMCSQMVGVALMVWILSIQLPTSTNWWMFQICQNARLITVVKSKFGGSGSGGNWSRWFYTSAAHSMFVISCLQWVMEGHMGTHKDT CANLIFAIGMEPWVAAICIKQRKQNTYEHDKIITSGSGSGSGAGVIYAIWTADEDQLWFFITNNQMHSQLYKNDEKVMSCRWCKIQSLTEDKTILSTSSAQEGHTREGDRR

[0282]

[0283] [Sequence No. 22] Amino acid sequence of scaffold protein #4

[0284] LVTLEFQGVYQEGLKLQCIQGQGDQMCSQMVGVALMVWILSIQLPTSTNWWMFQICQNARLITVVKSKFGGSGSGGQVFGNIFWSWAYSNGAYMGKADWRLARGRIRGMM EAGHICGRINPSGSYWSNKILLLFQNFRDSYGEDYGSGSGSGAGVIYAIWTADEDQLWFFITNNQMHSQLYKNDEKVMSCRWCKIQSLTEDKTILSTSSAQEGHTREGDRR

[0285]

[0286] [Sequence No. 15] Amino acid sequence of linker 1

[0287] GGSGSGG

[0288]

[0289] [Sequence No. 16] Amino acid sequence of linker 2

[0290] GSGSGSG

[0291]

[0292] Experimental Example 1: Confirmation of Increased MKK4 Phosphorylation by Selected Mini-proteins

[0293]

[0294] An experiment was conducted to determine whether the mini protein of SEQ ID NO. 2, which targets BRAF selected in Preparation Example 1-7-2 above, could perform phosphorylation of MKK4 by spatially positioning BRAF and MKK4 in close proximity.

[0295] Both the control sample and the sample containing the mini-protein of SEQ ID NO. 2 used the same BRAF plasmid having an N-terminal MBP in the kanamycin vector, and used either a single MKK4 construct having an N-terminal Trigger Factor in the pCold vector or a mini-protein of SEQ ID NO. 2 and an MKK4 construct linked with a SUMO tag. For the MKK4, an MKK4 containing a mutant site (K131M) to inhibit autophosphorylation activity was used.

[0296] For the co-expression of BRAF and MKK4, each plasmid DNA with different antibiotic resistances (pCold-TF vector and pET-28a vector) was added to BL21(DE3) competent E. coli cells. After incubating the two plasmid DNAs with the cells on ice for 10 minutes, a heat shock was applied at 42°C for 1.5 minutes, followed by incubation on ice for another 10 minutes.

[0297] Subsequently, 500 μL of LB was added to the cells and incubated in a 37°C shaker for 1 hour. After 1 hour of incubation, the cells were centrifuged at 13,500 rpm for 1 minute. 500 μL of the supernatant was removed, and the remaining 100 μL of supernatant was mixed with the pellet. 100 μL of cells were plated onto a plate mixed with ampicillin and kanamycin. The plate was incubated overnight at 37°C. A single colony was selected and incubated overnight in 5 mL of LB containing both antibiotics. The next day, 200 μL of the cultured cells were inoculated into a new 5 mL of LB and incubated until the OD 6000 reached 0.6 to 0.8. 1 mL of the pre-induction sample was recovered by centrifugation, and the remainder was cooled to 4°C.

[0298] Cells were induced with 0.05 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 4 °C for 2, 4, 6, and 8 hours. 1 mL of each of the five samples (pre-induction and post-induction at 2, 4, 6, and 8 hours) was centrifuged to remove the supernatant. Protein concentration was measured using a DC Protein Assay Kit (Bio-Rad). The pellets of the pre- and post-induction samples were resuspended in 200 μL of lysis buffer (50 mM Tris-HCl pH 7.5, 500 mM NaCl, and 5 mM β-mercaptoethanol). The samples were lysed using a Digital Sonifier (Branson) by switching the on / off cycle twice for 1 minute at 15% amplitude. 20 μL of each induction sample was mixed with 5 μL of 5X sample loading buffer and heated at 95 °C for 3 minutes. After electrophoresing the sample on a 10% sodium dodecyl sulfate-polyacrylamide gel at 90 V, it was transferred to a methanol-activated PVDF membrane (Merck) using a Trans-Blot SD semi-dry transfer device (Bio-Rad) at 20 V for 50 minutes. The membrane was blocked with 5 mL of SuperBlock (TBS) blocking buffer (Thermo Scientific) at room temperature for 2 hours.

[0299] Subsequently, the mixture was reacted overnight at 4°C with rabbit anti-pMKK4 antibody (against Ser257 / Thr261, 1:1000, Cell Signaling) diluted in 5% BSA / TBST, mouse anti-MBP antibody (1:10000, New England BioLabs) diluted in 5% (w / v) skim milk / TBST, and anti-His antibody (1:1000, Abbkine).

[0300] The next day, the samples were washed three times with TBST for 5 minutes each. For the rabbit-derived primary antibody, an HRP-conjugated goat anti-rabbit IgG antibody (1:500, AbClon) was diluted in 5% (w / v) skim milk / TBST and reacted at room temperature for 1 hour. For the mouse-derived primary antibody, an HRP-conjugated goat anti-mouse IgG antibody (1:1000, GenDEPOT) was diluted in 5% skim milk / TBST and reacted.

[0301] After repeating the washing step, the chemiluminescence signal from the membrane was detected using the ChemiDoc XRS+ System (Bio-Rad) with a Pierce ECL Western Blotting Substrate (ThermoFisher).

[0302] The results of the Western blot measured as above are shown in Figure 4a, the left side is the result when only MKK4 is expressed, and the right side is the result when MKK4 and the mini protein of sequence number 2 are expressed together.

[0303] In addition, the normalized result of MKK4 phosphorylation induced by the mini-protein of sequence number 2 is shown in Fig. 4b, and a schematic diagram of the process of the complex of MKK4 and the mini-protein phosphorylating MKK4 is shown in Fig. 4c.

[0304] As shown in Figure 4a, it was confirmed that the pMKK4 signal increased significantly in a time-dependent manner at the 110 kDa position under conditions containing the mini-protein, whereas the phosphorylation signal was very weak at the 82 kDa position in the control group. This indicated that the mini-protein brought BRAF and MKK4 closer together, thereby inducing the phosphorylation of MKK4. Additionally, the 82 kDa (anti-His) and 73 kDa (anti-MBP) bands remained similar across all time points, confirming that the observed increase in pMKK4 was due to MKK4 phosphorylation induced by the mini-protein, rather than differences in protein expression levels.

[0305] Furthermore, as shown in Figure 4b, it was found that the degree of phosphorylation was higher when mini-proteins were included compared to the control group, and it was confirmed that the degree of MKK4 phosphorylation by mini-proteins increased significantly over time.

[0306]

[0307] Experimental Example 2: Confirmation of Regulation of JNK1 Phosphorylation Levels of Scaffold Proteins in Cancer Cells

[0308]

[0309] An experiment was conducted to determine whether the scaffold protein #2 (sequence number 20) prepared in Preparation Examples 1-8 above could regulate the JNK1 phosphorylation level in cancer cells.

[0310] To this end, we confirmed the ability of a scaffold to mediate JNK1 phosphorylation by the regulated MAPK pathway in human cervical cancer HeLa cells. HeLa cells were cultured at 37°C and 5% CO2 conditions using Dulbecco's Modified Eagle's Medium (DMEM, Welgene) containing 10% FBS (Fetal Bovine Serum) and 1% antibiotic-antimycotic.

[0311] Cells were seeded into each well of a 24-well plate, and BRAF and MKK4 with N-terminal MBP tags, JNK1 with C-terminal myc-His tags, and Scaffold Protein #2 with C-terminal 3X flag tags were transfused to pcDNA using Lipofectamine 3000 transduction reagent (Invitrogen). TM 3.1 Subcloned into the / myc-His vector, and cells were harvested 24 hours after transduction.

[0312] pcDNA to verify the effect of scaffold proteins TM Empty vector control (pcDNA) TM An empty vector control was used, and protein quantification was performed using the DC Protein Assay Kit (Bio-Rad).

[0313] For SDS-PAGE, all proteins were loaded at 20 μg / mL. After electrophoresing the samples on a sodium dodecyl sulfate-polyacrylamide gel at 90 V, they were transferred to a methanol-activated PVDF membrane (Merck) using a Trans-Blot SD semi-dry transfer device (Bio-Rad) at 20 V for 50 minutes. The membrane was blocked with 5 mL of SuperBlock (TBS) blocking buffer (Thermo Scientific) at room temperature for 2 hours.

[0314] Subsequently, rabbit anti-p-JNK antibody (against T183 / Y185, 1:1000, Cell signaling) diluted in TBST containing 5% (w / v) BSA, mouse anti-MBP antibody (1:10000, New England BioLabs), anti-His antibody (1:1000, Abbkine), and mouse anti-β-Actin antibody (1:10000, Abbkine) diluted in TBST containing 5% (w / v) skim milk were treated to the membrane overnight at 4°C. After the overnight reaction, HRP-conjugated goat anti-mouse IgG antibody (1:5000, AbClon) diluted in 5% (w / v) skim milk / TBST was added to the membrane treated with the mouse-derived primary antibodies. HRP-conjugated IgG diluted in 5% (w / v) skim milk / TBST was applied to membranes treated with anti-His and anti-p-JNK primary antibodies, respectively. Κ BP antibody and HRP-conjugated goat anti-rabbit IgG antibody (1:500, AbClon) were used.

[0315] The results of JNK1 phosphorylation measured as above are shown in Figure 5.

[0316] As shown in Figure 5, pJNK signal was observed only when a scaffold protein was used (*1), and under conditions without a scaffold protein (*2), pJNK signal was hardly observed despite the presence of BRAF, MKK4, and JNK1, confirming that JNK1 activation is performed by the scaffold protein.

[0317]

[0318] Experimental Example 3: Confirmation of Cancer Cell Apoptosis Induction by Scaffold Protein

[0319]

[0320] An experiment was performed to verify whether the scaffold protein #2 (sequence number 20) prepared in Preparation Examples 1-8 above induced apoptosis in cancer cells through JNK1 phosphorylation resulting from rewiring to the BRAF-MKK4-JNK1 pathway.

[0321] 100 mM of human cervical cancer HeLa cells were cultured in each well of a 96-well plate at 37°C under 5% CO2 conditions. DMEM (Welgene) containing 10% FBS and 1% antibiotic-antimycotic was used for the culture of HeLa cells.

[0322] The next day, the medium was replaced with DMEM containing 1% FBS and antibiotic-antimycotic, and DNA constructs of MBP BRAF, MBP MKK4, JNK1, and scaffold proteins were transduced using Lipofectamine 3000 transduction reagent (Invitrogen).

[0323] After 48 hours had elapsed since transduction, 10 μL of the cell proliferation reagent WST-1 (EZ-Cytox) was added to each well. After the addition of WST-1, absorbance was repeatedly measured at 450 nm using a microplate (ELISA) reader at different time points in the experiment. Cell viability (%) was calculated at 165 minutes as the percentage of [(absorbance of wells containing cells and scaffold proteins) / (absorbance of wells containing only cells)].

[0324] The results of the relative cell viability of HeLa cells measured as above are shown in Figure 6. In Figure 6, the left side shows the results when scaffold proteins are not included, and the right side shows the results when scaffold proteins are included.

[0325] As shown in Figure 6, it was confirmed that the survival rate of HeLa cells decreases when the scaffold protein is expressed, and through this, it was confirmed that the prepared scaffold protein can induce apoptosis in cancer cells.

[0326]

[0327] Experimental Example 4: Confirmation of binding between scaffold protein and target kinase

[0328]

[0329] Co-immunoprecipitation (Co-IP) was performed to determine whether scaffold proteins #1 to #4 prepared in Preparation Examples 1-8 above interact with three target kinases.

[0330] Myc-tagged BRAF KD, MKK4, JNK1, and Flag-tagged scaffold plasmids were co-transduced into HEK293 cells using Lipofectamine3000 (Invitrogen, #L3000001) and cultured at 37°C for 24 hours. Cells were lysed in lysis buffer [50 mM Tris-HCl pH 7.5, 150 mM sodium chloride, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 2 mM EDTA, 1 mM sodium orthovanadate, 1 mM PMSF, 10 μg / mL leupeptin, 0.1% (v / v) NP-40] and centrifuged at 17,000 xg for 30 minutes at 4°C.

[0331] The supernatant was quantified by Bradford assay and reacted with anti-Flag M2 magnetic beads (Merck) at 4°C for 2 hours. After washing the resin to remove unbound components, the sample bound to the resin was eluted with 5X protein loading buffer [250 mM Tris-HCl pH 6.8, 5% SDS, 5% β-mercaptoethanol, 0.1% bromophenol blue, 50% glycerol]. The eluted protein was analyzed by Western blot using anti-Myc and anti-Flag antibodies, and the results are shown in Figures 7a to 7d.

[0332] As shown in Figures 7a to 7d, when the scaffold protein was immunoprecipitated with the Flag antibody (IP: Flag), BRAF, MKK4, and JNK1 were detected together, which indicated that the prepared scaffold protein physically interacted with each target kinase to form a protein complex.

[0333]

[0334] Experimental Example 5: Verification of Phosphorylation Signal Transduction Rewiring of Scaffold Proteins

[0335]

[0336] We conducted an experiment to determine whether the manufactured scaffold protein could increase the expression and phosphorylation levels of c-Jun, a downstream gene of JNK1, by rewiring intracellular signal transduction.

[0337] After co-transfecting HEK293 cells with a luciferase vector capable of quantifying the expression and activity of c-Jun, c-Jun, a prepared scaffold, and GFP for normalization, the medium was changed after 8 hours and the cells were cultured for a total of 48 hours.

[0338] After washing the cells with DPBS, they were lysed in reporter lysis buffer (Promega, #E1500). The lysate was collected in Eppendorf tubes and centrifuged at 17,000 xg for 20 minutes at 4°C. 40 μL of each supernatant was mixed with 10 μL of luciferin (Promega, #E1500) in a 96-well white plate, and luminescence was measured using a Varioskan LUX plate reader. For normalization, GFP expression values ​​in other wells were measured by fluorescence. The remaining lysate was used for Western blot analysis with the anti-Phospho c-Jun antibody.

[0339] Western blot results and the results of quantifying the expression and phosphorylation levels of c-Jun are shown in Figure 8.

[0340] As shown in Figure 8, it was confirmed that the expression and phosphorylation levels of c-Jun were significantly increased in cells introduced with the scaffold protein compared to cells that did not introduce the scaffold protein or c-Jun. Through this, it was confirmed that the prepared scaffold protein rewires intracellular signal transduction to increase the expression and phosphorylation levels of c-Jun, a downstream gene of JNK1.

[0341]

[0342] Experimental Example 6: Quantitative Analysis of Apoptosis Using Muse Cell Analyzer

[0343]

[0344] In HepG2 liver cancer cell lines, plasmids encoding scaffold proteins #1 to #4 of Preparation 1-8, tagged with Flag, were transduced. After 48 hours, cells were treated with 50 ng / mL EGF for 1 hour, or alternatively, serum-deprived overnight in a medium containing 0.5% fetal bovine serum (FBS) followed by stimulation with EGF for 48 hours.

[0345] Subsequently, 5 x 10⁵ cells were resuspended in 100 μL of analysis buffer and reacted with 100 μL of staining reagent containing Annexin V and 7-AAD. After reacting the sample at room temperature under dark conditions for 20 minutes, the Muse was prepared according to the manufacturer's instructions. TM The analysis was performed using a Cell Analyzer (Millipore).

[0346] Depending on fluorescence intensity, the apoptotic population is Annexin V - / 7-AAD - (Survival cells), Annexin V + / 7-AAD - (early apoptotic cells) and Annexin V + / 7-AAD + They were classified as (late-stage apoptotic cells), and the data is Muse TM It was quantified using analysis software.

[0347] In addition, the same experiment was performed on SW480 colon cancer cells.

[0348] The results for HepG2 liver cancer cells and SW480 colon cancer cells are shown in Figures 9a and 9b, respectively.

[0349] As can be seen in Fig. 9a, the prepared scaffold protein showed a maximum apoptosis effect of about 20% in HepG2 liver cancer cells, and as can be seen in Fig. 9b, the prepared scaffold protein showed a maximum apoptosis effect of 50% in SW480 colon cancer cells. Through this, it was confirmed that the prepared scaffold protein can induce cell death through the rewiring of signal transduction within the cell.

[0350]

[0351] From the foregoing description, those skilled in the art to which the present invention pertains will understand that the present invention may be implemented in other specific forms without altering its technical concept or essential features. In this regard, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. A first domain that specifically binds to a kinase involved in cell proliferation signaling, and A second domain that specifically binds to kinases involved in apoptosis signaling. A scaffold protein containing 2. In Paragraph 1, The above-mentioned first domain specifically binds to BRAF, and A scaffold protein in which the second domain specifically binds to one or more of MKK4 and MKK7 kinases.

3. In Paragraph 1, A scaffold protein comprising a third domain that specifically binds to JNK.

4. In Paragraph 1, A scaffold protein in which the first domain is composed of the amino acid sequence of SEQ ID NO. 1 or SEQ ID NO.

2.

5. In Paragraph 1, A scaffold protein in which the second domain is composed of the amino acid sequence of SEQ ID NO. 3, SEQ ID NO. 4, or SEQ ID NO.

5.

6. In Paragraph 3, A scaffold protein in which the third domain is composed of the amino acid sequence of SEQ ID NO. 6 or SEQ ID NO.

7.

7. In Paragraph 1, A scaffold protein in which the first domain and the second domain are connected by a linker.

8. In Paragraph 7, A scaffold protein in which the above linker is composed of the amino acid sequence of SEQ ID NO. 15 or SEQ ID NO.

16.

9. In Paragraph 1, A scaffold protein comprising the amino acid sequence of SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, or SEQ ID NO.

22.

10. A method for regulating signal transduction in mammalian cells other than humans using a scaffold protein of any one of claims 1 to 9.

11. In Paragraph 10, A method for regulating signal transduction within a cell, comprising the step of expressing the scaffold protein within the cell.

12. In Paragraph 10, A method for regulating intracellular signaling, wherein regulating the intracellular signaling involves converting a cell proliferation signal into a cell death signal.

13. In Paragraph 10, A method for regulating signal transduction within a cell, wherein the cell is a cancer cell.

14. A pharmaceutical composition for the prevention or treatment of cancer, comprising a scaffold protein according to any one of claims 1 to 9.

15. In Paragraph 14, A pharmaceutical composition for the prevention or treatment of cancer, wherein the above-mentioned cancer is one or more selected from the group consisting of colorectal cancer, liver cancer, cervical cancer, lung cancer, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, breast cancer, ovarian cancer, uterine cancer, pancreatic cancer, stomach cancer, skin cancer, bladder cancer, thyroid cancer, brain cancer, and prostate cancer.

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