Screening method of autophagy-inducing compound

Abstract

Provided is a screening method for exploring a novel autophagy-inducing compound. A novel autophagy-inducing compound is explored by using at least one of the following properties (1)-(3): (1) the novel autophagy-inducing compound has a high binding affinity for DEAD-box-type RNA helicase; (2) bringing the DEAD-box type RNA helicase into proximity to p62 to promote interaction; and (3) promoting the formation of droplets of the DEAD-box type RNA helicase and p62.

Description

Technical Field

[0001] This invention relates to a method for screening compounds that induce autophagy and a method for manufacturing compounds that induce the degradation of target intracellular molecules or organelles by autophagy. Background Technology

[0002] Autophagy is one of the intracellular molecular degradation mechanisms within cells, and it is a mechanism found in eukaryotes from yeast to humans. If autophagy is induced, membrane vesicles called autophagosomes are formed, which then fuse with lysosomes, thereby degrading the molecules taken in by the autophagosomes.

[0003] It is known that autophagy is induced by cellular starvation. Furthermore, it has been clarified that autophagy also participates in physiological functions such as production and differentiation, or in infection defense functions such as clearing viruses that have invaded cells.

[0004] The mechanism of induced autophagy has also been clarified through various reports.

[0005] In a review paper, Mohammad Omar Faruk et al. detailed a mechanism of selective autophagy that degrades specific soluble proteins, supramolecular complexes, liquid-phase separated droplets, abnormal or excess organelles, and pathogenic invading bacteria, thus contributing to cellular homeostasis (Non-Patent Literature 1). Specifically, they reported that p62 binds to ubiquitinated proteins, resulting in liquid-phase separation and the formation of droplets containing p62 and ubiquitinated proteins. These droplets recruit other proteins to form an isolation / phagocytic membrane, and the p62 and other proteins within the droplets are then degraded by lysosomes.

[0006] Lin Ma et al. suggested that in esophageal squamous cell carcinoma (ESCC), knockdown of DDX5 inhibits endoplasmic reticulum stress and promotes the recovery of autophagic flux (Non-Patent Literature 2).

[0007] Hao Zhang et al. reported that DDX5 interacts with the autophagy receptor p62 to stimulate autophagy and inhibit the occurrence of liver cancer (Non-Patent Literature 3). They also reported that DDX5 promotes the degradation of p62, significantly reducing its half-life.

[0008] Yongsang Jo et al. disclosed in their report on the essentially disordered protein-protein interactions within membrane-free protein droplets that droplets of DDX4 were formed by adding streptavidin (Non-Patent Document 4).

[0009] In addition, attempts to actively induce autophagy to provide new treatment methods (sometimes referred to as degraders) have also shown significant progress (e.g., non-patent literature 5-8).

[0010] Representative examples of degradative agents are protein degradation-targeting chimeras (PROTACs) / specific, non-hereditary IAP-dependent protein scavengers (SNIPERs), which are heterobifunctional molecules consisting of a ligand that binds to the target molecule and a degradation tag that induces degradation based on the ubiquitin-proteasome system (UPS) (Non-Patent Literature 5, 9, and 10). These heterobifunctional molecules have the advantage of targeting a wide range of substrates by optimizing the degradation tag and replacing the target ligand. However, degradation systems utilizing UPSs are limited to protein-targeting systems.

[0011] In response to this, in recent years, lysosomal degradation systems, lysosomal-targeted chimeras (LYTAC) and autophagy-targeted chimeras (AUTAC) that degrade the extracellular matrix have been developed (Non-Patent Literature 11 and 12), with the expectation of developing newer chimeric molecules.

[0012] Existing technical documents Non-patent literature Non-patent literature 1: Cancer Sci, 2021, 112(10): 3972-3978; Non-patent literature 2: Biochem Biophys Res Commun, 2020, 533(4): 1449-1456; Non-patent literature 3: Hepatology, 2019, 69(3): 1046-1063; Non-patent literature 4: Chem. Sci., 2020, 11, 1269; Non-patent literature 5: Samarasinghe, KTG; Crews, CM Targeted protein degradation: a promise for undruggable proteins. Cell Chem. Biol. 2021, 28, 934-951; Non-patent literature 6: Li, K.; M. Crews, C. PROTACs: Past, Present and Future. Chem. Soc. Rev. 2022. doi: 10. 1039 / d2cs00193d; Non-patent literature 7: Burslem, GM; Crews, CM. Proteolysis-targeting chimeras astherapeutics and tools for biological discovery. Cell 2020, 181, 102-114; Non-patent literature 8: Paiva, S.-L.; Crews, CM Targeted protein degradation: elements of PROTAC design. Curr. Opin. Chem. Biol. 2019, 50, 111-119; Non-patent literature 9: Hidesuke Tomoshige, Minoru Ishikawa PROTACs and other chemical proteindegradation technologies for the treatment of neurodegenerative disorders. Angew. Chem. Int. Ed. 2021, 60, 3346-3354; Non-patent literature 10: Naito Mikihiko, Ooka Nobumichi, Shibata Shikihito: SNIPERs-hijacking IAPactivity to induce protein degradation. Drug Discov. Today Technol. 2019, 31, 35-42; Non-patent literature 11: Banik, SM; Pedram, K.; Wisnovsky, S.; Ahn, G.; Riley, NM; Bertozzi, CR; Lysosome-targeting Chimaeras for degradation of extracellular proteins. Nature 2020, 584, 291-297; Non-patent literature 12: Daiki Takahashi and Hirokazu Arimoto: Selective autophagy as the basis of autophagy-base. Cell Chem. Biol. 2021, 28, 1061-1071; Non-Patent Document 13: Yoshinobu Ichimura, Masaki Komatsu p62 / SQSTM1: Its Role in Autophagy Journal of Japanese Biochemical Society 91(3): 380-387 (2019) doi:10. 14952 / SEIKAGAKU. 2019. 910380. Summary of the Invention

[0013] Problems to be Solved by the Invention However, the design of the degradation tags in these chimeric molecules that induce selective autophagy has been obtained empirically by trial and error, and in order to effectively create new chimeric molecules, it is desired to develop tools for designing degradation tags.

[0014] In response to this need, the present invention provides a tool for obtaining degradation tags of chimeric molecules that induce selective autophagy.

[0015] Means for Solving the Problems When the present inventors explored the mechanism of autophagy induction for the autophagy-inducing compounds and tag portions they developed, they found the following characteristics of these compounds. <​​​​​​​​​​​​​​​​​​​​​​​​​​​The interaction between the DEAD-box type RNA helicase and p62 was determined. The compound that induced the interaction was selected.

[0023] [3] According to the method of [2], wherein the DEAD-box RNA helicase and the p62 are expressed in cells, and the test compound is brought into contact with the cells.

[0024] [4] Methods for screening autophagy-inducing compounds, among which In the presence of p62, the test compound was brought into contact with a DEAD-box type RNA helicase. Determine the droplet formation of DEAD-box type RNA helicase with p62. The compound that induced the formation of the droplets was selected.

[0025] [5] The method according to any one of [1] to [4], wherein the DEAD-box type RNA helicase is DDX5 or DDX17.

[0026] [6] A method for manufacturing a compound that induces the degradation of target intracellular molecules or organelles by autophagy, comprising: The steps of implementing the method described in any one of [1] to [5]; and The step of attaching a ligand to the resulting compound via a linker to bind specifically to a molecule or organelle within the target cell.

[0027] [7] The method according to [6] further includes: The step of bringing a compound with the ligand into contact with a cell having the target intracellular molecule or organelle in vivo, in vitro, or in vitro to confirm the degradation of the target intracellular molecule or organelle by autophagy.

[0028] [8] A method of inducing the degradation of target intracellular molecules or target organelles by autophagy by contacting a compound obtained or available by performing the method described in [6] or [7] with a DEAD-box type RNA helicase in the presence of p62.

[0029] [9] According to the method of [8], wherein a compound obtained or available by performing the method of [6] or [7] is contacted in a cell with a DEAD-box RNA helicase in the presence of p62 to form a droplet containing the compound, the p62 and the DEAD-box RNA helicase.

[0030]

[10] The method described in [8] or [9] is used to prevent or treat cancer or cell degenerative diseases.

[0031] Invention Effects According to the method of the present invention, autophagy-inducing compounds can be effectively obtained and used as degradation tag portions to be linked via a linker to ligands that specifically bind to molecules within target cells, thereby enabling the synthesis of compounds that induce the degradation of target molecules or organelles by autophagy. Attached Figure Description

[0032] [ Figure 1 The value of light emitted is shown 10 minutes after the start of the light emission measurement in Examples 1-4.

[0033] [ Figure 2 The results show the amount of light emitted 10 minutes after the start of light emission measurement in Examples 5-33.

[0034] [ Figure 3 The image shows the EGFP fluorescence levels of cells transfected with pEGFP-FKBP12 when cultured in fresh DMEM containing 0.01 mM, 0.1 mM, 1 mM, and 10 mM AUTAC2-2G, and in fresh DMEM containing DMSO as a control.

[0035] [ Figure 4 [Image] is a fluorescence microscopy image showing the results of an assay for the degradation of mutant FKBP-tagged synuclein based on SLF'-AUTAC.

[0036] [ Figure 5 The display shows that GFP-FKBP will be transiently expressed. F36V HeLa cells were treated with SLF-AUTAC and fixed with paraformaldehyde, then immunostained with anti-DDX5 or anti-p62 antibody. The images were observed under a fluorescence microscope. The upper right square is a magnified view of the cytoplasm portion of the smaller square slightly to the upper left.

[0037] [ Figure 6 ] Figure 6 Image A shows a fluorescence microscope image of HeLa cells transiently expressing GFP-FKBP12 and RFP-DDX5 treated with AUTAC2-2G. Figure 6 Image B shows a fluorescence microscope image of HeLa cells transiently expressing GFP-FKBP12 and RFP-p62 treated with AUTAC2-2G. Detailed Implementation

[0038] The embodiments of the present invention will be described in detail below. However, the present invention should not be understood as limited to the following embodiments.

[0039] 1. Methods for screening autophagy-inducing compounds The screening method involved in this invention is a method for screening autophagy-inducing compounds using at least one of the following properties.

[0040] (1) It has a high binding affinity for DEAD-box type RNA helicases.

[0041] (2) Bring DEAD-box type RNA helicase and p62 close together to promote interaction.

[0042] (3) Promotes the formation of droplets of DEAD-box type RNA helicase and p62.

[0043] As demonstrated in the examples described below, since compounds with autophagy-inducing activity possess these properties, autophagy-inducing compounds can be obtained by selecting compounds having at least one of these properties.

[0044] 1-1. DEAD-box type RNA helicase DEAD-box RNA helicases are a superfamily of RNA helicases possessing the DEAD (Asp-Glu-Ala-Asp) motif. DEAD-box RNA helicases are known to utilize the energy from ATP hydrolysis to participate in the disintegration or formation of double-stranded RNA or RNA-protein complexes, playing important roles in transcription, splicing, extranuclear transport or degradation of RNA, translation, and ribosome biosynthesis.

[0045] Members of the DEAD-box RNA helicase family include DDX1 (DEAD box protein 1), DDX2A (DEAD box protein 2A), DDX3X (DEAD box protein 3X), DDX4 (DEAD box protein 4), DDX5 (DEAD box protein 5), DDX6 (DEAD box protein 6), DDX10 (DEAD box protein 10), DDX11 (DEAD box protein 11), DDX17 (DEAD box protein 17), DDX18 (DEAD box protein 18), DDX19B (DEAD box protein 19B), DDX20 (DEAD box protein 20), DDX21 (DEAD box protein 21), DDX23 (DEAD box protein 23), DDX24 (DEAD box protein 24), DDX25 (DEAD box protein 25), DDX27 (DEAD box protein 27), DDX28 (DEAD box protein 28), DDX31 (DEAD box protein 31), and DDX39B (DEAD box protein 31). DEAD-Box helicase 5 (DDX5) is also known as P68. The DEAD-Box helicase 5 (DDX5) is also known as DDX41 (DEAD box protein 41), DDX42 (DEAD box protein 42), DDX43 (DEAD box protein 43), DDX46 (DEAD box protein 46), DDX47 (DEAD box protein 47), DDX48 (DEAD box protein 48), DDX49 (DEAD box protein 49), DDX50 (DEAD box protein 50), DDX51 (DEAD box protein 51), DDX52 (DEAD box protein 52), DDX53 (DEAD box protein 53), DDX54 (DEAD box protein 54), DDX55 (DEAD box protein 55), DDX56 (DEAD box protein 56), DDX58 (DEAD box protein 58), and DDX59 (DEAD box protein 59).

[0046] The amino acid sequences and the nucleic acid sequences encoding these DEAD-box type RNA helicase members are as follows.

[0047] [Chemical Formula 1] DDX5 amino acid sequence (SEQ ID NO: 1) [Chemical Formula 2] DDX5 DNA sequence (SEQ ID NO: 2) [Chemical Formula 3] DDX17 amino acid sequence (SEQ ID NO: 3) [Chemical Formula 4] DDX17 DNA sequence (SEQ ID NO: 4) [Chemical Formula 5] DDX1 DNA sequence (SEQ ID NO: 22) [Chemical Formula 6] DDX2A DNA sequence (SEQ ID NO: 23) [Chemical Formula 7] DDX3X DNA sequence (SEQ ID NO: 24) [Chemical Formula 8] DDX4 DNA sequence (SEQ ID NO: 25) [Chemical Formula 9] DDX5 DNA sequence (SEQ ID NO: 26) [Chemical Formula 10] DDX6 DNA sequence (SEQ ID NO: 27) [Chemical Formula 11] DDX10 DNA sequence (SEQ ID NO: 28) [Chemical Formula 12] DDX11 DNA sequence (SEQ ID NO: 29) [Chemical Formula 13] DDX17 DNA sequence (SEQ ID NO: 30) [Chemical Formula 14] DDX18 DNA sequence (SEQ ID NO: 31) [Chemical Formula 15] DDX19B DNA sequence (SEQ ID NO: 32) [Chemical Formula 16] DDX20 DNA sequence (SEQ ID NO: 33) [Chemical Formula 17] DDX21 DNA sequence (SEQ ID NO: 34) [Chemical Formula 18] DDX23 DNA sequence (SEQ ID NO: 35) [Chemical Formula 19] DDX24 DNA sequence (SEQ ID NO: 36) [Chemical Formula 20] DDX25 DNA sequence (SEQ ID NO: 37) [Chemical Formula 21] DDX27 DNA sequence (SEQ ID NO: 38) [Chemical Formula 22] DDX28 DNA sequence (SEQ ID NO: 39) [Chemical Formula 23] DDX31 DNA sequence (SEQ ID NO: 40) [Chemical Formula 24] DDX39B DNA sequence (SEQ ID NO: 41) [Chemical Formula 25] DDX41 DNA sequence (SEQ ID NO: 42) [Chemical Formula 26] DDX42 DNA sequence (SEQ ID NO: 43) [Chemical Formula 27] DDX43 DNA sequence (SEQ ID NO: 44) [Chemical Formula 28] DDX46 DNA sequence (SEQ ID NO: 45) [Chemical Formula 29] DDX47 DNA sequence (SEQ ID NO: 46) [Chemical Formula 30] DDX48 DNA sequence (SEQ ID NO: 47) [Chemical Formula 31] DDX49 DNA sequence (SEQ ID NO: 48) [Chemical Formula 32] DDX50 DNA sequence (SEQ ID NO: 49) [Chemical Formula 33] DDX51 DNA sequence (SEQ ID NO: 50) [Chemical Formula 34] DDX52 DNA sequence (SEQ ID NO: 51) [Chemical Formula 35] DDX53 DNA sequence (SEQ ID NO: 52) [Chemical Formula 36] DDX54 DNA sequence (SEQ ID NO: 53) [Chemical Formula 37] DDX55 DNA sequence (SEQ ID NO: 54) [Chemical Formula 38] DDX56 DNA sequence (SEQ ID NO: 55) [Chemical Formula 39] DDX58 DNA sequence (SEQ ID NO: 56) [Chemical Formula 40] DDX59 DNA sequence (SEQ ID NO: 57) 1-2. Methods for determining binding affinity Surface plasmon resonance (SPR) measurements can be performed. For example, an autophagy-inducing compound is immobilized on a gold film on a sensor chip, and then a purified solution of DEAD-box RNA helicase protein is allowed to flow onto the surface of the sensor chip. The SPR signal, which varies according to the binding state of the two compounds, can be measured using BIAcore. Alternatively, the change in thermal stability of the DEAD-box RNA helicase protein due to binding with the autophagy-inducing compound can be detected by thermal displacement measurement called CETSA.

[0048] 1-3. p62 p62 is a receptor protein that directs specific proteins or small cellular organelles to autophagy. p62 is dispersed and localized in the cytoplasm or nucleus, with a half-life of approximately 10 hours, and is primarily degraded via autophagy. p62 possesses multiple domains, including an N-terminal Phox1 and Bem1p domain (PB1), a zinc finger (ZZ), a TRAF6-binding motif (TB), an LC3-interacting region (composed of LIR and DDDWTHL sequences), a Keap1-interacting region (KIR), and a C-terminal ubiquitin-associated domain (UBA). It directs autophagy by interacting with LC3, the autophagosome-localizing protein, in the LC3-interacting region (LIR) (see Non-Patent Literature 13). The amino acid sequence of p62 or the nucleic acid sequence of the SQSTM1 gene encoding it is shown below.

[0049] [Chemical Formula 41] p62 amino acid sequence (SEQ ID NO: 5) [Chemical Formula 42] p62 DNA sequence (SEQ ID NO: 6) 1-4. Methods for detecting the interaction between DEAD-box type RNA helicase and p62 (approximately) In this invention, there are no particular limitations on the method for detecting the interaction (proximity) between DEAD-box type RNA helicase and p62, as long as it is carried out by an available method. For example, NanoLuc (registered trademark) Binary Technology Assays (NanoBiT (registered trademark) Assays) can be used according to the reaction protocol shown below.

[0050] [Chemical Formula 43] (In the formula, LgBiT is the abbreviation for Large BiT, and SmBiT is the abbreviation for Small BiT).

[0051] In this assay, cells expressing modified DEAD-box RNA helicase (SmBiT-DDX or LgBiT-DDX) and p62 (LgBiT-p62 or SmBiT-p62) fused to DEAD-box RNA helicase and p62 respectively, forming complete luciferase peptides, were prepared. When these cells were brought into contact with the test compound, if the test compound brought DEAD-box RNA helicase and p62 closer together, the respective fused luciferase peptides SmBiT and LgBiT associated, restoring their luciferase activity. In this state, the addition of Furimazine, a substrate for luciferase, resulted in strong luminescence, allowing detection of the proximity (interaction) between DEAD-box RNA helicase and p62.

[0052] 1-5. Detection of droplet formation between DEAD-box type RNA helicase and p62. In this application specification, "droplet" refers to a liquid-like aggregate formed by the concentration of DEAD-box type RNA helicase and its associated compounds, as well as p62, and the liquid-liquid phase separation from the outside.

[0053] This "droplet" can be identified by first labeling DEAD-box type RNA helicases and / or p62 with fluorescent markers, then introducing a test compound into the system in which they are present. Preferably, cells expressing at least one of these substances are treated with the test compound, and the presence of the labeled substance is observed under a fluorescence microscope or similar method. Furthermore, for more detailed confirmation of the liquid's properties, it is preferable to combine it with photofading fluorescence recovery assay (FRAP).

[0054] 2. Methods for manufacturing compounds that induce the degradation of target intracellular molecules or organelles by autophagy. By attaching ligands that specifically bind to target intracellular molecules or organelles to compounds obtained through the screening methods described above, compounds that induce the degradation of target intracellular molecules by autophagy can be produced.

[0055] In this specification, "target intracellular molecule or organelle" refers to an intracellular molecule or organelle that becomes the target of degradation induced by autophagy. "Intracellular molecule" refers to a biomolecule, at least a portion of which is present within the cell, including not only naturally occurring biomolecules within the cell but also molecules artificially introduced into the cell or expressed within the cell. Furthermore, "organelle" includes not only naturally occurring organelles within the cell (e.g., dysfunctional mitochondria) but also foreign viruses and microorganisms that invade the cell.

[0056] "Intracellular molecules" can include, for example, lipids, glycolipids, proteins, and glycoproteins present within cells. Typical examples include aggregates of proteins such as β-amyloid (Aβ), tau protein, and α-synuclein. There are no particular restrictions on the cell type in which "intracellular molecules" exist; however, mammalian cells (e.g., mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey, human) are preferred, with human cells receiving the most attention in practical applications. As for intracellular proteins, several reports have already described intracellular proteins associated with pathological conditions (especially those associated with human pathological conditions) as potential targets for practical applications. Such pathologically associated intracellular proteins can be identified, for example, by UniProt or HumanProtein Atlas. A very small number of examples include: Aβ, tau protein, α-synuclein or their aggregates (associated with neurodegenerative diseases such as Alzheimer's and Parkinson's), BRD4 (associated with multiple myeloma, acute myeloid leukemia, etc.), Ras (associated with colorectal cancer, pancreatic cancer, etc.), calcineurin, CyP, FRB, FKBP, PyL, ABI1, GID1, GA1, FKBP F36V, IAA17 TIR1, EGFR, BTK (described in BZ Stanton et al. Science; 359: eaao5902 (2018), N. Shindo et al. Bioorg. Med Chem; 47: 116386 (2021), etc., the contents of which are incorporated herein by reference).

[0057] Additionally, intracellular molecules include, for example, intracellular proteins that can bind to ligands described later, by fusing a specified tag with any target intracellular molecule or organelle using tagging technologies such as HaloTag (registered trademark), SNAP-tag (registered trademark), and CLIP-tag (registered trademark). Intracellular proteins fused with HaloTag (registered trademark) have fusion properties derived from Rhodococcus purpureus (…). Rhodococcus rhodochrous The F272H mutant tag of the dehalogenase DhaA, fused with an intracellular protein containing a SNAP-tag (registered trademark), has O2 derived from human O2. 6 Tags for alkylguanine-DNA alkyltransferase hATG mutants, and tags for E30R mutants of intracellular proteins fused with CLIP-tag (registered trademark). These are outlined, for example, in Halo SNAP CLIP for Protein Labeling - BitesizeBio, the contents of which are incorporated herein by reference.

[0058] In addition, intracellular proteins such as α-synuclein or condensates tagged with mutant FKBP can also become intracellular molecules targeted for autophagy.

[0059] In this specification, "ligands that specifically bind to intracellular molecules or organelles" refers to structural units that constitute part of a compound that induces the degradation of target intracellular molecules or organelles by autophagy and have specific binding activity to target intracellular molecules (including the aforementioned tagged fusion proteins) or organelles. Therefore, substances constituting ligands are substances with specific binding activity to intracellular molecules or organelles, and several such substances have been reported. For example, as a very small subset of cases, as confirmed by BindingDB (https: / / www.bindingdb.org / rwd / bind / index.jsp), the following can be listed: FK506 (calcineurin), cyclosporine A (calcineurin, CyP), FKCsA (CyP, FKBP), rapamycin (FRB, FKBP), FK1012 (FKBP), abscisic acid (PyI, ABI1), gibberellin 3 (GID1, GA1), AP1903 / AP20187 (FKBP F36V), auxin (IAA17 TIR1), afatinib (EGFR), osimertinib (EGFR), dacomitinib (EGFR), neratinib (EGFR), sotoraraxib (EGFR), ibrutinib (BTK), acalabrutinib (BTK), zanubrutinib (BTK), tilabrutinib (BTK), adagraxib (BTK). (The parentheses show the combined objects).

[0060] In addition, examples of ligands targeting BRD4 include: [Chemical Formula 44] , Examples of ligands targeting FKBP12 include: [Chemical Formula 45] , Examples of ligands targeting MetAP2 include: [Chemical Formula 46] .

[0061] Examples of ligands targeting Ras include those described in the following literature: Nature, 2013, 503, 548-551; International Publication No. 2013 / 155223; Science, 2016, 351, 604-608; International Publication No. WO2014152588; International Publication No. 2015 / 054572; International Publication No. 2016 / 049524; Nat. Rev. DrugDiscov., 2014, 13, 828-851; Chem. Soc. Rev., 2016, advance article (DOI:10.1039 / C5CS00911A). Therefore, ligands targeting Ras can be composed of these compounds. Examples of ligands targeting Ras include: [Chemical Formula 47] [Chemical Formula 48] [Chemical Formula 49] [Chemical Formula 50] [Chemical Formula 51] .

[0062] As ligands for intracellular proteins fused with HaloTag (registered trademark), examples include: halogenated C 1-12 Alkyl groups (especially those with halogenated terminal carbons) 1-6 Alkyloxy group, preferably halogenated (especially halogenated terminal carbon) hexyloxy group (especially chlorohexyloxy group).

[0063] Examples of ligands targeting intracellular proteins fused with SNAP-tags (registered trademarks) include: O 6 -Benzylguanine.

[0064] Examples of ligands targeting intracellular proteins fused with CLIP-tags (registered trademarks) include: O 6 -Benzylcytosine.

[0065] It should be noted that the information on ligands for fused tagged intracellular proteins is outlined in Halo SNAP CLIP for Protein Labeling - Bitesize Bio, and its contents are incorporated herein by reference.

[0066] In addition, examples of ligands for target intracellular molecules tagged with mutant FKBP, such as α-synuclein or its condensates, can be listed as follows: [Chemical Formula 52] .

[0067] In this specification, "connector" refers to the atomic group that connects the "ligand" portion of the compound of the present invention to the tag portion that induces autophagy. The connector structure can utilize known connector structures used when connecting molecules with different functions. Furthermore, the structures of connectors connecting intracellular molecules and tag portions that induce autophagy are described in detail in WO2024 / 054876A, Japanese Patent No. 7161760, and non-patent documents 5, 9, and 10; the structures described in these documents can be used.

[0068] In addition, the connection between the "adapter" and the "ligand" and the tag portion can also be carried out using known reactions. For example, it can be based on Japanese Patent No. 7161760; Non-Patent Documents 5, 9, and 10; The 5th Edition of the Experimental Chemistry Lecture (New Experimental Chemistry Lecture), Volumes 13 to 19 (edited by the Chemical Society of Japan); New Experimental Chemistry Lecture (New Experimental Chemistry Lecture), Volumes 14 to 15 (edited by the Chemical Society of Japan); Precise Organic Chemistry (Precise Organic Chemistry) Revised 2nd Edition (L.F. Tietze, Th. Eicher, Nankodo); Revised Edition, Organic Name Reactions - Their Structure and Key Points (Revised Organic Name Reactions - Their Structure and Key Points) (written by Hideo Togo (Hideo Togo), published by Kodansha); ORGANIC SYNTHESES Collective Volume I to VII (John Wiley & Sons Inc); Modern Organic Synthesis in the Laboratory - A Collection of Standard Experimental Procedures (written by Jie Jack Li, published by OXFORD UNIVERSITY); Comprehensive Heterocyclic Chemistry III, Volumes 1 to 14 (Elsevier Japan K.K.); Learning Organic Synthesis Strategies from Name Reactions (translated by Kiyoshi Tomioka (Kiyoshi Tomioka), published by Kagaku Dojin); Comprehensive Organic Transformations (VCH Publishers Inc.), published in 1989, Wiley-Interscience, 2007 edition "Protective Groups in Organic Synthesis, 4th Ed." (written by Theodora W. Greene, Peter G.M. Wuts); Thieme, 2004 edition "Protecting Groups 3rd Ed." (written by P.J. Kocienski), etc. The methods described in these documents are incorporated into this specification by reference.

[0069] The reactions utilized include functional group protection or deprotection reactions, reduction reactions, oxidation reactions, aromatic nucleophilic substitution reactions, azidation reactions of alcohols, alkyl halides, and sulfonates, reductive amination reactions, photoelectrophoresis reactions, esterification reactions, amidation reactions or ureation reactions, coupling reactions, Wohl-Ziegler reactions, sulfonation reactions, hydrolysis reactions, bromination reactions, N-alkylation reactions, hydroxylation reactions via diazotization, deamination reactions, cyclization reactions, carbamate reactions, amidation reactions via carbon monoxide insertion reactions, esterification reactions, click reactions, etc. The reagents and reaction conditions required for these reactions are known to those skilled in the art to which this invention pertains.

[0070] The compounds obtained by the above manufacturing method are connected via a linker to a ligand that specifically binds to a target molecule or organelle and a tag that induces autophagy. Furthermore, the ligands that bind to the target molecule or organelle are replaced according to the target molecule or organelle, thus giving them the advantage of being able to target a wide range of substrates.

[0071] When cells are treated with the compound prepared by the above-described method, in the presence of p62 and in a state where it can contact DEAD-box RNA helicase, autophagy can be induced in a state where the target molecule or organelle is bound to the ligand, thereby degrading the target molecule or organelle. If the compound prepared by the above-described method is brought into contact with DEAD-box RNA helicase within the cell in the presence of p62, a droplet containing the compound, the target molecule or organelle, p62, and DEAD-box RNA helicase is formed. As a result, the septal membrane elongates, forming an autophagosome containing the droplet. The resulting autophagosome fuses with endosomes or lysosomes to digest the contents.

[0072] Therefore, the compounds of the present invention can be used as preventive or therapeutic agents for diseases involving targeted intracellular molecules or organelles. In particular, the compounds of the present invention, based on their AUTAC-based mechanism of action, are effective in preventing or treating any disease involving targeted intracellular molecules or organelles. Application in the treatment or prevention of cancer, inflammatory diseases, autoimmune diseases, and bone / joint degenerative diseases is anticipated. Example

[0073] The present invention will be further described in detail through the following examples, test examples and formulation examples, but they do not limit the present invention, and variations may be made without departing from the scope of the present invention.

[0074] Experiment 1. Screening method for constructing compounds that induce (close to) the interaction between DDX5 and p62. The NanoBiT system was used to determine whether certain compounds or their tag moieties (refer to Japanese Patent No. 7161760) developed by the inventors that degrade target intracellular molecules through autophagy induce the interaction between DDX5 and p62. In this system, the luminescence intensity increases when the test compound induces the interaction between DDX5 and p62, and the presence of compounds with this activity can be detected.

[0075] 1-1. Experimental Materials Production of pLgBiT-p62 and pSmBiT-DDX5 To generate pLgBiT-p62, the pBiT1.1-N [TK / LgBiT] vector (Promega#N2014) was linearized via polymerase chain reaction using oligoprims (5'-gcctccacctgctc-3' and 5'-gctagcagatcttagagtcgg-3'). Oligoprims (dT) were used. 15 Primers were synthesized from total RNA from HeLa cells to form p62 cDNA, which was then amplified using oligoprims (5'-agcggtaggcatggcgtcgctcaccg-3' and 5'-agaagatctgctagtacaacggcgggatgc-3') and inserted into the vector using the In-fusion cloning system (the underlined 15 nt is the In-fusion arm).

[0076] To generate pSmBiT-DDX5, the pBiT2.1-N [TK / SmBiT] vector (Promega#N2014) was linearized via polymerase chain reaction using oligoprims (5'-ggtggctttaccaacagtaccg-3' and 5'-gctagcagatcttctagtcgg-3'). The DDX5 cDNA was then generated using oligo(dT) primers. 15 Primers were synthesized from total RNA from HeLa cells and amplified using oligoprims (5'-agcggtggcatgtcgggattcgagtgaccg-3' and 5'-agaagatctgctagcttattgggaatcctgttggcattg-3'). The primers were then inserted into the vector using the In-fusion cloning system (the underlined 15 nt is the In-fusion arm).

[0077] The following shows the nucleic acid sequences of pLgBiT-p62 and pSmBiT-DDX5 carried by each vector.

[0078] [Chemical Formula 53] The nucleic acid sequence of pLgBiT-p62 (SEQ ID NO: 15) [Chemical Formula 54] The nucleic acid sequence of pSmBiT-DDX5 (SEQ ID NO: 16) Resources and reagents [Table 1] HeLa cells: Riken BRC Cell Bank #RCB0007 DMEM: Fujifilm Wako #043-30085 FBS: Thermo Fisher Scientific #10437028 Non-essential amino acids: Fujifilm Wako #139-15651 Opti-MEM I: Thermo Fisher Scientific #11058-021 96-well culture plate: Promega #E5650 Lipofectamine 3000 transfection reagent: Thermo Fisher Scientific #L3000008 Nano-Glo (registered trademark) live cell reagent: Promega#N2014 GloMax Discover Microplate Reader: Promega #GM3000 PrimeSTAR Max DNA Polymerase: TaKaRa #R045A TRIzol reagent (for total RNA extraction): Thermo Fisher Scientific #15596026 PrimeScript (trademark) RT Kit (for cDNA synthesis): TaKaRa #RR037A In-Fusion HD Cloning Kit: TaKaRa #Z9649N.

[0079] test compounds To verify the screening method of the present invention, the following compounds were used.

[0080] [Chemical Formula 55] AUTAC4-2G and 8-nitro-cGMP are compounds and their tag portions disclosed in Japanese Patent No. 7161760. AUTAC2-2G and FBnG are newly synthesized compounds and their tag portions for verifying whether compounds screened by the method of the present invention actually induce autophagy (the synthesis schemes of FBnG and AUTAC2-2G are as follows).

[0081] FBnG synthesis scheme [Chemical Formula 56] (a) Boc₂O, DMAP, DMSO, rt, 12 h; (b) NaH, THF, 0℃, 1 h, 62% (two steps); (c) 4-Fluorobenzyl alcohol, DEAD, PPh₃, THF, rt, 3 h, 72%; (d) 80% HCO₂H, 80℃, 16 h, 83%. Synthesis scheme of AUTAC2-2G [Chemical Formula 57] (a) Boc2O, CH2Cl2, rt, 3 hours; (b) NaNO2, 20% CH3COOH, rt, 3 hours, 40%; (c) TsCl, (CH3)3N·HCl, NEt3, CH2Cl2, rt, 2 hours, 96%; (d) 4-pyrazoloboronic acid pinacol, K2CO3, DMF, 75℃, overnight, 71%; (e) 6-chloro-9-(4-fluorobenzyl)-8-iodo-N-(tetrahydro-2-) H -pyran-2-yl)-9 H 2-Purine-2-amine, Pd(PPh3)4, K2CO3, 66%, N,N' (f) Dimethyl ethyl urea, 120°C, 2 hours, 61%; (g) 80% HCOOH, 80°C, overnight, 85%; (f) PyBOP, DIPEA, rt, 18 hours, 33%. 1-2. Test sequence [Example 1] Using Lipofectamine 3000 (registered trademark) transfection reagent (Thermo Fisher Scientific Inc.), pLgBiT-p62 and pSmBiT-DDX5 were transfected into HeLa cells proliferating in 96-well plates containing growth medium, as described in the appendix. After adding reagents, and after 24 hours, AUTAC2-2G was added to the wells to a concentration of 10 μM. Cells transfected for 12 hours were then treated with AUTAC2-2G. Subsequently, the growth medium was replaced with 100 μL of Opti-MEM (registered trademark) I (Thermo Fisher Scientific Inc.). 25 μL of 5×Nano-Glo live cell assay containing the substrate Furimazine was added, and luminescence was immediately measured using a GloMax Discover microplate reader (Promega Corporation).

[0082] [Chemical Formula 58] [Example 2] Except for adding AUTAC4-2G to the wells to achieve a concentration of 10 μM and treating cells transfected for 12 hours with AUTAC4-2G, the procedure was the same as in Example 1, and the cells were treated and luminescence was measured.

[0083] [Chemical Formula 59] [Example 3] Except for adding 8-nitro-cGMP to the wells to achieve a concentration of 10 μM and treating the cells 24 hours after transfection with 8-nitro-cGMP, the procedure was the same as in Example 1, and the cells were treated and the luminescence was measured.

[0084] [Chemical Formula 60] [Example 4] Except for adding FBnG to the wells to achieve a concentration of 10 μM and treating the cells 24 hours after transfection with FBnG, the procedure was the same as in Example 1, and the cells were treated and the luminescence was measured.

[0085] [Chemical Formula 61] [Comparative Example 1] Except for adding DMSO (dimethyl sulfoxide) to the wells to a concentration of 10 μM and treating the cells 24 hours after transfection with DMSO, the procedure was the same as in Example 1, and the cells were treated and the luminescence was measured.

[0086] 1-3. Measurement Results Figure 1 The graphs show the luminescence levels 10 minutes after the start of the luminescence measurement in Examples 1-4. Data are expressed as the average of three independent experiments. The upper limit for the graph is 10. 7 The actual luminescence was much greater than indicated. Error bars represent standard deviation. Statistical analysis was performed using the Turkey-Kramer multiple comparison test. A p-value < 0.01 was considered statistically significant.

[0087] Compared with cells treated with DMSO, cells treated with AUTAC2-2G, AUTAC4-2G, 8-nitro-cGMP, and FBnG showed more than a thousand-fold increase in luminescence, confirming that p62 is close to DDX5 in the presence of these compounds, and demonstrating that compounds that make p62 close to DDX5 can be screened by using this method.

[0088] Experiment 2. Additional verification of the screening method for compounds that induce (close to) the interaction between DDX5 and p62. The NanoBiT system was used to determine whether the additional compound developed by the inventors that degrades target intracellular molecules through autophagy (refer to J Med Chem. 2023 Sep 14;66(17):12342-12372) induced the interaction between DDX5 and p62.

[0089] 2-1. Experimental Materials Except for the following compounds used to verify the screening method of the present invention, the test materials used were the same as those used in Test 1 (pLgBiT-p62 and pSmBiT-DDX5, resources and reagents).

[0090] [Chemical Formula 62] The above compounds were synthesized according to the descriptions in Second-Generation AUTACs for Targeted Autophagic Degradation, Daiki Takahashi et al., Journal of Medicinal Chemistry 66 12342-12372.

[0091] 2-2. Test sequence [Examples 5-33] Except for adding compounds 5-33 to the wells to achieve a concentration of 10 μM, and treating the cells 24 hours after transfection with each compound, the procedure was the same as in Example 1, and the cells were treated and the luminescence was measured.

[0092] 2-3. Experimental Results Figure 2 The graphs simultaneously show the luminescence levels 10 minutes after the start of the luminescence assay in Examples 5–33 and the luminescence levels 10 minutes after the start of the assay in transfected cells treated with AUTAC2-2G. Data are expressed as the average of three independent experiments. The upper limit of the graph is 10. 7 The actual luminescence was much greater than indicated. Error bars represent standard deviation. Statistical analysis was performed using the Turkey-Kramer multiple comparison test. A p-value < 0.01 was considered statistically significant.

[0093] Similar to cells treated with AUTAC2-2G, cells treated with compounds 5–33 showed more than a thousand-fold increase in luminescence compared to cells treated with DMSO, confirming that p62 and DDX5 are similar even in the presence of these compounds. Compounds 5–33 were identified by the inventors as inducing autophagy-induced degradation of target intracellular molecules (see Daiki Takahashi et al., Journal of Medicinal Chemistry, 2023 Sep 14;66(17):12342-12372), demonstrating that by screening for compounds that bring p62 and DDX5 similarly, compounds inducing autophagy-induced degradation of target intracellular molecules can be obtained.

[0094] Experiment 3. Constructing a formula that induces (closer to) the interaction between other DEAD-box type RNA helicase members and p62. Screening methods for compounds As a method for detecting compounds or their tag moieties that degrade target intracellular molecules via autophagy, a screening method for detecting compounds that induce interaction with p62 has also been constructed for DEAD-box RNA helicase members other than DDX5. In this system, when the test compound induces the interaction between the DEAD-box RNA helicase member and p62, the luminescence intensity increases, allowing the detection of compounds with this activity.

[0095] 3-1. Experimental Materials Preparation of pLgBiT-DDX (DDX is short for DEAD-box type RNA helicase) and pSmBiT-p62 To generate pLgBiT-DDX, the pBiT1.1-N [TK / LgBiT] vector (Promega#N2014) was linearized via polymerase chain reaction using oligoprims (5'-gcctccacctgctc-3' and 5'-gctagcagatcttagagtcgg-3'). 15Primers were synthesized from total RNA from HeLa cells to form DDX cDNA, which was then amplified using oligoprims (5'-agcggtaggcatggcgtcgctcaccg-3' and 5'-agaagatctgctagtacaacggcgggatgc-3') and inserted into the vector using the In-fusion cloning system (the underlined 15nt indicates the In-fusion arm).

[0096] To generate pSmBiT-p62, the pBiT2.1-N [TK / SmBiT] vector (Promega#N2014) was linearized via polymerase chain reaction using oligoprims (5'-ggtggctttaccaacagtaccg-3' and 5'-gctagcagatcttctagtcgg-3'). The p62 cDNA was then generated using oligo(dT) primers. 15 Primers were synthesized from total RNA from HeLa cells and amplified using oligoprims (5'-agcggtggcatgtcgggattcgagtgaccg-3' and 5'-agaagatctgctagcttattgggaatcctgttggcattg-3'). The primers were then inserted into the vector using the In-fusion cloning system (the underlined 15nt indicates the In-fusion arm).

[0097] The following shows the nucleic acid sequences of pSmBiT-p62 and pLgBiT-DDX carried by each vector.

[0098] [Chemical Formula 63] The nucleic acid sequence of pSmBiT-p62 (SEQ ID NO: 57) [Chemical Formula 64] The nucleic acid sequence of pLgBiT-DDX1 (SEQ ID NO: 58) [Chemical Formula 65] The nucleic acid sequence of pLgBiT-DDX2A (SEQ ID NO: 60) [Chemical Formula 66] The nucleic acid sequence of pLgBiT-DDX3X (SEQ ID NO: 61) [Chemical Formula 67] The nucleic acid sequence of pLgBiT-DDX4 (SEQ ID NO: 62) [Chemical Formula 68] The nucleic acid sequence of pLgBiT-DDX5 (SEQ ID NO: 63) [Chemical Formula 69] The nucleic acid sequence of pLgBiT-DDX6 (SEQ ID NO: 64) [Chemical Formula 70] The nucleic acid sequence of pLgBiT-DDX10 (SEQ ID NO: 65) [Chemical Formula 71] The nucleic acid sequence of pLgBiT-DDX11 (SEQ ID NO: 66) [Chemical Formula 72] The nucleic acid sequence of pLgBiT-DDX17 (SEQ ID NO: 67) [Chemical Formula 73] The nucleic acid sequence of pLgBiT-DDX18 (SEQ ID NO: 68) [Chemical Formula 74] The nucleic acid sequence of pLgBiT-DDX19B (SEQ ID NO: 69) [Chemical Formula 75] The nucleic acid sequence of pLgBiT-DDX20 (SEQ ID NO: 70) [Chemical Formula 76] The nucleic acid sequence of pLgBiT-DDX21 (SEQ ID NO: 71) [Chemical Formula 77] The nucleic acid sequence of pLgBiT-DDX23 (SEQ ID NO: 72) [Chemical Formula 78] The nucleic acid sequence of pLgBiT-DDX24 (SEQ ID NO: 73) [Chemical Formula 79] The nucleic acid sequence of pLgBiT-DDX25 (SEQ ID NO: 74) [Chemical Formula 80] The nucleic acid sequence of pLgBiT-DDX27 (SEQ ID NO: 75) [Chemical Formula 81] The nucleic acid sequence of pLgBiT-DDX28 (SEQ ID NO: 76) [Chemical Formula 82] The nucleic acid sequence of pLgBiT-DDX31 (SEQ ID NO: 77) [Chemical Formula 83] The nucleic acid sequence of pLgBiT-DDX39B (SEQ ID NO: 78) [Chemical Formula 84] The nucleic acid sequence of pLgBiT-DDX41 (SEQ ID NO: 79) [Chemical Formula 85] The nucleic acid sequence of pLgBiT-DDX42 (SEQ ID NO: 80) [Chemical Formula 86] The nucleic acid sequence of pLgBiT-DDX43 (SEQ ID NO: 81) [Chemical Formula 87-1] The nucleic acid sequence of pLgBiT-DDX46 (SEQ ID NO: 82) [Chemical Formula 87-2] [Chemical Formula 88] The nucleic acid sequence of pLgBiT-DDX47 (SEQ ID NO: 83) [Chemical Formula 89] The nucleic acid sequence of pLgBiT-DDX48 (SEQ ID NO: 84) [Chemical Formula 90] The nucleic acid sequence of pLgBiT-DDX49 (SEQ ID NO: 85) [Chemical Formula 91] The nucleic acid sequence of pLgBiT-DDX50 (SEQ ID NO: 86) [Chemical Formula 92] The nucleic acid sequence of pLgBiT-DDX51 (SEQ ID NO: 87) [Chemical Formula 93] The nucleic acid sequence of pLgBiT-DDX52 (SEQ ID NO: 88) [Chemical Formula 94] The nucleic acid sequence of pLgBiT-DDX53 (SEQ ID NO: 89) [Chemical Formula 95] The nucleic acid sequence of pLgBiT-DDX54 (SEQ ID NO: 90) [Chemical Formula 96] The nucleic acid sequence of pLgBiT-DDX55 (SEQ ID NO: 91) [Chemical Formula 97] The nucleic acid sequence of pLgBiT-DDX56 (SEQ ID NO: 92) [Chemical Formula 98] The nucleic acid sequence of pLgBiT-DDX58 (SEQ ID NO: 93) [Chemical Formula 99] The nucleic acid sequence of pLgBiT-DDX59 (SEQ ID NO: 94) Resources and reagents Use the same resources and reagents as in Experiment 1.

[0099] 3-2. Test sequence [Examples 34-70] Using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific Inc.), pLgBiT-DDX (SEQ ID NO: 58-93) and pSmBiT-p62 (SEQ ID NO: 57) were transfected into HeLa cells proliferating in 96-well plates containing growth medium, as described in the appendix. After adding the reagents, and after 24 hours, the test compound was added to the wells to a concentration of 10 μM. Cells transfected for 12 hours were then treated with the test compound. Subsequently, the growth medium was replaced with 100 μL of Opti-MEM (Thermo Fisher Scientific Inc.), and 25 μL of 5× Nano-Glo live cell assay containing the substrate Furimazine was added. Immediately, luminescence assay was performed using a GloMax Discover microplate reader (Promega Corporation), and the luminescence intensity over time was measured.

[0100] Experiment 4. Verification of the autophagy-inducing effect of the screened test compounds To verify the effectiveness of the screening method of the present invention, cells transfected with FKBP12 were treated with the newly synthesized compound AUTAC2-2G to test whether FKBP12 could be degraded.

[0101] 4-1. Modulation of pEGFP-FKBP12 To create pEGFP-FKBP12, pEGFP-HaloTag [Takahashi et al., Mol. Cell 76 797-810.e10 (2019)] was linearized using polymerase chain reaction with oligoprims (5'-cgcaaatctagacttgtacagctcgtcc-3' and 5'-ggccgcttcgagcagatg-3'). FKBP12 cDNA was synthesized from total RNA in HeLa cells using oligo(dT)15 primers, amplified with oligoprims (5'-aagtctagatttgcgatgggagtgcaggtgg-3' and 5'-ctgctcgaagcggcctcattccagttagaagc-3'), and inserted into the vector using the Infusion cloning system (the underlined 15 nt indicates the Infusion arm).

[0102] [Chemical Formula 100] The coding sequence of pEGFP-FKBP12 is as follows (SEQ ID NO: 21).

[0103] 4-2. Resources and Reagents HeLa cells: Riken BRC Cell Bank #RCB0007 Dulbecco's Modified Eagle Medium (DMEM): Fujifilm & Light #043-30085 FBS: Thermo Fisher Scientific #10437028 Non-essential amino acids: Fujifilm Wako #139-15651 Opti-MEM I: Thermo Fisher Scientific #11058-021 96-well culture plate: Thermo Fisher Scientific #165305 Lipofectamine 3000 transfection reagent: Thermo Fisher Scientific #L3000008 Live cell imaging solution: Thermo Fisher Scientific #A14291DJ Fluorescence microscope: KEYENCE #BZ-X800 PrimeSTAR Max DNA Polymerase: TaKaRa #R045A TRIzol reagent (for total RNA extraction): Thermo Fisher Scientific #15596026 PrimeScript (trademark) RT Kit (for cDNA synthesis): TaKaRa #RR037A In-Fusion HD Cloning Kit: TaKaRa #Z9649N.

[0104] 4-3. Test sequence [Example 71] HeLa cells were proliferated in 96-well plates and transfected with pEGFP-FKBP12 using Lipofectamine 3000 transfection reagent. After 24 hours, the transfected cells were further incubated for 24 hours in fresh DMEM containing 0.01 mM AUTAC2-2G and in fresh DMEM containing DMSO as a control. The culture medium was then removed and replaced with 100 μL of live-cell imaging solution. EGFP-FKBP12 levels were analyzed by measuring EGFP fluorescence.

[0105] [Example 72] Except for further incubating the transfected cells in fresh DMEM containing 0.1 mM AUTAC2-2G for 24 hours, the cells were treated and EGFP fluorescence was measured in the same manner as in Example 71.

[0106] [Example 73] Except for further incubating the transfected cells in fresh DMEM containing 1 mM AUTAC2-2G for 24 hours, the cells were treated in the same manner as in Example 71, and EGFP fluorescence was measured.

[0107] [Example 74] Except for further incubating the transfected cells in fresh DMEM containing 10 mM AUTAC2-2G for 24 hours, the cells were treated in the same manner as in Example 71, and EGFP fluorescence was measured.

[0108] [Comparative Example 2] Except for further incubating the transfected cells in fresh DMEM containing DMSO for 24 hours, the cells were treated in the same manner as in Example 72, and EGFP fluorescence was measured.

[0109] 4-4. Measurement Results Figure 3 Shows EGFP fluorescence levels under various conditions. Each value represents the mean EGFP fluorescence level from three independent experiments. Data without identical text were statistically significant using the Turkey-Kramer multiple comparison test. A p-value < 0.01 was considered statistically significant. Data were normalized relative to the DMSO control mean.

[0110] When cells transfected with pEGFP-FKBP12 are cultured in a medium containing AUTAC2-2G, the fluorescence level decreases in a concentration-dependent manner according to AUTAC2-2G, confirming that AUTAC2-2G induces autophagy-based degradation of FKBP12. Therefore, novel compounds that induce autophagy can be explored using the screening method of the present invention.

[0111] Experiment 5. Determine the formation of droplets containing DEAD-box type RNA helicase and p62. [Example 75] Using the newly synthesized compound SLF'-AUTAC, which has been confirmed to degrade synuclein via autophagy, we tested whether it induced the formation of droplets containing DDX5 and p62. The structure and synthetic protocol of SLF'-AUTAC, as well as the synuclein degradation assay, are summarized below.

[0112] SLF'-AUTAC structural formula [Chemical Formula 101] Synthesis scheme of SLF'-AUTAC [Chemical Formula 102] Summary of SLF'-AUTAC-based mutant FKBP-tagged synuclein degradation assay Transient expression of GFP-α-synuclein (GFP-αSyn-FKBP) tagged with mutant FKBP F36V HeLa cells were treated with SLF-AUTAC for 48 hours. GFP fluorescence was captured in wells containing untreated and treated cells using a fluorescence microscope. GFP-αSyn-FKBP was evaluated based on GFP fluorescence intensity. F36V Degradation. For example... Figure 4 As shown in the fluorescence microscopy images, GFP-αSyn-FKBP was detected in cells treated with 1 μM and 10 μM SLF'-AUTAC compared to the untreated group (DMSO). F36V Degradation.

[0113] Test sequence Transient expression of GFP tagged with mutant FKBP (GFP-FKBP) F36V HeLa cells were treated with 1 μM SLF-AUTAC for 12 hours. After treatment, the cells were fixed with 4% paraformaldehyde, then permeated with 0.1% Triton X-100 / PBS, blocked with 1% BSA / PBS, and incubated overnight at 4°C in PBS containing 1% rabbit anti-DDX5 antibody and mouse anti-p62 antibody, respectively. Subsequently, they were incubated for 1 hour at room temperature in PBS containing 1% each of Alexa Fluor 555-labeled anti-rabbit IgG and Alexa Fluor 555-labeled anti-mouse IgG, respectively. The cells after immunostaining were observed under a fluorescence microscope.

[0114] Test results Figure 5Images observed under a fluorescence microscope (scale bar: 10 μm).

[0115] By adding SLF'-AUTAC containing the FBnG structure to HeLa cells, droplets of DDX5 and p62 were observed to form within the cells. In this experiment, after treatment with SLF'-AUTAC, the cells were fixed, and then DDX5 and p62 were observed. DDX5 and p62 were observed at the same location within the cells, thus proving that they exist in the same droplet.

[0116] [Example 76] The AUTAC2-2G test was used to determine whether droplets containing DDX5 and p62 were induced.

[0117] Test sequence HeLa cells transiently expressing GFP-FKBP12 and RFP-DDX5 or GFP-FKBP12 and RFP-p62 were treated with AUTAC2-2G at a concentration of 10 μM for 13 hours. Live-cell observation was performed using a fluorescence microscope, continuously monitoring fluorescence from GFP and RFP. Five minutes after the start of observation, 5% 1,6-hexanediol was added, and the disappearance of punctate structures in the field of view was observed.

[0118] 1,6-Hexanediol is known to dissolve droplets, which can confirm whether the dot-like structures present in the microscope field of view are droplets.

[0119] Test results Figure 6 Images observed under a fluorescence microscope (scale bar: 10 μm). Figure 5 Image A is an image observed under a fluorescence microscope in HeLa cells transiently expressing GFP-FKBP12 and RFP-DDX5 after treatment with AUTAC2-2G. Figure 5 Image B is a fluorescence microscopy image observed in HeLa cells transiently expressing GFP-FKBP12 and RFP-p62 after treatment with AUTAC2-2G.

[0120] Droplets containing GFP-FKBP12 and RFP-DDX5, as well as dot-like structures containing GFP-FKBP12 and RFP-p62, were observed in various HeLa cells. These structures disappeared upon the addition of 1,6-hexanediol, thus confirming them as droplets.

Claims

1. A method for screening autophagy-inducing compounds, among which... The test compound was brought into contact with a DEAD-box type RNA helicase, and the binding affinity of the test compound to the DEAD-box type RNA helicase was determined. Choose compounds with a binding affinity above the specified value.

2. Methods for screening autophagy-inducing compounds, among which... In the presence of p62, the test compound was brought into contact with a DEAD-box type RNA helicase. The interaction between the DEAD-box type RNA helicase and p62 was determined. The compound that induced the interaction was selected.

3. The method of claim 2, wherein the DEAD-box type RNA helicase and the p62 are expressed in cells, thereby contacting the test compound with the cells.

4. Methods for screening autophagy-inducing compounds, among which... The test compound was introduced in the presence of p62 and DEAD-box type RNA helicase. The formation of droplets containing the DEAD-box type RNA helicase and the p62 was determined. The compound that induced the formation of the droplets was selected.

5. The method according to any one of claims 1 to 4, wherein the DEAD-box type RNA helicase is DDX5 or DDX17.

6. A method for manufacturing a compound that induces the degradation of target intracellular molecules or organelles by autophagy, comprising: The steps of implementing the method according to any one of claims 1 to 5; as well as The step of attaching a ligand to the resulting compound via a linker to bind specifically to a molecule or organelle within the target cell.

7. The method of claim 6, further comprising: The step of contacting a compound with the ligand attached to a cell having the target intracellular molecule or organelle to confirm the degradation of the target intracellular molecule or organelle by autophagy.

8. A method for inducing the degradation of target intracellular molecules or target organelles by autophagy by contacting a compound obtained by the method of claim 6 or 7 with a DEAD-box type RNA helicase in the presence of p62 within cells.

9. The method of claim 8, wherein the compound obtained by the method of claim 6 or 7 is contacted intracellularly with a DEAD-box RNA helicase in the presence of p62 to form a droplet comprising the compound, the p62 and the DEAD-box RNA helicase.

10. The method according to claim 8 or 9, for the prevention or treatment of cancer or cellular degenerative diseases.

Images