Fluorescence resonance energy transfer biosensor for detecting interaction between TEAD isoforms and YAP / taz

Abstract

The present invention relates to a fluorescence resonance energy transfer biosensor for detecting interaction between TEAD isomers and YAP / TAZ, and more specifically, to a method for screening an inhibitor against interaction between TEAD isomers and YAP / TAZ using the biosensor. The biosensor according to the present invention can precisely detect the interaction between TEAD isomers and YAP / TAZ in real time in a living state of an individual. In addition, it was experimentally confirmed that the biosensor of the present invention can sensitively detect an inhibitor against the interaction between TEAD isomers and YAP / TAZ. Therefore, the biosensor of the present invention can be variously utilized for screening drugs targeting the interaction between YAP / TAZ and TEAD.

Description

Fluorescence resonance energy transfer biosensor for detection of TEAD isomers and YAP / TAZ interactions

[0001] The present invention relates to a fluorescence resonance energy transfer biosensor for detecting the interaction between TEAD isomers and YAP / TAZ, and more particularly, to a method for screening TEAD isomers and YAP / TAZ interaction inhibitors using the biosensor.

[0002] The normal Hippo signaling pathway is an evolutionarily conserved pathway that regulates organ size by inducing cell proliferation, apoptosis, and self-renewal. However, in the development of tumors and cancer, abnormal Hippo signaling is a dangerous signaling pathway that indiscriminately induces tumor growth and metastasis through oncogenesis. The Hippo signaling pathway is achieved through interactions between various proteins, and ultimately, dephosphorylated YAP / TAZ translocates into the nucleus and binds to the transcription factors TEADs, thereby increasing the transcription of genes that induce cell proliferation.

[0003] Conventional methods for identifying the Hippo signaling pathway have either quantitatively confirmed YAP phosphorylation through Western blot or determined whether YAP / TAZ is cytoplasmically or nuclearly localized through immunostaining. Another method has been to identify genes transcribed by TEADs using reverse transcription polymerase chain reaction (RT-PCR). However, these techniques have limitations in spatiotemporal analysis of living samples due to the potential for damage and modification during sample fixation and extraction of required molecules (proteins or RNA). Consequently, the development of new anticancer drugs is time-consuming and requires significant labor from skilled researchers.

[0004] Accordingly, the inventors of the present invention developed eight types of fluorescence resonance energy transfer biosensors based on YAP / TAZ-TEADs interactions, and confirmed that these can precisely observe the interaction between YAP / TAZ and TEAD isomers in real time in living cells, thereby completing the present invention.

[0005] Accordingly, an object of the present invention is to provide a fluorescence resonance energy transfer (FRET) biosensor comprising a fluorescence acceptor; a YAP (Yes associated protein) / TAZ (transcriptional coactivator with PDZ-binding motif) binding domain; a TEAD (transcriptional enhancer associate domain) isoform binding domain; and a fluorescence donor.

[0006] Another object of the present invention is to provide a cell expressing the biosensor.

[0007] Another object of the present invention is to provide a composition for screening TEAD isomers and YAP / TAZ interaction inhibitors including the biosensor.

[0008] Another object of the present invention is to provide a method for screening TEAD isomers and YAP / TAZ interaction inhibitors using the biosensor.

[0009] To achieve the above object, the present invention provides a fluorescence resonance energy transfer biosensor comprising: a fluorescence receptor; a YAP / TAZ binding domain; a TEAD isoform binding domain; and a fluorescence donor.

[0010] The present invention also provides a cell expressing the biosensor.

[0011] The present invention also provides a composition for screening TEAD isomers and YAP / TAZ interaction inhibitors, including the biosensor.

[0012] In addition, the present invention provides a method for screening inhibitors of the interaction between TEAD isomers and YAP / TAZ, comprising the steps of: (a) transfecting cells with a plasmid expressing the biosensor; (b) treating the transfected cells of step (a) with a test substance; and (c) measuring a fluorescence signal in cells treated with the test substance.

[0013] The biosensor according to the present invention can precisely detect TEAD isomers and YAP / TAZ interactions in real time in a living organism. Furthermore, it has been experimentally confirmed that the biosensor of the present invention can sensitively detect TEAD isomers and YAP / TAZ interaction inhibitors. Therefore, the biosensor of the present invention can be widely utilized in the screening of drugs targeting the YAP / TAZ and TEAD interactions.

[0014]

[0015] Figure 1 is a diagram showing eight types of YAP / TAZ-TEADs interaction-based fluorescence resonance energy transfer biosensors according to the present invention.

[0016] FIG. 2 is a diagram showing the operating principle of a YAP / TAZ-TEADs interaction-based fluorescence resonance energy transfer biosensor according to the present invention.

[0017] Figure 3a is a diagram showing the results of measuring the interaction between the TEAD binding domains of four types of biosensor TEADs-YAP and TEAD isomers according to the present invention (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0018] Figure 3b is a diagram showing the results of measuring the interaction between the TEAD binding domains of four types of biosensor TEADs-TAZ and TEAD isomers according to the present invention (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0019] Figure 3c is a diagram showing the results of measuring the interaction between the YAP / TAZ binding domain of the biosensor TEAD1-YAP / TAZ according to the present invention and YAP / TAZ (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0020] Figure 3d is a diagram showing the results of measuring the interaction between the YAP / TAZ binding domain of the biosensor TEAD2-YAP / TAZ according to the present invention and YAP / TAZ (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0021] Figure 3e is a diagram showing the results of measuring the interaction between the YAP / TAZ binding domain of the biosensor TEAD3-YAP / TAZ according to the present invention and YAP / TAZ (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0022] Figure 3f is a diagram showing the results of measuring the interaction between the YAP / TAZ binding domain of the biosensor TEAD4-YAP / TAZ according to the present invention and YAP / TAZ (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0023] Figure 4 is a diagram showing the results of detecting YAP / TAZ-TEADs specific interactions of eight types of fluorescence resonance energy transfer biosensors based on YAP / TAZ-TEADs interactions according to the present invention according to the cell nutritional environment (**p<0.01, ****p<0.0001).

[0024] Figure 5 is a diagram showing the results of screening YAP / TAZ-TEADs interaction inhibitors according to a concentration gradient using four types of TEADs-YAP biosensors according to the present invention.

[0025] Figure 6 is a diagram showing the results of screening YAP / TAZ-TEADs interaction inhibitors according to a concentration gradient using four types of TEADs-TAZ biosensors according to the present invention.

[0026] Figure 7 is a diagram showing the results of calculating the YAP / TAZ-TEADs activity of four TEADs-YAP biosensors of the present invention in cells treated with K-975 (10 μM) (****p<0.0001).

[0027] Figure 8 is a diagram showing the results of calculating the YAP / TAZ-TEADs activity of four TEADs-TAZ biosensors of the present invention in cells treated with K-975 (10 μM) (****p<0.0001).

[0028] Figure 9 is a diagram showing the results of calculating the YAP / TAZ-TEADs activity of four TEADs-YAP biosensors of the present invention in cells treated with TED-347 (25 μM) (****p<0.0001).

[0029] Figure 10 is a diagram showing the results of calculating the YAP / TAZ-TEADs activity of four TEADs-TAZ biosensors of the present invention in cells treated with TED-347 (25 μM) (****p<0.0001).

[0030] Hereinafter, the present invention will be described in detail.

[0031] According to an aspect of the present invention, the present invention provides a fluorescence resonance energy transfer biosensor comprising a fluorescence receptor; a YAP / TAZ binding domain; a TEAD isoform binding domain; and a fluorescence donor. The present invention also provides a cell expressing the fluorescence resonance energy transfer biosensor.

[0032] In the present invention, TEAD isoforms refer to proteins that are primarily derived from the same gene and have slight differences in amino acid sequence. TEAD isoforms include TEAD1, TEAD2, TEAD3, and TEAD4.

[0033]

[0034] In a specific embodiment of the present invention, the YAP / TAZ binding domain is a part of a TEAD isoform, meaning a domain within TEAD to which YAP or TAZ binds. In an embodiment of the present invention, the YAP / TAZ binding domain was obtained through PCR using a TEAD isoform as a template. The obtained YAP / TAZ binding domain can be represented by one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1, 3, 5, and 7.

[0035] In a specific embodiment of the present invention, the YAP / TAZ binding domain may be represented by one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1, 3, 5 and 7, and includes functional equivalents thereof.

[0036] The above "functional equivalent" refers to a peptide having at least 80%, preferably 90%, and more preferably 95% sequence homology (i.e., identity) with the peptides of SEQ ID NOs: 1, 3, 5, and 7 as a result of addition, substitution, or deletion of amino acids, including, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence homology, and exhibiting substantially the same physiological activity. In this specification, sequence homology and identity are defined as the percentage of amino acid residues in the candidate sequence relative to the amino acid sequence of SEQ ID NOs: 1, 3, 5, and 7, after aligning the candidate sequence with the amino acid sequence and introducing gaps. If necessary, conservative substitutions are not considered as part of the sequence identity to obtain the maximum percentage sequence identity. N-terminal, C-terminal, or internal extensions, deletions, or insertions of the amino acid sequences of SEQ ID NOs: 1, 3, 5, and 7 are not construed as sequences that affect sequence identity or homology.

[0037] Furthermore, the sequence identity can be determined by commonly used standard methods for comparing similar portions of the amino acid sequences of two polypeptides. Computer programs such as BLAST or FASTA align two polypeptides so that each amino acid matches optimally (along the full length of one or both sequences or along predicted portions of one or both sequences). These programs provide default opening penalties and default gap penalties, and provide scoring matrices such as PAM250 (a standard scoring matrix) that can be used in conjunction with the computer program. For example, the percent identity can be calculated as follows: the total number of identical matches is multiplied by 100, and then divided by the sum of the length of the longer sequence within the matched span and the number of gaps introduced into the longer sequence to align the two sequences.

[0038] Additionally, the scope of "functional equivalents" of the present invention includes derivatives in which some of the chemical structures of the peptides of SEQ ID NOs: 1, 3, 5, and 7 are modified while maintaining substantially the same physiological activity as the basic backbone of the peptides. For example, this includes structural modifications to alter the stability, storability, volatility, or solubility of the peptides.

[0039] In addition, functional equivalents of the peptides of SEQ ID NOS: 1, 3, 5 and 7 of the present invention can be prepared by site-directed mutagenesis, and examples thereof can be peptides represented by the amino acid sequences of SEQ ID NOS: 2, 4, 6 and 8. The peptides represented by the amino acid sequences of SEQ ID NOS: 2, 4, 6 and 8 have tyrosine (Y), an amino acid conserved at the C-terminus of the YAP / TAZ binding domain, substituted with histidine (H) residues, and TEAD1 has tyrosine at position 406, TEAD2 has tyrosine at position 446, TEAD3 has tyrosine at position 361 and TEAD4 has tyrosine at position 386.

[0040] In a specific embodiment of the present invention, the TEAD isoform binding domain refers to a domain within YAP or TAZ, which is part of YAP or TAZ, to which the TEAD isoform binds. In an embodiment of the present invention, it is obtained through PCR using YAP or TAZ as a template. The obtained TEAD isoform binding domain can be represented by the amino acid sequence of SEQ ID NO: 9 or 11.

[0041] In a specific embodiment of the present invention, the TEAD isoform binding domain may be represented by the amino acid sequence of SEQ ID NO: 9 or 11, and includes functional equivalents thereof.

[0042] In addition, a functional equivalent of the peptide of SEQ ID NO: 9 or 11 of the present invention can be prepared by site-directed mutagenesis, and an example thereof may be a peptide represented by the amino acid sequence of SEQ ID NO: 10 or 12. The peptide represented by the amino acid sequence of SEQ ID NO: 10 is one in which both arginine (R) residue at position 89 and leucine (L) residue at position 91 in the TEAD binding domain of YAP are substituted with alanine (A) residues (RL89, 91AA). In addition, the peptide represented by the amino acid sequence of SEQ ID NO: 12 is one in which both lysine (K) residue at position 46 and leucine (L) residue at position 48 in the TEAD binding domain of TAZ are substituted with alanine (A) residues (KL46, 48AA).

[0043] In the present invention, "Fluorescence resonance energy transfer (FRET)" is a mechanism related to energy transfer between two chromophores that are close to each other, and refers to a non-radiative energy transfer phenomenon that occurs between two fluorescent substances of different emission wavelengths. It is a phenomenon in which the excitation level energy of a fluorescent donor in an excited state is transferred to a fluorescent acceptor, and emission is observed from the fluorescent acceptor, or quenching of the fluorescent donor is observed.

[0044] In the present invention, a fluorescent donor means a fluorescent substance that acts as a donor in the FRET phenomenon, and a fluorescent acceptor means a fluorescent substance that acts as an acceptor in the FRET phenomenon.

[0045] In a specific embodiment of the present invention, the fluorescent donor may be at least one selected from the group consisting of fluorescent proteins, fluorescent dyes, bioluminescent proteins, and quantum dots. In addition, the fluorescent protein may be at least one cyan fluorescent protein (CFP) selected from the group consisting of Cerulean, Turquoise2-GL, mTurquoise, mTFP, ECFP, CyPet, and ECGFP, but the scope of the present invention is not limited thereto.

[0046] In a specific embodiment of the present invention, the fluorescent receptor may be at least one selected from the group consisting of a fluorescent protein, a quencher, and an Au-nano particle. In addition, the fluorescent protein may be at least one yellow fluorescent protein (YFP) selected from the group consisting of LanYFP, mNeonGreen, YPet, mEYFP, SEYFP, phiYFP, Citrine, mCitrine, Venus, mVenus, iq-mVenus, cp50Venus, cp157Venus, cp172Venus, cp195Venus, and cp229Venus, but the scope of the present invention is not limited thereto.

[0047] In a specific embodiment of the present invention, it is preferable that the biosensor is operably connected in the form of the following structural formula.

[0048] [constitutional formula]

[0049] Fluorescent receptor - YAP / TAZ binding domain - TEAD isoform binding domain - Fluorescent donor

[0050] In a preferred embodiment of the present invention, the biosensor of the present invention may further include a nuclear localization signal (NLS) and may be coupled to the C-terminus of the fluorescent donor within the biosensor. The nuclear localization signal is preferably represented by the amino acid sequence of SEQ ID NO: 16.

[0051] In a preferred embodiment of the present invention, the biosensor of the present invention may further comprise an EV linker, which may be bound to the C-terminus of the YAP / TAZ binding domain or the N-terminus of the TEAD isoform binding domain in the biosensor. The EV linker is preferably represented by the amino acid sequence of SEQ ID NO: 14.

[0052] In a specific embodiment of the present invention, the biosensor can be represented by one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 17, 20, 23, 26, 29, 32, 35, and 38, and includes functional equivalents thereof. In addition, the functional equivalents of one or more amino acid sequences selected from the group consisting of 17, 20, 23, 26, 29, 32, 35, and 38 of the present invention can be produced by site-directed mutagenesis, and an example thereof can be a peptide represented by an amino acid sequence of SEQ ID NOs: 18, 19, 21, 22, 24, 25, 27, 28, 30, 31, 33, 34, 36, 37, 39, or 40.

[0053] In a specific embodiment of the present invention, a biosensor represented by one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 17, 20, 23, 26, 29, 32, 35 and 38 may be used for detecting YAP / TAZ position switching.

[0054] In a preferred embodiment of the present invention, the biosensor represented by one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 29, 32, 35, and 38 may be used for detecting YAP / TAZ position switching. In addition, the biosensor represented by one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 29, 32, 35, and 38 may be used for detecting the nutritional status of cells.

[0055] Fetal bovine serum (FBS) contains growth factors important for cell growth and induces the localization of YAP or TAZ from the cytoplasm to the nucleus. In an embodiment of the present invention, a biosensor represented by one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 29, 32, 35, and 38 can sensitively detect the localization of YAP or TAZ from the cytoplasm to the nucleus, thereby detecting the cellular nutritional status.

[0056] The biosensor of the present invention is designed based on fluorescence resonance energy transfer to measure the interaction between YAP / TAZ and TEAD isomers (TEAD1, TEAD2, TEAD3, and TEAD4) as an optical signal. The biosensor of the present invention may be characterized by having an integrated structure that includes binding domain sequences for YAP, TAZ, and TEADs within the biosensor and can bind to both YAP / TAZ and TEADs. This has the advantage of being able to exhibit high specificity because it can target the interaction between YAP / TAZ and TEADs in a 1:1 combination within the cell nucleus.

[0057] The biosensor of the present invention can be transduced into a subject and expressed as a protein with the sequence encoding it. The biosensor according to the present invention can precisely detect the interaction between TEAD isoforms and YAP / TAZ in real time in a living subject. Furthermore, it has been experimentally confirmed that the biosensor of the present invention can sensitively detect TEAD isoforms and YAP / TAZ interaction inhibitors. Therefore, the biosensor of the present invention can be utilized in various ways for screening drugs targeting the interaction between YAP / TAZ and TEAD.

[0058] A cell expressing a biosensor according to the present invention may be transformed with a vector containing a base sequence encoding the biosensor of the present invention.

[0059] In the present invention, a "vector" means a genetic construct comprising a base sequence of a gene operably linked to a suitable regulatory sequence so as to express a target gene in a suitable host, wherein the regulatory sequence may include a promoter capable of initiating transcription, an arbitrary operator sequence for regulating such transcription, and a sequence for regulating the termination of transcription and translation. The vector of the present invention is not particularly limited as long as it is capable of replicating in a cell, and any vector known in the art may be used, for example, a plasmid, cosmid, phage particle, or viral vector.

[0060] In the present invention, the term "transformation" refers to the introduction of DNA into a cell. The introduced DNA is typically in the form of a vector containing the inserted DNA fragment. The introduced DNA sequence may be from the same species as the host cell, a different species, or a hybrid DNA sequence containing some DNA from the host species and some foreign DNA.

[0061]

[0062] According to another aspect of the present invention, the present invention provides a composition for screening TEAD isomers and YAP / TAZ interaction inhibitors, which comprises the biosensor described above.

[0063] The screening composition of the present invention may further include, in addition to the biosensor, a buffer or reaction solution that stably maintains the structure or physiological activity of a protein or nucleic acid.

[0064] In addition, the screening composition of the present invention may include the biosensor described above in the form of (i) a cell expressing the biosensor; or (ii) a plasmid expressing the biosensor under a promoter capable of controlling the transcription rate.

[0065] In a specific embodiment of the present invention, the screening composition may include a first compartment containing the aforementioned biosensor; and a second compartment containing a YAP / TAZ interaction inhibitor. The YAP / TAZ interaction inhibitor in the second compartment may be used for cross-validation of test substances. Examples of the YAP / TAZ interaction inhibitor may be known YAP / TAZ interaction inhibitors such as K-975, TED-347, MGH-CP1, and YAP-TEAD inhibitor 1.

[0066]

[0067] According to another aspect of the present invention, the present invention provides a method for screening inhibitors of the interaction between TEAD isomers and YAP / TAZ, comprising the steps of: (a) transfecting a cell with a plasmid expressing the biosensor; (b) treating the transfected cell of step (a) with a test substance; and (c) measuring a fluorescence signal in the cell treated with the test substance.

[0068] In the present invention, the test substance may be selected randomly or is assumed to have the potential to be an inhibitor of the interaction between TEAD isomers and YAP / TAZ. Examples of the test substance may be selected from the group consisting of nucleic acids, compounds, microbial cultures or extracts, natural product extracts, peptides, substrate analogs, aptamers, and antibodies.

[0069] In a specific example of the present invention, the fluorescence signal may be 'the ratio of the emission intensity of the fluorescence donor to the emission intensity of the fluorescence acceptor'.

[0070] In a specific embodiment of the present invention, it is preferable that the method further comprises a step of selecting a TEAD isomer and YAP / TAZ interaction inhibitor when the ratio of the emission intensity of the fluorescent donor to the emission intensity of the fluorescent acceptor in the cells treated with the test substance measured in step (c) decreases compared to the untreated group.

[0071] In a specific embodiment of the present invention, the method may further include a step of (e) treating the transduced cells of step (a) with a verified YAP / TAZ interaction inhibitor and then measuring the ratio of the luminescence intensity to the luminescence intensity of the fluorescent receptor, thereby cross-validating the inhibitor selected in step (d).

[0072]

[0073] Duplicate contents are omitted in consideration of the complexity of this specification, and terms not otherwise defined in this specification have meanings commonly used in the technical field to which the present invention belongs.

[0074] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0075]

[0076] Example 1. Fabrication of a fluorescence resonance energy transfer biosensor based on YAP / TAZ-TEADs interaction.

[0077] A fluorescence resonance energy transfer (FRET)-based biosensor was designed to measure the interaction of YAP / TAZ and TEAD isomers (TEAD1, TEAD2, TEAD3, and TEAD4) as an optical signal.

[0078]

[0079] 1-1. Production of gene fragments

[0080] The YAP / TAZ binding domain (hereinafter referred to as 'TEAD') of the gene sequence of the TEAD isoform was amplified by polymerase chain reaction (PCR) using the gene sequence as a template. The YAP / TAZ binding domain of the amplified TEAD isoform encodes peptides of sequence numbers 1, 3, 5, and 7, respectively.

[0081] The gene sequences of YAP and TAZ were obtained, and polymerase chain reaction was performed using them as templates to amplify the TEAD binding domains of the corresponding gene sequences (hereinafter referred to as 'YAP' or 'TAZ'). The amplified TEAD binding domain of YAP encodes a peptide of SEQ ID NO: 9, and the amplified TEAD binding domain of TAZ encodes a peptide of SEQ ID NO: 11.

[0082] In addition, mutant sequences of the amplified YAP / TAZ binding domain and TEAD binding domain were also constructed to be used as controls in the experiments described below. The constructed YAP-TAZ binding domain mutants of TEAD isoforms encode peptides of SEQ ID NOs: 2, 4, 6, and 8, respectively. The constructed TEAD binding domain of YAP and TEAD binding domain of TAZ encode peptides of SEQ ID NOs: 10 and 12, respectively.

[0083] The amplified YAP / TAZ binding domain and TEAD binding domain encode proteins in Table 1, respectively.

[0084] 서열번호명칭서열1YAP / TAZ binding domain of TEAD1RSIGTTKLRLVEFSAFLEQQRDPDSYNKHLFVHIGHANHSYSDPLLESVDIRQIYDKFPEKKGGLKELFGKGPQNAFFLVKFWADLNCNIQDDAGAFYGVTSQYESSENMTVTCSTKVCSFGKQVVEKVETEYARFENGRFVYRINRSPMCEYMINFIHKLKHLPEKYMMNSVLENFTILLVVTNRDTQETLLCMACVFEVSNSEHGAQHHIYRLVKD2YAP / TAZ binding domain of TEAD1_mutantSIGTTKLRLVEFSAFLEQQRDPDSYNKHLFVHIGHANHSYSDPLLESVDIRQIYDKFPEKKGGLKELFGKGPQNAFFLVKFWADLNCNIQDDAGAFYGVTSQYESSENMTVTCSTKVCSFGKQVVEKVETEYARFENGRFVYRINRSPMCEYMINFIHKLKHLPEKYMMNSVLENFTILLVVTNRDTQETLLCMACVFEVSNSEHGAQHHIHRLVKD3YAP / TAZ binding domain of TEAD2RGLGTARLQLVEFSAFVEPPDAVDSYQRHLFVHISQHCPSPGAPPLESVDVRQIYDKFPEKKGGLRELYDRGPPHAFFLVKFWADLNWGPSGEEAGAGGSISSGGFYGVSSQYESLEHMTLTCSSKVCSFGKQVVEKVETERAQLEDGRFVYRLLRSPMCEYLVNFLHKLRQLPERYMMNSVLENFTILQVVTNRDTQELLLCTAYVFEVSTSERGAQHHIYRLVRD4YAP / TAZ binding domain ofTEAD2_mutantRGLGTARLQLVEFSAFVEPPDAVDSYQRHLFVHISQHCPSPGAPPLESVDVRQIYDKFPEKKGGLRELYDRGPPHAFFLVKFWADLNWGPSGEEAGAGGSISSGGFYGVSSQYESLEHMTLTCSSKVCSFGKQVVEKVETERAQLEDGRFVYRLLRSPMCEYLVNFLHKLRQLPERYMMNSVLENFTILQVVTNRDTQELLLCTAYVFEVSTSERGAQHHIHRLVRD5YAP / TAZ binding domain of TEAD3RTIASSRLRLLEYSAFMEVQRDPDTYSKHLFVHIGQTNPAFSDPPLEAVDVRQIYDKFPEKKGGLKELYEKGPPNAFFLVKFWADLNSTIQEGPGAFYGVSSQYSSADSMTISVSTKVCSFGKQVVEKVETEYARLENGRFVYRIHRSPMCEYMINFIHKLKHLPEKYMMNSVLENFTILQVVTSRDSQETLLVIAFVFEVSTSEHGAQHHVYKLVKD6YAP / TAZ binding domain of TEAD3_mutantRTIASSRLRLLEYSAFMEVQRDPDTYSKHLFVHIGQTNPAFSDPPLEAVDVRQIYDKFPEKKGGLKELYEKGPPNAFFLVKFWADLNSTIQEGPGAFYGVSSQYSSADSMTISVSTKVCSFGKQVVEKVETEYARLENGRFVYRIHRSPMCEYMINFIHKLKHLPEKYMMNSVLENFTILQVVTSRDSQETLLVIAFVFEVSTSEHGAQHHVHKLVKD7YAP / TAZ binding domain ofTEAD4RSVASSKLWMLEFSAFLEQQQDPDTYNKHLFVHIGQSSPSYSDPYLEAVDIRQIYDKFPEKKGGLKDLFERGPSNAFFLVKFWADLNTNIEDEGSSFYGVSSQYESPENM IITCSTKVCSFGKQVVEKVETEYARYENGHYSYRIHRSPLCEYMINFIHKLKHLPEKYMMNSVLENFTILQVVTNRDTQETLLCIAYVFEVSASEHGAQHHIYRLVKE8YAP / TAZ binding domain of TEAD4_mutantRSVASSKLWMLEFSAFLEQQQDPDTYNKHLFVHIGQSSPSYSDPYLEAVDIRQIYDKFPEKKGGLKDLFERGPSNAFFLVKFWADLNTNIEDEGSSFYGVSSQYE SPENMIITCSTKVCSFGKQVVEKVETEYARYENGHYSYRIHRSPLCEYMINFIHKLKHLPEKYMMNSVLENFTILQVVTNRDTQETLLCIAYVFEVSASEHGAQHHIHRLVKE9TEAD binding domain of YAPAGHQIVHVRGDSETDLEALFNAVMNPKTANVPQTVPMRLRKLPDSFFKPPE10TEAD binding domain of YAP_mutantAGHQIVHVRGDSETDLEALFNAVMNPKTANVPQTVPMRLAKAPDSFFKPPE11TEAD binding domain of TAZPGQQVIHVTQDLDTDLEALFNSVMNPKPSSWRKKILPESFFKEPD12TEAD binding domain of TAZ_mutantPGQQVIHVTQDLDTDLEALFNSVMNPKPSSWRKAIAPESFFKEPD

[0085] 상기 중합효소연쇄반응에 사용한 프라이머 서열은 표 2와 같다.

[0086] 시이번번명치서열 (5'->3')41TEAD1_Forward primerCATGACCGGTCGCTCCATTGCACAA42TEAD1_Reverse primerGTACGGTACCGTCCTTTACAAGCCTGTAAATATGATG43TEAD2_Forward primerCAAAGTCGACACCGGTCGGGGCCTGGGCACC44TEAD2_Reverse primerGTTTGGTACCGTCCCTGACCAGGCGGTAAATG45TEAD3_Forward primerCATGACCGGTCGTACCATTGCCTCCTCCCG46TEAD3_Reverse primerGTAAGGTACCGTCTTTGACGAGCTTGTAGACATGG47TEAD4_Forward primerCAAACTCGAGCGCAGCTGGCCAGCTC48TEAD4_Reverse primerGTACGGTACCTTCTTTCACCAGCCTGTAGATGTGG49YAP_Forward primerCATGACCGGTGCTAGCGCCGGGCATCAGATCG50YAP_Reverse primer primerGTACGCGGCCGCGCTCCGGCGGCTTGAAGAAG51TAZ_Forward primerCATGTCCGGACCTGGGCAGCAAGTGATCCAC52TAZ_Reverse primerGTACGCGGCCGCAATCAGGCTCCTTAAAGAAAGACTCCG

[0087] 1-2. YAP / TAZ-TEADs 에성사용 마리스타고공명에서스트 및수서 한국어

[0088] Eight fluorescence resonance energy transfer biosensors were fabricated in which the YAP / TAZ binding domains, TEAD isoforms (SEQ ID NOs: 1, 3, 5, and 7); CFP (SEQ ID NO: 15); mNeonGreen (SEQ ID NO: 13); the TEAD binding domain, YAP (SEQ ID NO: 9) or the TEAD binding domain, TAZ (SEQ ID NO: 11); an EV linker (SEQ ID NO: 14) (Komatsu, Naoki, et al., 2011), and a nuclear localization signal (NLS) (SEQ ID NO: 16) were sequentially and operably linked (Fig. 1). The fabricated biosensors were designated TEAD1-YAP, TEAD2-YAP, TEAD3-YAP, TEAD4-YAP, TEAD1-TAZ, TEAD2-TAZ, TEAD3-TAZ, and TEAD4-TAZ, respectively.

[0089] The gene sequence encoding the above biosensor was inserted into a DNA carrier (FRET vector) to construct a plasmid containing the gene sequence encoding the biosensor. When the plasmid containing the gene sequence encoding the constructed biosensor was transfected into cells, the introduced gene sequence was expressed as a protein in the transfected cells after 1 to 2 days. The expressed biosensor protein is located in the cellular organelles, and the interaction between YAP / TAZ and TEAD isoforms can be observed through fluorescence signals.

[0090]

[0091] Meanwhile, biosensors can be made into mutant biosensors by utilizing each mutant of TEAD isomers, YAP, and TAZ. Specifically, mutants were made in which residues that play an important role in interaction were substituted with other residues in biosensors made using site-directed mutagenesis. Specifically, in each of the eight biosensors, four mutants were made in which both arginine (R) residue 89 and leucine (L) residue 91 in the TEAD binding domain of YAP were substituted with alanine (A) residues (RL89, 91AA), and four mutants were made in which both lysine (K) residue 46 and leucine (L) residue 48 in the TEAD binding domain of TAZ were substituted with alanine (A) residues (KL46, 48AA). They are homologous to the YAP / TAZ binding domain of TEAD isoforms, and the conserved amino acid tyrosine (Y) at the C-terminus was substituted with histidine (H) residue. TEAD1 has tyrosine at position 406, TEAD2 at position 446, TEAD3 at position 361, and TEAD4 at position 386. Two mutants for each were created.

[0092]

[0093] The amino acid sequences of the eight fluorescence resonance energy transfer biosensors based on the fabricated YAP / TAZ-TEADs interaction and the 16 mutant biosensors are shown in Table 3.

[0094] 시이벨벨명치서열17TEAD1-YAP *18TEAD1-YAP biosensor_TEAD1*19TEAD1-YAP biosensor_YAP*20TEAD2-YAP*21TEAD2-YAP biosensor_TEAD2*22TEAD2-YAP biosensor_YAP*23TEAD3-YAP*24TEAD3-YAP biosensor_TEAD3*25TEAD3-YAP biosensor_YAP*26TEAD4-YAP*27TEAD4-YAP biosensor_TEAD4*28TEAD4-YAP biosensor_YAP*29TEAD1-TAZ*30TEAD1-TAZ biosensor_TEAD1*31TEAD1-TAZ biosensor_TAZ*32TEAD2-TAZ*33TEAD2-TAZ biosensor_TEAD2*34TEAD2-TAZ biosensor_TAZ*35TEAD3-TAZ*36TEAD3-TAZ biosensor_TEAD3*37TEAD3-TAZ biosensor_TAZ*38TEAD4-TAZ*39TEAD4-TAZ biosensor_TEAD4*40TEAD4-TAZ biosensor_TAZ*

[0095] Example 2. 바수서 8종을 더이한 YAP / TAZ-TEADs 정리작용 정다

[0096] As shown in Fig. 2, the biosensor fabricated in Example 1 can detect the interaction between YAP / TAZ and TEAD isomers through changes in optical signals. More specifically, when a structural change occurs due to a change in the binding site as a result of the interaction, the interaction between YAP / TAZ-TEAD isomers present in the nucleus of a cell can be measured through changes in the 'emission intensity ratio of the fluorescent protein pair of the biosensor (FRET emission intensity / CFP emission intensity)'. In addition, this can be utilized to select drugs that modulate the interaction.

[0097]

[0098] 2-1. Measurement of the interaction between the TEAD binding domain of YAP / TAZ and TEAD isoforms

[0099] The interaction levels of the TEAD binding domains (TEADs) of YAP / TAZ and TEAD isoforms of the eight biosensors of the present invention were confirmed in the basal state of HEK293T cells. The interaction levels were visualized and quantified using the FRET / CFP emission ratio. The results of visualizing and quantifying the interaction levels of the TEAD binding domains of YAP and TEAD isoforms are shown in Fig. 3a. In addition, the results of visualizing and quantifying the interaction levels of the TEAD binding domains of TAZ and TEAD isoforms are shown in Fig. 3b.

[0100] As shown in Figure 3a, the interaction between the TEAD binding domain of YAP and TEAD isoforms was confirmed to have a distinct level of interaction depending on the type of TEAD isoform.

[0101] As shown in Fig. 3b, the interaction between the TEAD binding domain of TAZ and TEAD isomers was confirmed to have a distinct level of interaction depending on the type of TEAD isomer.

[0102]

[0103] 2-2. Measurement of YAP / TAZ binding domains of TEAD isomers and YAP / TAZ interaction

[0104] The interaction levels of the YAP / TAZ binding domains of the TEAD isoforms (TEAD1, TEAD2, TEAD3, and TEAD4) of the biosensor of the present invention and YAP / TAZ were confirmed in the basal state of HEK293T cells. The interaction levels were visualized and quantified using the FRET / CFP emission ratio. The results of visualizing and quantifying the YAP / TAZ binding domains of the TEAD isoforms and the YAP / TAZ interaction levels are shown in Figures 3c to 3f, respectively.

[0105] As shown in Figures 3c to f, the YAP / TAZ binding domains of TEAD1, TEAD2, TEAD3, and TEAD4 were confirmed to have significantly different levels of interaction with YAP and TAZ.

[0106] The above results indicate that TEAD isomers have different degrees of interaction with YAP / TAZ despite their high homology to each other, and demonstrate that the biosensor of the present invention can measure the difference in interaction between TEAD isomers and YAP / TAZ, which could not be measured using conventional techniques.

[0107]

[0108] Example 3. Ability to detect specific interactions of YAP / TAZ-TEADs according to cell nutritional environment

[0109] Fetal bovine serum (FBS) contains growth factors important for cell growth and induces the relocation of YAP or TAZ from the cytoplasm to the nucleus. Therefore, in this example, it was confirmed whether the eight YAP / TAZ-TEADs interaction-based fluorescence resonance energy transfer biosensors manufactured in Example 1 could detect the YAP / TAZ relocation by FBS. Specifically, cells transduced with each of the eight YAP / TAZ-TEADs interaction-based fluorescence resonance energy transfer biosensors were cultured in a medium containing 10% FBS to express the biosensors in protein form. Thereafter, the FRET / CFP ratio was visualized and quantified, and is shown in Figs. 4a to 4h.

[0110] As shown in Figures 4a to d, the YAP-TEADs interaction-based fluorescence resonance energy transfer biosensors (TEAD1-YAP, TEAD2-YAP, TEAD3-YAP, and TEAD4-YAP) showed no significant change in the FRET / CFP ratio depending on the presence or absence of FBS.

[0111] As shown in Figures 4e to h, the TAZ-TEADs interaction-based fluorescence resonance energy transfer biosensors (TEAD1-TAZ, TEAD2-TAZ, TEAD3-TAZ, and TEAD4-TAZ) showed significant changes in the FRET / CFP ratio depending on the presence or absence of FBS.

[0112] The above results imply that the TAZ-TEADs interaction-based fluorescence resonance energy transfer biosensor can be utilized to determine the nutritional status of cells.

[0113]

[0114] Example 4. Drug screening platform using YAP / TAZ-TEADs interaction inhibitors.

[0115] Using the eight TAZ-TEADs interaction-based fluorescence resonance energy transfer biosensors fabricated in Example 1, TEAD isomers and four YAP / TAZ interaction inhibitors (K-975, TED-347, MGH-CP1, and YAP-TEAD inhibitor 1) were screened. Specifically, HEK293T cells transfected with each of the eight YAP / TAZ-TEADs interaction-based fluorescence resonance energy transfer biosensors fabricated in Example 1 were seeded and cultured in a 96-well plate. After cell attachment, each well was treated with the four inhibitors at various concentrations. After drug treatment, the 96-well plate was mounted on a microplate reader every 12, 24, and 48 hours to analyze the YFP / CFP emission ratio. The quantified ratios were normalized using the solvent of each drug (e.g., dimethyl sulfoxide, water), and the change in value for each concentration was measured to calculate the YAP / TAZ-TEADs activity. The results of calculating the YAP / TAZ-TEADs activity of the TEADs-YAP biosensor are shown in Fig. 5. In addition, the results of calculating the YAP / TAZ-TEADs activity of the TEADs-TAZ biosensor are shown in Fig. 6.

[0116] As shown in FIGS. 5 and 6, it was confirmed that the eight YAP / TAZ-TEADs interaction-based fluorescence resonance energy transfer biosensors fabricated in Example 1 react sensitively to inhibitors K-975, TED-347, and MGH-CP1. The above results indicate that the eight TAZ-TEADs interaction-based fluorescence resonance energy transfer biosensors of the present invention can be used to screen candidate drugs. Furthermore, this means that the precision of screening candidate drugs can be improved by using a verified YAP / TAZ interaction inhibitor during candidate drug screening.

[0117]

[0118] Example 5. Cross-validation of YAP / TAZ-TEADs interaction inhibitors using high-resolution microscopy.

[0119] In this example, high-resolution real-time screening of inhibitors K-975 or TED-347 identified in Example 5 was performed using a high-resolution microscope. Specifically, HEK293T cells were cultured in a confocal dish, and eight kinds of YAP / TAZ-TEADs interaction-based fluorescence resonance energy transfer biosensors manufactured in Example 1 were transduced and expressed. Then, the cells expressing the biosensors were treated with K-975 (10 μM) or TED-347 (25 μM), and real-time imaging (0, 20, and 40 min) was performed using a high-resolution microscope. The YAP / TAZ-TEADs activity was calculated by measuring the change value, and the results are shown in Figures 7 to 10. More specifically, in cells treated with 10 μM of K-975, the TEADs-YAP biosensor; The results of calculating the YAP / TAZ-TEADs activity of the TEADs-YAP biosensor and the TEADs-TAZ biosensor in cells treated with 25 μM of TED-347 are shown in Figs. 7 and 8, respectively. In addition, the results of calculating the YAP / TAZ-TEADs activity of the TEADs-YAP biosensor and the TEADs-TAZ biosensor in cells treated with 25 μM of TED-347 are shown in Figs. 9 and 10, respectively.

[0120] As shown in FIGS. 7 to 10, the eight TAZ-TEADs interaction-based fluorescence resonance energy transfer biosensors fabricated in Example 1 were sensitive to inhibitors K-975 and TED-347. In particular, among the eight biosensors, TEAD4-YAP and TEAD4-TAZ were sensitive to inhibitors K-975 and TED-347. The above results indicate that the eight TAZ-TEADs interaction-based fluorescence resonance energy transfer biosensors of the present invention are sensitive to inhibitors and can be usefully utilized for cross-validation in the discovery of candidate drugs.

[0121]

[0122] In summary, the present inventors have developed eight fluorescence resonance energy transfer biosensors based on the YAP / TAZ-TEADs interaction. Cells transfected with sequences encoding the biosensors of the present invention express the biosensors as proteins, enabling precise, real-time observation of the interaction between YAP / TAZ and TEAD isomers in living cells. Therefore, the eight fluorescence resonance energy transfer biosensors based on the YAP / TAZ-TEADs interaction of the present invention can be utilized in various ways for screening drugs targeting the interaction between YAP / TAZ and TEAD.

[0123]

[0124] While specific aspects of the present invention have been described in detail, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Flourescence acceptor; Yes associated protein (YAP) / transcriptional coactivator with PDZ-binding motif (TAZ) binding domain; TEAD (transcriptional enhancer associate domain) isoform binding domain; and A fluorescence resonance energy transfer (FRET) biosensor comprising a fluorescence donor.

2. A biosensor according to claim 1, wherein the YAP / TAZ binding domain is represented by at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, and 7.

3. A biosensor according to claim 1, wherein the TEAD isomer binding domain is represented by the amino acid sequence of SEQ ID NO: 9 or 11.

4. A biosensor according to claim 1, wherein the fluorescent donor is at least one selected from the group consisting of a fluorescent protein, a fluorescent dye, a bioluminescent protein, and a quantum dot.

5. A biosensor according to claim 1, wherein the fluorescent receptor is at least one selected from the group consisting of a fluorescent protein, a quencher, and a gold nanoparticle.

6. A biosensor according to claim 1, wherein the biosensor is operably connected in the form of the following structural formula. [constitutional formula] Fluorescent receptor - YAP / TAZ binding domain - TEAD isoform binding domain - Fluorescent donor 7. In the first paragraph, the biosensor is represented by one or more amino acid sequences selected from the group consisting of sequence numbers 17, 20, 23, 26, 29, 32, 35, and 38.

8. In the 7th paragraph, a biosensor represented by at least one amino acid sequence selected from the group consisting of sequence numbers 29, 32, 35, and 38 is a biosensor for detecting YAP / TAZ position switching.

9. A cell expressing the biosensor of paragraph 1.

10. A composition for screening TEAD isomers and YAP / TAZ interaction inhibitors, comprising the biosensor of claim 1. 11.(a) A step of transfecting a cell with a plasmid expressing the biosensor of paragraph 1; (b) a step of treating the transduced cells of step (a) with a test substance; and (c) a step of measuring a fluorescence signal in cells treated with the test substance; a method for screening inhibitors of the interaction between TEAD isomers and YAP / TAZ.

12. In the 11th paragraph, the method further comprises a step of selecting as an inhibitor of the interaction between TEAD isomers and YAP / TAZ, if the ratio of the emission intensity of the fluorescent donor to the emission intensity of the fluorescent acceptor in the cells treated with the test substance measured in the step (c) decreases compared to the untreated group.

13. In the 12th paragraph, the method further comprises the step of (e) treating the transduced cells of the step (a) with a verified YAP / TAZ interaction inhibitor and then measuring the fluorescence intensity to cross-validate the inhibitor selected in the step (d).

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