Peptide Design to Inhibit the Formation of YAP-TEAD Protein Complex

Abstract

The invention relates to peptides designed using a peptidomimetic approach to inhibit the formation of the YAP-TEAD (Yes-Associated Protein – TEA Domain) protein complex.

Description

1 TARIFF To prevent the formation of the YAP-TEAD protein complex. PEPTIDE DESIGN Technical Field to Which the Invention Relates The invention relates to the development of a 5-protein complex of the YAP-TEAD (Yes-Associated Protein – TEA Domain) protein complex. peptides designed using a peptidomimetic approach to prevent its formation It is related to the invention through the peptidomimetic approach and the use of unnatural amino acids. High selectivity and efficacy against the YAP-TEAD complex thanks to its use. Inhibitory structures that can exhibit this behavior are obtained. State of the Art The YAP / TEAD complex plays a role in cancer biology in proliferation and evasion of apoptosis. key tumorigenic processes such as angiogenesis, epithelial-mesenchymal transition (EMT), and metastasis It plays a critical role in controlling these processes. Under normal cellular conditions, Hippo signaling... This pathway phosphorylates YAP and TAZ via LATS1 / 2 kinase, converting them into 14-3-3 proteins. It enables binding and retention in the cytoplasm, thus leading to nuclear activation. is inhibited [1]. However, in many solid tumors, the tumor suppressor Hippo pathway is inhibited [1]. components (NF2, LATS1 / 2, MST1 / 2) genetically or epigenetically inactivated When this happens, YAP / TAZ undergoes phosphorylation loss, translocates to the nucleus, and By binding to TEAD family transcription factors, it inhibits oncogenic gene expression. is initiated [2]. This complex includes CTGF, CYR61, AXL, MYC and BIRC5. genes that support proliferation, cell survival, angiogenesis, and invasion It activates promoter / enhancer regions [3]. Also YAP / TEAD, AP-1 Co- with transcription factors such as (FOS / JUN) in super-enhancer regions. By exhibiting coupulation, it strengthens transcriptional programs and 25 tumor cells It increases its plasticity [4]. At the clinical level, nuclear YAP expression high altitude hepatocellular carcinoma, mesothelioma, non-small cell lung cancer and poor prognosis and treatment in aggressive tumors such as triple-negative breast cancer It has been associated with resistance [5]. Furthermore, YAP / TEAD activity is associated with DNA damage repair. By regulating genes and drug transporters, adaptive resistance to chemotherapy 30 strengthens their mechanisms [6]. For these reasons, the YAP / TEAD axis strengthens both its mechanisms [6]. Cancer treatment as both a prognostic biomarker and a target of pharmacological intervention. It is a strategic focus point in their research. 2 Preventing the formation of the YAP / TEAD complex in current treatment approaches, with the direct aim of reducing the proliferative and invasive potential of cancer cells It is related, but achieving this is a challenging process. The known aspects of the technique... in this case, the palmitoylation (fatty acid binding) site at the center of TEAD The aim is to prevent YAP from connecting to TEAD by targeting TEAD 5. Palmitoylation has been shown to be critical for stability and YAP binding, Therefore, inhibitors targeting this region indirectly affect the interaction between TEAD and YAP. It was thought that this could block the pathway. Furthermore, the protein pathway between YAP and TEAD... Strategies aimed at directly blocking protein interactions have also been tried. 10 in the strategy to develop molecules targeting YAP / TEAD binding sites focuses on. In this approach, the company called Inventiva developed the YAP-TEAD complex. We used both fragment-based screening and high-frequency screening to find inhibitory compounds. It has used efficient screening (HTS) methods [6]. However, these small molecules Because modulating the complex interaction is difficult, developing effective inhibitors is challenging. It is still limited. Apart from that, the binding sites of YAP on the TEAD surface are 15 Peptide inhibitors targeting cystine-dense peptides have been developed. For example, "cystine-dense peptides" and Some YAP-derived peptides bind to surface pockets on TEAD, forming a complex. It tries to prevent the formation of peptides in the cell [7]. This approach, in particular, prevents the formation of peptides in the cell. It faces challenges such as low entry capacity. The YAP / TEAD complex is activated by suppression of the Hippo signaling pathway, leading to cancer. It increases oncogenic gene expression in many pathological processes, including this. Natural amino acids and short-term derivatives of them prevent the formation of the complex. Peptides are used as protein-protein interaction inhibitors (PPI inhibitors). can be evaluated. Especially hydrophobic (leucine, isoleucine, valine) and aromatic 25 (phenylalanine, tyrosine) amino acids, α-helix and Ω-loop where YAP binds to TEAD by mimicking critical side chains in their regions, competitively attaching to the TEAD surface. It can bind. In addition, some amino acids such as proline and methylated arginine bind to TEAD. or by creating allosteric conformational changes in YAP to increase binding affinity It can lower it. Hydrophobic side-chain amino acid derivatives, on the other hand, are TEAD's palmitate 30 It can disrupt structural stability by targeting the attachment pocket. This type of natural The advantage of amino acid-based inhibitors is their biocompatibility and low risk of toxicity. Their advantages include their transportability and the ease with which they can be chemically modified. However, alone... Natural amino acids generally have a low capacity to block the YAP / TEAD interface; 3 They are rapidly degraded by intracellular proteases, have difficulty crossing the cell membrane, and Limitations such as low binding affinity restrict their effectiveness. Therefore, clinical In order to achieve a meaningful effect, these amino acids must be in the form of short peptides. regulation, modification by chemical stabilization techniques or cell It must be used with carrier systems that will facilitate its intake. 5 Various methods are used in the synthesis of peptides. For example; solid-phase peptides. Its synthesis forms the basis of modern peptide synthesis and is short to medium length. It is preferred for peptides. Methods such as Fmoc-SPPS and Boc-SPPS are used. It is applied. Among the advantages of SPPS is the easy 10 that increases the purity of peptides. purification processes and the use of automated synthesis equipment However, the chemical synthesis of long peptides (>50 amino acids) is still challenging. (especially in large-scale production). Chemical modifications are also involved in peptide synthesis. For example, proteolytic activity in peptides is used in spinal modification techniques. Replacing L-amino acids with D-amino acids or β-15 to increase stability Methods such as the addition of amino acids are used. Similarly, cyclization. in the technique, to increase the proteolytic stability and cell permeability of peptides various cyclization methods (e.g., linking from side chain to side chain) It is applied. The synthesis of peptides is also carried out using recombinant DNA technology. It is possible. This method is particularly useful for long and complex bonds with disulfide bonds. It is used in the production of peptides. Developing peptide inhibitors is quite difficult due to the complexity of peptide synthesis. It is one of the methods. The synthesis of peptides involves the reactive groups of each amino acid. 25 This is necessary to prevent side reactions and ensure the effective formation of peptide bonds. This constitutes a critical step in enabling its formation. Furthermore, natural peptides... It is susceptible to enzymatic degradation and tends to be rapidly eliminated from the body, which by reducing their bioavailability, they make oral administration difficult and parenteral administration This requires the use of application methods [8]. Peptide synthesis, especially 30 This presents a number of challenges for therapeutic purposes. First, hydrophobic amino acids. It is stated that peptides containing these substances may cause folding and solubility problems. This situation affects the solubility of peptides during the synthesis and purification stages. This leads to a decrease in productivity and an increase in aggregation tendencies, which in turn causes efficiency to decrease. 4 This reduces and complicates the synthesis process. Furthermore, classical synthesis... methods, especially solid-phase peptide synthesis (SPPS), harm the environment and It is emphasized that there is a significant cost in terms of waste production. Therefore, green More environmentally friendly methods based on chemical principles are needed. Patent application number US2023242506A1, which is included in the prior art, Under the heading of heterobifunctional molecules as TEAD inhibitors, cancer treatment This document focuses on the design of next-generation drugs for TEAD proteins. specially designed heterobifunctional molecules that can inhibit TEAD proteins play a critical role in cell growth and proliferation. Because of this, these inhibitors prevent cancer cells from multiplying uncontrollably. It aims to stop this. The developed molecules belong to two different functional groups. It has a molecule with one end that binds to the TEAD protein, while the other end binds to different molecules or They can interact with surfaces. Thanks to these properties, inhibitors of TEAD By targeting proteins with high selectivity, it minimizes side effects and simultaneously targets 15 It can be combined with more than one therapeutic strategy. The drugs presented in this document... Its design is particularly suitable for the treatment of cancer types in which TEAD proteins are overactive. while offering a promising solution, it also contributes to biological research. This allows for the development of new tools that can be used. Amino acids residues are limited to 20 naturally occurring amino acids, commonly represented by three-letter symbols. although not, phosphoserine, phosphothreonine, phosphotyrosine, 4-hydroxyproline, hydroxylysine, demosine, isodemosine, gamma-carboxyglutamate, hippuric acid, octahydroindole-2-carboxylic acid, statin, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, penicillamine, ornithine, 3-methylhistidine, norvaline, beta-alanine, gamma-aminobutyric acid acid, citrulline, homocysteine, homoserine, methyl-alanine, para-benzolphenylalanine, 25 amino acids such as phenylglycine, propargylglycine, sarcosine, methionine sulfone, and tert-butylglycine It also contains acids. Current techniques have been developed to inhibit the YAP / TEAD complex. These approaches face significant limitations. Small molecule inhibitors, especially in the 30s, Compounds that target the palmitoylation pocket of TEAD indirectly inhibit complex formation. Although it aims to disrupt protein-protein interactions in this way, the broad and straightforward nature of protein-protein interactions... Their effectiveness is limited because targeting their surfaces with high specificity is difficult. Therefore, these types of inhibitors remain clinically strong and reliable candidates. Conversion is still difficult. In peptide-based inhibitors, YAP / TEAD binding is also difficult. Although promising results have been obtained using arrays that mimic their regions Peptides exhibit low efficiency in crossing the cell membrane, making them resistant to proteases. Problems such as fragility and rapid degradation, leading to decreased bioavailability, arise. It is also used to increase the proteolytic stability of short peptides. Although modification methods such as D-amino acid addition or cyclization have been suggested These approaches complicate the production process and increase costs. The peptide synthesis process also presents significant challenges in itself. Solid-phase peptides. Although synthesis (Fmoc-SPPS, Boc-SPPS) is preferred in short and medium-length sequences Efficiency and purity problems are encountered in long peptides (>50 amino acids), 10 The presence of hydrophobic amino acids causes solubility and aggregation problems. This increases the amount of organic solvents and waste generated during chemical synthesis processes. It produces recombinant DNA, which creates environmental costs. While peptide production using this technology offers a solution for complex structures, Challenges are encountered in scalability and purification processes. 15 Therefore, these limitations in the current technique apply to both small molecule and peptide molecules. There are significant obstacles to the transition of YAP / TEAD-based inhibitors to clinical practice. This creates a more stable, highly bioavailable, intracellular environment. new strategies that can be effectively implemented and are more environmentally friendly in terms of production It is needed. 20 Brief Description and Objectives of the Invention The invention involves the YAP-TEAD (Yes-Associated Protein – TEA Domain) protein complex. Peptides designed using a peptidomimetic approach to prevent formation 25 It is explained that the first of the first interface (S1) peptides subject to the invention has an N-terminal It contains the 'GH' sequence, followed by 3-methyl- which is inserted in place of natural isoleucine. It contains the residue L-alloisoleucine (X24). After this modified amino acid The peptide continues with the amino acid sequence 'IVHVR'. It is the second of the S1 peptides. It contains the sequence 'GH' at the N-terminus, followed by 30 amino acids in place of the natural amino acid. It contains the norvaline (X59) residue. After this modified amino acid, the peptide, It continues with the amino acid sequence 'IVHVR'. It is the first of the second interface (S2) peptides. It contains a 'T' amino acid residue at the n-terminus, followed by naturally occurring aspartic acid. 3,3-dimethyl aspartic acid (W82) has been substituted for the amino acid. From this modified amino acid 6 The peptide then continues with the amino acid sequence SEK ID NO: 1. Second interface (S2) The second of these peptides contains the amino acid sequence 'TDLEALFN' at its N-terminus. and subsequently, 2-(trifluoromethyl)-phenylalanine (L00) in place of natural phenylalanine is inserted. After this modified amino acid, the peptide has the amino acid sequence 'VMNP'. It continues. The last of the second interface (S2) peptides has 5 at the N-terminus. It contains the amino acid sequence 'TDLEALFN', followed by natural glutamic acid. (3S)-3-methyl-L-glutamic acid (X43) has been substituted in its place. From this modified amino acid The peptide then continues with the amino acid sequence 'VMNP'. Third interface (S3) The first of its peptides is norleucine (X58), an unnatural amino acid at the N-terminus. It contains the residue, followed by the amino acid sequence SEK ID NO:2. 10 Thus, norleucine (X58) replaces the natural amino acid in the first position of the sequence. It has been used. The second of the third interface (S3) peptides is 'TVP' at the N-terminus. It includes the extension, and following this extension, 5-methyl-arginine replaces natural arginine. Residue (W32) has been inserted. After this modified amino acid, the peptide has SEK ID NO:3 It continues with the amino acid sequence. Another of the third interface (S3) peptides is 15 It contains the 'TVP' extension at the n-terminus, followed by 2- instead of natural arginine. The amino-3-guanidinopropionic acid (X01) residue has been inserted. This modification After the amino acid, the peptide continues with the amino acid sequence SEK ID NO:3. Thus, 2-amino-3- is used instead of the natural arginine residue at the fifth position of the sequence. Guanidinopropionic acid (X01) was used. One of the third interface (S3) peptides is 20. The other contains the amino acid sequence 'TVPMRLRK' at the N-terminus, followed by 3-methyl-L-alloisoleucine (X24) has been substituted for natural isoleucine. This modified After the amino acid, the peptide continues with the sequence 'PDSFFKPPE'. Thus... 3-methyl-L- is used instead of the natural isoleucine residue at the ninth position of the sequence. Alloisoleucine (X24) was used. Another of the third interface (S3) peptides is N-25. It contains the SEK ID NO: 4 amino acid sequence at its terminal, followed by the natural It contains a diethylalanine (W80) residue that has replaced alanine. This modified After the amino acid, the peptide continues with the amino acid sequence 'FFKPPE'. Thus... Diethylalanine (W80) is used in place of natural alanine at the twelfth position of the sequence. Another of the third interface (S3) peptides is SEK ID NO: 4 30 at the N-terminus. It contains an amino acid sequence followed by a natural amino acid substitute The homocysteine ​​(X08) residue has been inserted. After this modified amino acid, the peptide, It continues with the amino acid sequence 'FFKPPE'. Thus, the twelfth amino acid in the sequence is... In the third position, homocysteine ​​(X08) was used instead of a natural amino acid. 7 Another of the interface (S3) peptides has the amino acid sequence SEK ID NO: 5 at its N-terminus. It contains, subsequently, 2-fluoro-phenylalanine (W94) instead of natural phenylalanine. a residue has been inserted. After this modified amino acid, the peptide has the sequence 'FKPPE'. It continues. Another of the third interface (S3) peptides, at the N-terminus SEK ID NO: Contains a sequence of 6 amino acids, followed by 2-5 amino acids in place of natural glycine. The fluoro-phenylglycine (W91) residue has been inserted. After this modified amino acid The peptide continues with the 'PE' sequence. Another of the third interface (S3) peptides is, It contains the amino acid sequence SEK ID NO: 7 at the N-terminus, followed by the natural sequence. Glycine has been replaced by a phenylglycine (W00) residue. This is a modified amino acid. Then the peptide continues with the amino acid sequence 'PE'. Thus, the fifteenth amino acid in the sequence is 10 Natural glycine at position [position] has been replaced with phenylglycine (W00). Third interface (S3) Another of its peptides contains the amino acid sequence SEK ID NO: 8 at its N-terminus. and subsequently, 2-cyano-phenylalanine (W62) residue in place of natural phenylalanine. is inserted. After this modified amino acid, the peptide has the amino acid sequence 'PE'. It continues. Another of the third interface (S3) peptides has 15 at the N-terminus. SEK ID NO: Contains a sequence of 7 amino acids, followed by 4- amino acid instead of natural glycine. The hydroxy-phenylglycine (W75) residue has been inserted. After this modified amino acid The peptide continues with the amino acid sequence 'PE'. It is one of the third interface (S3) peptides. Another one contains the amino acid sequence SEK ID NO: 7 at its N-terminus, which Subsequently, the residue 4-iodo-phenylalanine (X74) was substituted for natural phenylalanine. This 20 After the modified amino acid, the peptide continues with the amino acid sequence 'PE'. Another of the third interface (S3) peptides is SEK ID NO: 7 at the N-terminal. It contains the amino acid sequence followed by 4-bromo- instead of natural phenylalanine. A phenylalanine (W18) residue has been inserted. Following this modified amino acid, the peptide, 'PE' continues with the amino acid sequence. Thus, the fifteenth amino acid in the sequence is 25. Natural phenylalanine at this position has been replaced with 4-bromo-phenylalanine (W18). Another of the third interface (S3) peptides is SEK ID NO: 7 at the N-terminal. It contains the amino acid sequence followed by 4-chloro- instead of natural phenylalanine. A phenylalanine (W04) residue has been inserted. Following this modified amino acid, the peptide, It continues with the amino acid sequence 'PE'. It is one of the third interface (S3) peptides. The other contains the amino acid sequence SEK ID NO: 7 at the N-terminus, followed by 4-fluoro-phenylalanine (X72) residue has been substituted for natural phenylalanine. This modified Following the amino acid, the peptide continues with the amino acid sequence 'PE'. Third interface. Another of the (S3) peptides has the SEK ID NO: 8 amino acid sequence at its N-terminus. 8 It contains, subsequently, a residue of 2-fluoro-phenylglycine (W91) instead of natural glycine. is inserted. After this modified amino acid, the peptide is connected with the 'E' amino acid residue. It continues. Another of the third interface (S3) peptides, at the N-terminus SEK ID NO: Contains an 8-amino acid sequence followed by a substitute for natural glycine. The phenylglycine (W00) residue has been inserted. After this modified amino acid, the peptide is labeled 'E' 5. It continues with an amino acid residue. One of the third interface (S3) peptides. The other contains the amino acid sequence SEK ID NO: 8 at the N-terminus, followed by Natural phenylalanine has been replaced with a residue of 2-cyano-phenylalanine (W62). This After the modified amino acid, the peptide continues with an 'E' amino acid residue. Another of the third interface (S3) peptides is SEK ID NO: 8 10 at the N-terminal. It contains the amino acid sequence followed by 4-hydroxy- instead of natural glycine. The phenylglycine (W75) residue is inserted. After this modified amino acid, the peptide is labeled 'E'. It continues with amino acid residue. The aim of the invention is to determine the YAP-TEAD protein interaction using a peptidomimetic approach. The primary aim of this invention is to prevent the interaction between YAP and TEAD proteins. By directly targeting protein-protein interactions, complex formation is achieved. The goal is to develop peptide-based inhibitors that can block these small molecules. In cases where it is insufficient to effectively disrupt the large interaction surface, Thanks to the peptidomimetic design approach, a specific and 20-bit design was created for the YAP-TEAD interface. Larger molecular structures capable of strong binding are being formed. Peptides, YAP mimics the binding sites (e.g., S1, S2, and S3 pockets) on TEAD. By doing so, it acts with higher selectivity. In this way, the invention, protein by simultaneously targeting contact points spread across its interface, small It aims to provide more effective inhibition compared to molecules. 25 Inhibitors developed using the peptidomimetic approach inhibit the natural interactions of the target protein. By mimicking their motifs, YAP-TEAD minimizes unwanted side interactions. It offers an intervention specific to the complex. One aim of the invention is to improve the stability of peptides with unnatural amino acids and 30 The goal is to increase its affinity. Unnatural amino acids are added to the sequences of the designed peptide inhibitors. By integrating acids, the metabolic stability and target binding of these peptides are improved. The goal is to increase its power. Non-natural amino acids (for example, D-amino acids or β-amino acids), when incorporated into the peptide structure, are easily processed by proteases. 9 prevents degradation and thus maintains the proteolytic stability of peptides. This increases the advantage. Furthermore, these unique amino acids benefit from the different steric properties provided by their side chains. and enhances interaction with the target protein thanks to its chemical properties. For example, a non-natural amino acid that is more hydrophobic or sterically bulky. Adding it to a critical position makes the peptide more securely attached to the hydrophobic pocket on TEAD. By enabling it to sit securely, it can increase attachment affinity. This technical element, The peptide can be used not only in laboratory settings but also in challenging biological environments such as intracellular environments. The aim is to ensure that it remains stable and effective for a longer period of time. Another aim of the invention is to determine the optimum peptide 10 through computational design and modeling. The selection of sequences. Within the scope of the invention, predicting the effectiveness of peptide candidates. and advanced computational modeling and design methods to select the best ones Molecular modeling techniques are used to model the YAP-TEAD complex. By examining the binding sites in detail, it is possible to understand how peptides bind to these sites. It is designed that they will settle there. In particular, the possible 15 of the unnatural amino acids. By creating a large rotamer library that includes various conformations, different The effect of amino acid combinations on peptide structures is being evaluated. Thanks to the computational screening process, the most suitable one is selected from hundreds of possible peptide derivatives. Several candidates with high coupling energy gain and stability are identified. In conclusion, peptide 20 was optimized before proceeding to the experimental phase. Time and resources are saved by selecting the sequences. One aim of the invention is to simulate long-term molecular dynamics with peptide-protein The aim is to verify the stability of the complex. The peptides selected as the subject of the invention are YAP-TEAD. After connecting to the interface, their structural stability in the long term is 25 The aim is to demonstrate that they can be sustained. In this context, peptide-protein complexes... by performing long-duration molecular dynamics simulations such as 100 ns The system's behavior is monitored over time. Throughout the simulations, the peptide's behavior is observed. Maintaining its position in the binding pockets on TEAD, key interactions (hydrogen bonds, hydrophobic interactions, etc.) maintaining continuity and the complex's 30 It is expected not to disintegrate. The results obtained indicate that the designed peptide only It not only provides strong connectivity in real-time but also exhibits stability under dynamic conditions. This reveals that if there is a peptide-position shift or If a weakening of interactions is observed, these findings should be considered for peptide redesign. It is providing guidance. Therefore, long-term molecular dynamics analyses, the durability of inhibitor peptides under realistic biological conditions and their binding to the target protein This supports the technical success of the invention by confirming the tendency to remain attached. Figure Description 5 Figure 1. Development of peptides that can inhibit the YAP-TEAD complex. workflow followed during the process. Explanation of References in Figures 1000. Drawing of unnatural amino acids and optimization of 3D structures 10 to be done. 1001. Compilation of the rotamer library of molecules. 1002. Interaction of amino acids in the YAP / TEAD interface region with non-natural amino acids. change. 1003. Calculation of binding scores (REU) using the Flex_ddg protocol. 15 1004. Tensile molecular dynamics simulation and umbrella sampling method Calculation of binding free energies. 1005. Long molecular dynamics simulations (100 ns) of selected peptides. Determining the stabilization in the binding region. Detailed Description of the Invention The invention relates to the YAP-TEAD (Yes-Associated Protein – TEA Domain) protein complex. peptides designed using a peptidomimetic approach to prevent its formation This is related. The peptides included in the invention are intended for individual use. The invention describes the YAP-TEAD protein-protein interaction using a peptidomimetic approach. It aims to block and is more effective than inhibitory peptides compared to small molecules. It develops them. For this purpose, unnatural amino acids are added to peptide sequences. Proteolytic stability and binding affinity are increased. Unnatural Peptides are formed thanks to the unique steric and chemical properties provided by amino acids. It can adhere more tightly to the hydrophobic pockets on TEAD. Also, 30 Hundreds of candidates were identified using computational modeling and rotamer libraries. The most suitable peptide sequences are selected from among them. The selected candidates undergo long-term testing. In terms of binding stability using molecular dynamics simulations. 11 This confirms the durability of peptides under realistic biological conditions. Inhibitory efficacy is technically supported. The first of the interface (S1) peptides subject to the invention has the 'GH' sequence at its N-terminus. It contains, followed by 3-methyl-L-alloisoleucine 5, which is a substitute for natural isoleucine. It contains residue (X24). After this modified amino acid, the peptide is called 'IVHVR'. It continues with the amino acid sequence. The second of the S1 peptides is at the N-terminus. It contains the sequence 'GH' followed by norvaline (X59) instead of the natural amino acid. It contains the residue. After this modified amino acid, the peptide is called 'IVHVR'. It continues with the amino acid sequence. The first of the second interface (S2) peptides is N-10. It contains a 'T' amino acid residue at its terminal, followed by natural aspartic acid. 3,3-dimethyl aspartic acid (W82) has been substituted for the amino acid. From this modified amino acid The peptide then continues with the amino acid sequence SEK ID NO: 1. Second interface (S2) The second of these peptides contains the amino acid sequence 'TDLEALFN' at its N-terminus. and subsequently, 2-(trifluoromethyl)-phenylalanine (L00) 15 is used in place of natural phenylalanine. is inserted. After this modified amino acid, the peptide has the amino acid sequence 'VMNP'. It continues. The last of the second interface (S2) peptides is located at the N-terminal. It contains the amino acid sequence 'TDLEALFN', followed by natural glutamic acid. (3S)-3-methyl-L-glutamic acid (X43) has been substituted in its place. From this modified amino acid The peptide then continues with the amino acid sequence 'VMNP'. The third interface (S3) is 20. The first of its peptides is norleucine (X58), an unnatural amino acid at the N-terminus. It contains the residue, followed by the amino acid sequence SEK ID NO:2. Thus, norleucine (X58) replaces the natural amino acid in the first position of the sequence. It has been used. The second of the third interface (S3) peptides is 'TVP' at the N-terminus. It includes the extension, followed by 5-methyl-arginine 25 in place of natural arginine. Residue (W32) has been inserted. After this modified amino acid, the peptide has SEK ID NO:3 It continues with the amino acid sequence. Another of the third interface (S3) peptides, It contains the 'TVP' extension at the n-terminus, followed by 2- instead of natural arginine. The amino-3-guanidinopropionic acid (X01) residue has been inserted. This modification After the amino acid, the peptide continues with the amino acid sequence SEK ID NO:3. 30 Thus, 2-amino-3- is used instead of the natural arginine residue at the fifth position of the sequence. Guanidinopropionic acid (X01) was used. One of the third interface (S3) peptides. The other contains the amino acid sequence 'TVPMRLRK' at the N-terminus, followed by 3-methyl-L-alloisoleucine (X24) has been substituted for natural isoleucine. This modified 12 After the amino acid, the peptide continues with the sequence 'PDSFFKPPE'. Thus... 3-methyl-L- is used instead of the natural isoleucine residue at the ninth position of the sequence. Alloisoleucine (X24) was used. Another of the third interface (S3) peptides, N- It contains the SEK ID NO: 4 amino acid sequence at its terminal, followed by the natural It contains a diethylalanine (W80) residue that has replaced alanine. This modified 5 After the amino acid, the peptide continues with the amino acid sequence 'FFKPPE'. Thus... Diethylalanine (W80) is used in place of natural alanine at the twelfth position of the sequence. Another of the third interface (S3) peptides is SEK ID NO: 4 at the N-terminal. It contains an amino acid sequence followed by a natural amino acid substitute The homocysteine ​​(X08) residue has been inserted. After this modified amino acid, the peptide is 10 It continues with the amino acid sequence 'FFKPPE'. Thus, the twelfth amino acid in the sequence is... In the third position, homocysteine ​​(X08) was used instead of a natural amino acid. Another of the interface (S3) peptides has the amino acid sequence SEK ID NO: 5 at its N-terminus. It contains, subsequently, 2-fluoro-phenylalanine (W94) instead of natural phenylalanine. a residue has been inserted. After this modified amino acid, the peptide has the sequence 'FKPPE' 15 It continues. Another of the third interface (S3) peptides, at the N-terminus SEK ID NO: Contains a sequence of 6 amino acids, followed by 2- amino acid instead of natural glycine. The fluoro-phenylglycine (W91) residue has been inserted. After this modified amino acid The peptide continues with the 'PE' sequence. Another of the third interface (S3) peptides is, It contains the amino acid sequence SEK ID NO: 7 at the N-terminus, followed by 20 naturally occurring amino acids. Glycine has been replaced by a phenylglycine (W00) residue. This is a modified amino acid. Then the peptide continues with the amino acid sequence 'PE'. Thus, the fifteenth in the sequence... Natural glycine at position [position] has been replaced with phenylglycine (W00). Third interface (S3) Another of its peptides contains the amino acid sequence SEK ID NO: 8 at its N-terminus. and subsequently, 2-cyano-phenylalanine (W62) residue 25 in place of natural phenylalanine. is inserted. After this modified amino acid, the peptide has the amino acid sequence 'PE'. It continues. Another of the third interface (S3) peptides, at the N-terminus SEK ID NO: Contains a sequence of 7 amino acids, followed by 4- amino acid instead of natural glycine. The hydroxy-phenylglycine (W75) residue has been inserted. After this modified amino acid The peptide continues with the amino acid sequence 'PE'. It is one of the 30 third interface (S3) peptides. Another one contains the amino acid sequence SEK ID NO: 7 at its N-terminus, which Subsequently, the residue 4-iodo-phenylalanine (X74) was substituted for natural phenylalanine. This After the modified amino acid, the peptide continues with the amino acid sequence 'PE'. Another of the third interface (S3) peptides is SEK ID NO: 7 at the N-terminal. 13 It contains the amino acid sequence followed by 4-bromo- instead of natural phenylalanine. A phenylalanine (W18) residue has been inserted. Following this modified amino acid, the peptide, The sequence continues with the amino acid 'PE'. Thus, the fifteenth amino acid in the sequence... Natural phenylalanine at this position has been replaced with 4-bromo-phenylalanine (W18). Another of the third interface (S3) peptides is SEK ID NO: 7 5 at the N-terminal. It contains the amino acid sequence followed by 4-chloro- instead of natural phenylalanine. A phenylalanine (W04) residue has been inserted. Following this modified amino acid, the peptide, It continues with the amino acid sequence 'PE'. It is one of the third interface (S3) peptides. The other contains the amino acid sequence SEK ID NO: 7 at the N-terminus, followed by 4-fluoro-phenylalanine (X72) residue has been substituted for natural phenylalanine. This modified 10 Following the amino acid, the peptide continues with the amino acid sequence 'PE'. Third interface. Another of the (S3) peptides has the SEK ID NO: 8 amino acid sequence at its N-terminus. It contains, subsequently, a residue of 2-fluoro-phenylglycine (W91) instead of natural glycine. is inserted. After this modified amino acid, the peptide is connected with the 'E' amino acid residue. It continues. Another of the third interface (S3) peptides has 15 at the N-terminus. SEK ID NO: Contains an 8-amino acid sequence followed by a substitute for natural glycine. A phenylglycine (W00) residue has been inserted. After this modified amino acid, the peptide is labeled 'E'. It continues with an amino acid residue. One of the third interface (S3) peptides. The other contains the amino acid sequence SEK ID NO: 8 at the N-terminus, followed by Natural phenylalanine has been replaced by a residue of 2-cyano-phenylalanine (W62). This 20 After the modified amino acid, the peptide continues with an 'E' amino acid residue. Another of the third interface (S3) peptides is SEK ID NO: 8 at the N-terminal. It contains the amino acid sequence followed by 4-hydroxy- instead of natural glycine. The phenylglycine (W75) residue is inserted. After this modified amino acid, the peptide is labeled 'E'. It continues with amino acid residue. 25 1. Peptide Design Use of Natural and Non-Natural Amino Acids: The peptides used in the invention are derived from both naturally occurring and unnatural amino acids. It consists of unnatural amino acids, which reduce the metabolic stability of peptides by 30. While increasing its effectiveness, natural amino acids preserve the peptide's biological function. Unnatural amino acids... amino acids can provide greater hydrophobicity or lower polarity. They can enhance the interactions of peptides with target proteins. 14 Peptidomimetic Approach: The peptidomimetic approach enhances the desired biological properties of peptide structures. This involves modification for the purpose of modifying the peptide backbone or sides. Modifications made to their chains confer resistance to proteases, and It increases the affinity for binding. 5 2. Modification of Peptides Determining Attachment Sites: Three main binding sites (S1, S2, S3) have been identified in the YAP-TEAD complex. The first interface (S1) peptides are amino acid intervals 51 and 58 of the YAP sequence. (GHQIVHVR), second interface (S2) peptides with amino acid ranges 63 and 75. (SEK ID NO:9) and third interface (S3) peptides amino acids 83 and 100 It was created using the range (SEK ID NO:10). Natural amino acids are found in these regions. Their original sequences have been modified with unnatural amino acids. This Modifications resulted in tighter peptide binding and increased inhibition potential. 15 It aims to increase it. Specific Amino Acid Changes: In the binding sites, modified amino acids (e.g., 3-methyl-l-alloisoleucine, D- Ornithine (4-methoxy-phenylalanine) has been used as a substitute for natural amino acids. 20 designed... the interaction of new peptides with other amino acids located in the interface region where they are inserted The interaction amounts have been calculated. These changes affect the binding free energy. By reducing its effectiveness, it increases the efficacy of inhibitor candidate peptides. 3. Molecular Modeling and Simulation 25 Connection Free Energy Calculations: Binding free energies between the designed peptides and the TEAD protein wham module included in GROMACS 2020.2 software [9] for calculation It has been used. Peptide designed using umbrella sampling method and The free energy of protein binding has been calculated. These calculations... Modified peptides, when compared to their natural form, are more effective against the target protein. It shows whether the connection is stronger or weaker. Umbrella sampling. The values ​​obtained from the simulation, the YAP-TEAD complex and 5 from this structure Compared to derived related interaction interface peptides, and higher Peptides with binding valencies have been identified. Additionally, those found in the literature... also compared with peptides obtained using unnatural amino acids. For example, in the study by Furet and his colleagues

[10] YAP’s 86th tryptophan, 6-chlorotryptophan and norleucine 10 performed for this position modifications, binding free energy calculation simulations This was carried out. Subsequently, the molecular dynamics of these modified peptides were subjected to pull-ups. ΔG binding energies with simulations and umbrella sampling simulations It has been calculated. As a result of the simulations, the tryptophan at position 86 is 0.430, 6- ΔΔG scores of 0.466 for chlorotryptophan and -0.083 for norleucine were obtained. 15 Binding obtained from umbrella sampling method Their free energies are -16.16 kcal / mol for tryptophan and -16.53 kcal / mol for 6-chlorotryptophan. A ΔG binding energy of -9.98 kcal / mol was obtained for kcal / mol and for norleucine. According to this situation, the results we obtained from the umbrella sampling method are consistent with Furet and with the results obtained from the TR-FRET analysis in the work of his colleagues 20 It shows consistency. For example, in TR-FRET analysis, it has the lowest IC50 value. The modification, 6-chlorotryptophan, was found in ΔG samples obtained from umbrella sampling. The binding energy is also low. These results indicate that the binding affinity of 6-chlorotryptophan is low. This indicates that it is higher compared to the other two modifications. Similarly Norleucine, the modification with the highest IC50 value, is 25 from umbrella sampling. The ΔG binding energy obtained is quite high, and these results indicate that norleucine that the binding affinity is lower compared to the other two modifications It shows. Molecular Dynamics Simulations: 30 Peptides with higher binding statistics have different interaction mechanisms. more detailed examination and assessment of stability in the binding site 16 for the purpose of 100 nanosecond (ns) long molecular dynamics simulations This has been carried out. These simulations, performed using GROMACS, show that peptides revealing the binding tendencies of the target protein and the stability at the binding sites. This is the molecular dynamics for all protein-peptide complexes. Simulations; 310 Kelvin temperature, 1 atmosphere pressure, 0.15 M sodium and chloride ions. This was carried out using concentration and a type 3p water model. Industrial Applicability of the Invention The invention relates to the YAP-TEAD (Yes-Associated Protein – TEA Domain) protein complex. 10 peptides designed using a peptidomimetic approach to prevent its formation It is relevant and applicable to industry. The invention is not limited to the above descriptions, and a person skilled in the field can easily make further discoveries. It can demonstrate different applications of the invention. These are the claims and demands of the invention. It should be evaluated within the scope of the protection granted. 15 25 17 REFERENCES [1] Calses, P. C., Crawford, J. J., Lill, J. R., & Dey, A. (2019). Hippo pathway in cancer: Aberrant regulation and therapeutic opportunities. Nature Reviews Cancer, 19(11), 671–685. https: / / doi.org / 10.1038 / s41568-019-0206-7 [2] Harvey, K. F., Zhang, X., & Thomas, D. M. (2013). The Hippo pathway and 5 human cancer. Nature Reviews Cancer, 13(4), 246–257. https: / / doi.org / 10.1038 / nrc3458 [3] Kim, M. H., Kim, J., Hong, H., Lee, S. H., Lee, J. K., Jung, E., ... & Jang, S. Y. (2019). Actin remodeling confers BRAF inhibitor resistance to melanoma cells through YAP / TAZ activation. Clinical Cancer Research, 25(3), 1022–1035. 10 https: / / doi.org / 10.1158 / 1078-0432.CCR-18-1100 [4] Zanconato, F., Forcato, M., Battilana, G., Azzolin, L., Quaranta, E., Bodega, B., ... & Piccolo, S. (2015). Genome-wide association between YAP / TAZ / TEAD and AP-1 at enhancers drives oncogenic growth. Nature Cell Biology, 17(9), 1218–1227. https: / / doi.org / 10.1038 / ncb3216 15 [5] Zhao, B., Wei, X., Li, W., Udan, R. S., Yang, Q., Kim, J., ... & Guan, K. L. (2007). Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control. Genes & Development, 21(21), 2747–2761. https: / / doi.org / 10.1101 / gad.1602907 [6] Crawford, J. J., Bronner, S. M., Zbieg, J. R. (2018). Hippo pathway inhibition by 20 blocking the YAP / TAZ-TEAD interface: a patent review. Expert opinion on therapeutic patents. [7] Pobbati, A. V., & Rubin, B. P. (2020). Protein-Protein Interaction Disruptors of the YAP / TAZ-TEAD Transcriptional Complex. Molecules. [8] Rossino, G., Marchese, E., Galli, G., Verde, F., Finizio, M., Serra, M., Linciano, 25 P., & Collina, S. (2023). Peptides as Therapeutic Agents: Challenges and Opportunities in the Green Transition Era. Molecules. [9] Hub, J. S., De Groot, B. L., & Van Der Spoel, D. (2010). g_wham A Free Weighted Histogram Analysis Implementation Including Robust Error and Autocorrelation Estimates. Journal of chemical theory and computation, 6(12), 30 3713-3720.

[10] Furet, P., Salem, B., Mesrouze, Y., Schmelzle, T., Lewis, I., Kallen, J., & Chène, P. (2019). Structure-based design of potent linear peptide inhibitors of the YAP- TEAD protein-protein interaction derived from the YAP omega-loop 18 sequence. Bioorganic & medicinal chemistry letters, 29(16), 2316–2319. https: / / doi.org / 10.1016 / j.bmcl.2019.06.022 10 20 30

Claims

19 REQUESTS 1. YAP-TEAD (Yes-Associated Protein – TEA Domain) protein complex Its characteristic feature is that it is an inhibitor designed to prevent its formation; • Containing a sequence of 'GH' at the N-terminus, followed by a substitute for natural isoleucine. containing the 3-methyl-L-alloisoleucine (X24) residue, these 5 followed by the modified amino acid, then the 'IVHVR' amino acid sequence. peptide or, • Containing the sequence 'GH' at the N-terminus, followed by a natural amino acid from this modified amino acid which contains norvaline (X59) residue instead then a peptide followed by the amino acid sequence 'IVHVR' or, 10 • Containing a 'T' amino acid residue at the n-terminus, followed by a natural This contains 3,3-dimethyl aspartic acid (W82) instead of aspartic acid. After the modified amino acid, continue with the amino acid sequence SEK ID NO:

1. peptide or, • Containing the amino acid sequence 'TDLEALFN' at the N-terminus, followed by 15 Containing 2-(trifluoromethyl)-phenylalanine (L00) instead of natural phenylalanine, After this modified amino acid, continue with the 'VMNP' amino acid sequence. peptide or, • Containing the amino acid sequence 'TDLEALFN' at the N-terminus, followed by (3S)-3-methyl-L-glutamic acid (X43) 20 instead of natural glutamic acid containing, after this modified amino acid, the amino acid sequence 'VMNP'. ongoing peptide or, • Norleucine (X58), an unnatural amino acid at the N-terminus containing the residue followed by the amino acid sequence SEK ID NO:2 peptide or, 25 • Containing the 'TVP' extension at the n-terminal and followed by a natural letter. This modified version contains a 5-methyl-arginine (W32) residue instead of arginine. after the amino acid, the sequence continues with SEK ID NO:3 amino acid sequence. peptide or, • Containing the 'TVP' extension at the n-terminus, followed by natural arginine 30 containing 2-amino-3-guanidinopropionic acid (X01) residue instead, After the modified amino acid, the peptide has the SEK ID NO:3 amino acid sequence. ongoing peptide or, • Containing the amino acid sequence 'TVPMRLRK' at the N-terminus, followed by containing 3-methyl-L-alloisoleucine (X24) instead of natural isoleucine, followed by the modified amino acid and then the sequence 'PDSFFKPPE' peptide or, • Containing a sequence of 4 amino acids at the N-terminus, followed by 5 diethylalanine (W80) residue substituted for natural alanine containing, after this modified amino acid, the amino acid sequence is 'FFKPPE'. peptide or, • Containing the SEK ID NO: 4 amino acid sequence at the N-terminus, followed by These 10 contain a homocysteine ​​(X08) residue instead of a natural amino acid. After the modified amino acid, continue with the amino acid sequence 'FFKPPE'. peptide or, • Containing the SEK ID NO: 5 amino acid sequence at the N-terminus, followed by containing 2-fluoro-phenylalanine (W94) residue instead of natural phenylalanine, peptide 15, which is followed by the sequence 'FKPPE' after this modified amino acid. or, • Containing the SEK ID NO: 6 amino acid sequence at the N-terminus, followed by This contains a residue of 2-fluoro-phenylglycine (W91) instead of natural glycine. A peptide that starts with a modified amino acid followed by a 'PE' sequence, or, • Containing the amino acid sequence SEK ID NO: 7 at the N-terminus, followed by 20 This modified glycine contains a phenylglycine (W00) residue instead of natural glycine. A peptide that follows an amino acid with the amino acid sequence 'PE', or, • Containing the SEK ID NO: 8 amino acid sequence at the N-terminus, followed by containing 2-cyano-phenylalanine (W62) residue instead of natural phenylalanine, This modified amino acid is followed by the amino acid sequence 'PE', which is 25. peptide or, • Containing the amino acid sequence SEC ID NO: 7 at the N-terminus, followed by This contains a 4-hydroxy-phenylglycine (W75) residue instead of natural glycine. followed by a modified amino acid, then the amino acid sequence 'PE'. peptide or, 30 • Containing the amino acid sequence SEC ID NO: 7 at the N-terminus, followed by containing 4-iodo-phenylalanine (X74) residue instead of natural phenylalanine, 21 this modified amino acid is followed by the amino acid sequence 'PE'. peptide or, • Containing the amino acid sequence SEC ID NO: 7 at the N-terminus, followed by containing 4-bromo-phenylalanine (W18) residue instead of natural phenylalanine, This modified amino acid is followed by the amino acid sequence 'PE', which is the next 5. peptide or, • Containing the amino acid sequence SEC ID NO: 7 at the N-terminus, followed by containing 4-chloro-phenylalanine (W04) residue instead of natural phenylalanine, this modified amino acid is followed by the amino acid sequence 'PE'. peptide or, 10 • Containing the amino acid sequence SEC ID NO: 7 at the N-terminus, followed by containing 4-fluoro-phenylalanine (X72) residue instead of natural phenylalanine, this modified amino acid is followed by the amino acid sequence 'PE'. peptide or, • Containing an amino acid sequence with SEC ID NO: 8 at the N-terminus, followed by 15 This contains a residue of 2-fluoro-phenylglycine (W91) instead of natural glycine. followed by a modified amino acid, then an 'E' amino acid residue. peptide or, • Containing the SEK ID NO: 8 amino acid sequence at the N-terminus, followed by This modified 20 contains a phenylglycine (W00) residue instead of natural glycine. A peptide that is followed by an 'E' amino acid residue after an amino acid, or, • Containing the SEK ID NO: 8 amino acid sequence at the N-terminus, followed by containing 2-cyano-phenylalanine (W62) residue instead of natural phenylalanine this modified amino acid is followed by an 'E' amino acid residue. peptide or, 25 • Containing the SEK ID NO: 8 amino acid sequence at the N-terminus, followed by This contains a 4-hydroxy-phenylglycine (W75) residue instead of natural glycine. followed by a modified amino acid, then an 'E' amino acid residue. peptide That is. 30

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