CN112047935A

CN112047935A - Autophagy targeting protein degradation technology and application thereof

Info

Publication number: CN112047935A
Application number: CN201910486769.1A
Authority: CN
Inventors: 汪义朋
Original assignee: Shanghai Qiangrei Biotechnology Co ltd
Current assignee: Wang Yipeng
Priority date: 2019-06-05
Filing date: 2019-06-05
Publication date: 2020-12-08
Anticipated expiration: 2039-06-05
Also published as: CN112047935B

Abstract

The invention relates to the technical field of targeted protein degradation, discloses an autophagy targeted protein degradation technology and application thereof, and particularly relates to an autophagy targeted chimera, application thereof and a targeted protein degradation method for degrading a target protein by autophagy. The method utilizes an autophagy targeting chimera to mediate degradation of a target protein through autophagy, wherein the autophagy targeting chimera is a bifunctional molecule and has a chemical structure of TBM-L-ABM or a pharmaceutically acceptable salt, enantiomer, stereoisomer, solvate, polymorph or N-oxide thereof, TBM is a target protein binding part, L is a connector group, ABM is an autophagy receptor binding part, and the target protein binding part is connected with the autophagy receptor binding part through the connector group. The autophagy targeting chimera disclosed by the invention can mediate various proteins needing to be degraded in an organism to be degraded through autophagy.

Description

Autophagy targeting protein degradation technology and application thereof

Technical Field

The invention relates to the technical field of targeted protein degradation, in particular to an autophagy targeted protein degradation technology and application thereof, and more particularly relates to an autophagy targeted chimera and application thereof as well as a targeted protein degradation method for degrading a target protein by autophagy.

Background

A targeted protein degradation technique for selective degradation of target proteins using protein degradation Targeting Chimera (PROTAC) is a new technique developed in recent years (see Neklesa TK, Winkler JD, Crews CM. targeted protein degradation by PROTACs. Pharmacol Ther.2017 Jun; 174: 138-. ProTAC is a bifunctional molecule with one end capable of specifically binding to a target protein and the other end specifically binding to a specific ubiquitin ligase, which are connected via a linker group (linker). ProTAC can simultaneously bind a target protein and ubiquitin ligase to bring the target protein into proximity with the ubiquitin ligase, thereby enhancing ubiquitination of the target protein for eventual degradation by Proteasome (proteosomes) (see Neklesa TK, Winkler JD, Crews CM. targeted protein degradation by PROTACs. Pharmacol Ther.2017 Jun; 174: 138-. ProTAC is considered to be a potential alternative to monoclonal antibodies for the next best therapy because it is selective for the target protein, acts on many traditionally poorly druggable targets, and can be reused in cells with catalytic-like effects, thus eliminating the need for high concentrations (refer to Desheies RJ. protein degradation: Prime time for ProTACs. Nat Chem biol. 2015Sep; 11(9):634-5, the entire contents of the corresponding parts are incorporated herein by reference). However, ProTAC also has the following disadvantages or application limitations: (1) PROTAC recruits target proteins to the vicinity of ubiquitin ligase, but this is sometimes not sufficient to cause target proteins to become ubiquitinated, since ubiquitination depends on the spatial structure of the ternary complex formed by the target protein, the PROTAC and the ubiquitin ligase. The length of the linker in ProTAC has a crucial effect on its activity. At present, it is still very challenging to construct a well functioning PROTAC. (2) Even if PROTAC mediates ubiquitination of a target protein due to the presence of deubiquitinase within a cell, if the ubiquitinated target protein is not degraded in time, it may be deubiquitinated by deubiquitinase and eventually not degraded by proteasome. (3) The polymer of the protein cannot be degraded by proteasomes, and some oligomers of the protein even have toxic effects on proteasomes, so that PROTAC cannot be used to directly degrade the protein polymer. (4) In some pathological situations, proteasome itself is not able to efficiently degrade proteins due to impaired function, and thus PROTAC is not used to enhance target protein clearance.

In addition to proteases, another pathway of protein degradation within cells is autophagy (see gateway D, Lahiri V, Klionsky DJ. Cargo recognition and degradation by selective autophagy. Nat. Cell biol.2018 Mar; 20(3):233-242, the entire contents of the corresponding sections of which are incorporated herein by reference). Unlike proteasomes, autophagy degrades protein polymers and even organelles. Autophagy is both nonselective and selective (see, gateway D, Lahiri V, Klionsky DJ. Cargo recognition and degradation by selective autophagy. Nat Cell biol.2018 Mar; 20(3): 233-. Nonselective autophagy is a reaction of cells in the absence of nutrients, and is characterized by random uptake of a portion of the cytoplasm into phagocytes (phagophores), subsequent formation of phagosomes (autophagosomes), and finally, binding of the phagosomes to lysosomes (lysosomes), degradation of the contained cargo, provision of amino acids, etc., necessary for protein synthesis to the cells. Selective autophagy is a way for cells to select for degradation of Aggregation-prone, abnormally folded proteins (aggregate-Protein misfolded proteins), Protein polymers (Protein aggregates), or damaged organelles. In which autophagy, which selectively degrades abnormally folded proteins (Aggregation-Protein misfolded proteins) or Protein polymers (Protein aggregates), is referred to as autophagy (agrephhagy) (see gateway D, Lahiri V, Klionsky DJ. Cargorgement and degradation by selective autophagy. Nat. Cell biol.2018 Mar; 20(3):233-242, the entire contents of the respective sections being incorporated herein by reference). Selective autophagy is mediated by autophagy receptors such as P62, NBR1 and OPTN, among others. The C-terminal UBA (ubiquitin associated) segment of an autophagy receptor (e.g., P62) binds to a Cargo (Cargo), while its LC3 active segment (LC3-interacting region motif, LIR) binds to a protein of the ATG8 family (e.g., LC3) that is covalently attached to the surface of the growing phagocytic vesicle's intimal membrane. Phagocytic bleb formation relies on the aggregation of autophagy receptors loaded with a load to form sufficiently large structures. The phagocytic vesicles eventually close, forming phagosomes (autophagosomes) with a double-layered membrane structure. Phagosomes bind to lysosomes (lysomes) and cause degradation of the target proteins contained therein, autophagy receptors and LC3, among other proteases, by lysosomes (see, Gatica D, Lahiri V, Klionsky dj.cartoon recognition and degradation by selective autophagy. nat Cell biol.2018mar; 20(3):233-242, the entire contents of the corresponding portions of which are incorporated herein by reference). To date, no report has been made on techniques for selectively degrading a specific target protein using autophagy.

Alzheimer's Disease (AD) is the most common dementia, accounting for about 50-70% of dementia. Statistically, in 2016, there are about 1000 million AD patients in China and 4400 million AD patients in the world. With the aging population, the incidence of AD will rise further, with an expectation that about 4000 million AD patients will be present in china by the year 2050. At present, all the drugs for treating AD are characteristic drugs (Symptomatic drugs), and can only relieve symptoms temporarily but cannot delay the progress of the disease. There is a global need for new drugs (Disease-modifying drugs) that can actually change the progress of AD.

Two characteristic pathological changes in AD are Senile Plaques (SPs) and Neuronal Fibrillar Tangles (NFTs), which are polymers of beta-amyloid (a β) and highly phosphorylated tau proteins, respectively, in which tauopathy, but not a β lesions, are positively correlated with dementia in AD (see Wang Y, mantelkow e. tau in physiology and pathology. nat Rev neurosci.2016 Jan; 17(1):5-21, the entire contents of which are incorporated herein by reference). Recent studies have shown that tau mediates A β -induced neurotoxicity, necessary for A β neurotoxicity (see Roberson ED, research-Levie K, palap JJ, Yan F, Cheng IH, Wu T, Gerstein H, Yu GQ, Mucke L. reducing endogenes tau amides beta-induced deficits in Alzheimer's disease mouse model 2007. May 4; 316(5825):750-4, the entire contents of the corresponding parts being incorporated herein by reference); and Tau is a Prion-like protein (Prion-like protein) (see Goedert M, Eisenberg DS, crown RA. propagation of Tau Aggregates and neuro-genesis. Annu Rev neurosci.2017 Jul 25; 40:189-210, the entire contents of the corresponding parts of which are incorporated herein by reference), and is capable of propagating between neurons to cause the spread of tauopathy, suggesting that it may be a necessary drug target for AD.

In addition to AD, tau aggregation is also seen in frontotemporal dementia (frontotemporal dementia, FTDP-17) linked to chromosome 17 with Parkinson's Disease, Pick's Disease (PiD), Progressive Supranuclear Palsy (PSP), corticobasal degeneration (CBD), primary age-related tauopathy (PART), argyrophilia (argentati) granule Disease, AGD), aging-related tauopathies (AG), chronic traumatic encephalopathy (chronic ischemic encephalopathy, Huntington), glial cell tau (GLUCE), and Parkinson's Disease (HD), Parkinson's Disease, etc. Such diseases including AD are collectively referred to as tau diseases (Tauopathies). tau protein is an important cause of such diseases and therefore an important therapeutic target for such diseases.

Currently, although there are a number of tau-based therapeutic proposals, one of the most attractive approaches is to reduce the amount of intracellular tau protein. This solution is favored mainly for the following reasons: (1) there is a great deal of evidence that reducing the level of intracellular tau protein causes fewer side effects in animal models; (2) reducing the content of tau protein can inhibit the aggregation of tau protein, which is an important reason for causing neuron degeneration; (3) reducing the level of tau protein reduces the effects of neuronal excitotoxicity caused by a number of factors, such as a β. Therefore, reducing tau protein is also considered as a new potential treatment for epilepsy and stroke.

There are two common techniques for reducing intracellular target proteins. (1) The expression of the target protein is reduced with siRNA, miRNA or antisense oligonucleotide. Because of the poor distribution of these oligonucleotides in tissues, the poor pharmacokinetics, and the potential for off-target, their clinical use is currently limited and still further improvements are desired. (2) Enhancing the degradation of the target protein. A common approach is to enhance the activity of protein degradation systems, including protease systems and autophagy systems. However, since the non-specific enhancement of the activity of the protein degradation system is likely to cause the degradation of other non-target proteins to cause serious side effects, no drug activating the protein degradation system is approved for clinical application at present. It is desirable to selectively enhance the degradation of only the target protein while avoiding degradation of non-target proteins by enhancing the activity of the protein degradation system.

Disclosure of Invention

The invention aims to construct an autophagy targeting protein degradation technology. Recent studies have shown that the ZZ segment of the autophagy receptor P62, when bound to a specific ligand, promotes aggregation and increases phagocyte formation, thereby enhancing autophagy (see Cha-Molstad H, Yu JE, Feng Z, Lee SH, Kim JG, Yang P, Han B, Sung KW, Yoo YD, Hwang J, McGuire T, Shim SM, Song HD, Ganipisetti S, Wang N, Jang JM, Lee MJ, Kim SJ, Lee KH, Hong JT, Ciechanover A, Mook-Jung I, Kim KP, Xie XQ, Kwon TYm BY. p62/SQSTM1/Sequestosome-1is N-log of the N-endcanal waist tissue half 2018, Nature 102, incorporated herein by reference). In order to achieve the above object, the present inventors have found through intensive studies that a bifunctional molecular compound having one end capable of specifically binding to a target protein and the other end specifically binding to an autophagy receptor, which are linked via a linker group (linker), can be constructed to selectively degrade the target protein by autophagy. The binding of the bifunctional molecule to the autophagy receptor can promote the aggregation of the autophagy receptor and the binding of the autophagy receptor to LC3, so that the autophagy receptor and a load thereof and a target protein connected with the autophagy receptor through a connector are transported to an autophagy corpuscle (Autophagosome), and finally the autophagy corpuscle is bound with a lysosome, so that the target protein is degraded. Unlike protein degradation TArgeting Chimeras (PROTAC), which mediate degradation of a target protein by proteasomes, this bifunctional molecule mediates degradation of the target protein by AUtophagy, and we therefore refer to it as an AUtophagy TArgeting Chimera (AUTAC).

Similar to the ProTAC technology, AUTAC technology has another advantage of being able to act on many traditionally hard-to-use targets, in addition to its selectivity for target proteins. Many conventional small molecule drugs must act on specific binding pockets of the target protein in order to exert an inhibitory effect. The AUTAC technology is not limited as long as it can interact with any segment of the target protein and does not need high affinity, so that the AUTAC technology can lead to the degradation of the target protein to inhibit the function of the target protein, and can act on many targets which are difficult to prepare drugs conventionally. Compared with ProTAC, AUTAC has the advantage of directly mediating the degradation of protein polymers.

To this end, the present invention provides a targeted protein degradation method for degrading a target protein by autophagy, which mediates the degradation of the target protein by autophagy using an autophagy-targeting chimera, which is a bifunctional molecule having a chemical structure of TBM-L-ABM or a pharmaceutically acceptable salt, enantiomer, stereoisomer, solvate, polymorph or N-oxide thereof, wherein TBM is a target protein binding moiety, L is a linker group, and ABM is an autophagy receptor binding moiety, and the target protein binding moiety and the autophagy receptor binding moiety are linked by a linker.

The invention also provides an autophagy targeting chimera, which has a chemical structure of TBM-L-ABM or pharmaceutically acceptable salts, enantiomers, stereoisomers, solvates, polymorphs or N-oxides thereof, wherein TBM is a target protein binding part, L is a linker group, ABM is an autophagy receptor binding part, and the target protein binding part and the autophagy receptor binding part are connected through the linker group.

Optionally, the target protein to which the TBM is capable of binding is tau protein, alpha-synuclein, polyglutamine proteins including huntingtin, copper/zinc superoxide dismutase, TDP-43, C9orf72, FUS, or a polymer of one or more thereof.

Alternatively, the autophagy receptor to which ABM binds is P62, NBR1, OPTN, CALCOCO2/NDP₅₂TAX1BP1, NIX, BNIP3, FUNDC1, Bcl2L13 or FKBP 8.

Preferably, the autophagy receptor P62 is a ZZ segment of P62, and the amino acid sequence of the ZZ segment of P62 is shown in SEQ ID No. 1.

In a preferred embodiment, ABM is a group having a structure represented by formula (1),

wherein R is¹And R²Is H or C1-C4 alkyl;

R³is-R⁴-M-, ABM is linked to a linker group L through M, wherein R⁴is-O-or C1-C4 alkylene, M is a bond, C1-C4 alkylene, -NH-or-R⁵-CH(OH)-R⁶-NH-R⁷-, wherein R⁵、R⁶And R⁷Is C1-C4 alkylene.

In a preferred embodiment, L is a group-X-Y-Z-, X is attached to TBM, Z is attached to ABM,

wherein X is a bond, alkylene of C1-C4, or-NH-;

y is-R⁸-(R¹⁰-E-R¹¹)_n-R⁹-, wherein R⁸And R⁹Each being a bond or alkylene of C1-C8, R¹⁰And R¹¹Each is C1-C4 alkylene, n is an integer of 0-10, E is O, S, amido, piperazinyl, NR¹²、S(O)、S(O)₂、-S(O)₂O、-OS(O)₂、OS(O)₂O、

Wherein E¹Is O, S, CHR¹²Or NR¹²，R¹²Is H orC1-C3 alkyl optionally substituted with one or two hydroxy groups;

z is-A-B-wherein A is a bond, O or S, B is a bond, C1-C4 alkylene or-NH-R¹³-, wherein R¹³Is C1-C4 alkylene.

In a preferred embodiment, TBM is a group having a structure shown in formula (2), or a group further modified by a substituent group at the position of (c), (,

wherein R is¹⁴Is C1-C4 alkylene, R¹⁵And R¹⁶Each is C1-C4 alkyl, R¹⁷Is a bond, H, C1-C4 alkyl or-R¹⁸-O-, wherein R¹⁸Is C1-C4 alkylene.

In a preferred embodiment, the structures of the autophagy-targeting chimera compound 1 and compound 2 are as follows.

Compound 1

Compound 2

The invention also provides a targeted protein degradation method for degrading a target protein by utilizing autophagy, which comprises the following steps: the degradation of the target protein by autophagy is mediated by an autophagy targeting chimera, which is the autophagy targeting chimera described above.

The invention also provides a method of degrading tau protein in a patient in need thereof comprising administering to the patient an effective amount of an autophagy-targeting chimera as described above.

In a preferred embodiment, the autophagy-targeting chimera is administered to the patient by at least one means selected from the group consisting of: nasal, inhalation, topical, oral, intramuscular, subcutaneous, transdermal, intraperitoneal, epidural, intrathecal and intravenous routes.

The invention also provides application of the autophagy targeting chimera in preparation of a medicine for treating or preventing tau protein related diseases.

Optionally, the disease is at least one of alzheimer's disease, frontotemporal dementia with parkinson's disease linked to chromosome 17, pick's disease, progressive supranuclear palsy, corticobasal degeneration, primary age-related tauopathies, silvery particle disease, aging-related tau astrocytosis, chronic traumatic encephalopathy, globoid tauopathy, parkinson's disease, huntington's disease, stroke, and epilepsy.

The autophagy targeting protein degradation method provided by the invention is characterized in that autophagy target chimera is utilized to mediate target protein to degrade through autophagy. One end of the autophagy targeting chimera can be specifically combined with a target protein, the other end of the autophagy targeting chimera is specifically combined with an autophagy receptor, and the autophagy targeting chimera and the autophagy receptor are connected through a connecting body group. The binding of the autophagy targeting chimera to the autophagy receptor promotes the aggregation of the autophagy receptor and the binding to LC3, thereby causing the transport of the autophagy receptor and its cargo to its target protein linked via a linker to autophagosome, which finally binds to lysosomes, resulting in the degradation of the target protein.

The autophagy targeting chimera compound 1 and compound 2 for tau protein constructed by the invention are proved to be capable of enhancing the degradation of tau protein in cells through an immunoblotting test, so that the content of tau protein is reduced. In addition, subcutaneous injection of the autophagy-targeting chimeric into normal mice significantly reduced the amount of tau protein in the mouse brain. Therefore, the autophagy targeting chimera provided by the invention can mediate autophagy degradation of the target protein, and can play a role in preventing and treating a series of tau diseases including Alzheimer disease.

Drawings

Figure 1 shows the mechanism by which autophagy targeting chimera (AUTAC) mediates the selective degradation of target proteins by autophagy;

FIG. 2 is a nuclear magnetic resonance spectrum of compound C090019 prepared in example 1 of the present invention;

FIG. 3 is a nuclear magnetic resonance spectrum of Compound C080019 prepared in example 2 of the present invention;

FIG. 4 results of immunoblot hybridization (a) and semi-quantitative analysis (b) of the degradation of intracellular tau protein using different concentrations of the compound C090019 provided by the present invention;

FIG. 5 results of immunoblot hybridization (a) and semi-quantitative analysis (b) of the degradation of intracellular tau protein using different concentrations of compound C080019 provided by the present invention;

FIG. 6 results of immunoblot hybridization (a, C) and semi-quantitative analysis (b, d) of the effect of subcutaneous injection of compound C090019 provided by the present invention on tau content in cerebral cortex and hippocampus of mice.

Detailed Description

The following detailed description of embodiments of the invention refers to the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

The invention provides a targeted protein degradation method for degrading a target protein by autophagy, which utilizes an autophagy targeting chimera to mediate the degradation of the target protein by autophagy, wherein the autophagy targeting chimera is a bifunctional molecule, and the chemical structure of the bifunctional molecule is TBM-L-ABM or pharmaceutically acceptable salt, enantiomer, stereoisomer, solvate, polymorph or N-oxide thereof, wherein TBM is a target protein binding part, L is a connector group, ABM is an autophagy receptor binding part, and the target protein binding part is connected with the autophagy receptor binding part through the connector group.

Figure 1 shows the mechanism by which autophagy targeting chimera (AUTAC) mediates the selective degradation of target proteins by autophagy. Referring to fig. 1, the chemical structure of the autophagy targeting chimera of the present invention is TBM-L-ABM or a pharmaceutically acceptable salt, enantiomer, stereoisomer, solvate, polymorph or N-oxide thereof, wherein TBM is a target protein binding moiety, L is a linker group, ABM is an autophagy receptor binding moiety, and the target protein binding moiety and the autophagy receptor binding moiety are linked through a linker group.

The autophagy targeting chimeras can mediate autophagy degradation of various proteins in an organism which need to be degraded.

Alternatively, the target protein to which the TBM is capable of binding is tau protein, alpha-synuclein (NP _000336.1), Polyglutamine proteins (Polyglutamine proteins) including Huntington protein (NP _002102.4), copper/zinc superoxide dismutase (Cu/Zn superoxide dismutase (SOD1)) (NP _000445.1), TDP-43(TAR DNA-binding protein 43) (NP _031401.1), C9orf72(NP _001242983.1), FUS (fused sarcoma, fused in sarcoma), or a polymer of one or more thereof.

Alternatively, the autophagy receptors to which ABM can bind are P62(SQSTM1, sequenstosome 1) (AAH17222.1), NBR1 (the gene adjacent to the BRCA1gene, Neighbor of BRCA1gene) (EAW60953.1), OPTN (optinin) (NP-001008213.1), CALCOCO2 (calcium binding and coild-coil domain 2)/NDP₅₂(AAH15893.1), TAX1BP1, NIX (AAD03589.1), BNIP3(Bcl 2-related immortalized Gene, Bcl2-associated alkane) (AAH01936.2), FUNDC1 (NP-776155.1), Bcl2L13(Gene ID:23786), FKBP8(AAQ 84561.1).

Preferably, the autophagy receptor to which the ABM is capable of binding is the ZZ segment of P62, and the amino acid sequence of the ZZ segment of P62 is shown in seq.id No. 1.

SEQ.ID.NO:1：

Cys Asp Gly Cys Asn Gly Pro Val Val Gly Thr Arg Tyr Lys Cys Ser Val Cys Pro Asp Tyr Asp Leu Cys Ser Val Cys Glu Gly Lys Gly Leu His Arg Gly His

In a more preferred embodiment, ABM is a group having a structure represented by formula (1),

wherein R is¹And R²H or C1-C4 alkyl (such as methyl, ethyl, n-propyl, isopropyl or n-butyl and isomers thereof);

R³is-R⁴-M-, ABM is linked to a linker group L via "-M-", wherein R is⁴is-O-, C1-C4 alkylene (e.g., methylene, ethylene, propylene or butylene), M is a bond, C1-C4 alkylene (e.g., methylene, ethylene, propylene or butylene), -NH-, or-R⁵-CH(OH)-R⁶-NH-R⁷-, wherein R⁵、R⁶And R⁷Is C1-C4 alkylene (such as methylene, ethylene, propylene or butylene). When "-M-" is-R⁵-CH(OH)-R⁶-NH-R⁷When is, "-R⁷- "is directly attached to the linker group L.

Further preferably, in the structure represented by the formula (1), R¹And R²Is H, R³is-R⁴-NH-or-O-R⁵-CH(OH)-R⁶-NH-R⁷-, in which R⁴、R⁵And R⁶Is methylene, R⁷Is an ethylene group.

In a more preferred embodiment, L is a group-X-Y-Z-, X is attached to TBM and Z is attached to ABM.

Wherein X is a bond, an alkylene group of C1 to C4 (e.g., methylene, ethylene, propylene, or butylene), or-NH-.

Y is-R⁸-(R¹⁰-E-R¹¹)_n-R⁹-, wherein R⁸And R⁹Each independently a bond or C1-C8 alkylene (e.g. methylene, ethylene, propylene, butylene, pentylene)Hexyl, heptylene or octylene), R¹⁰And R¹¹Each is C1-C4 alkylene (e.g., methylene, ethylene, propylene or butylene), n is an integer from 0 to 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9or 10), E is O, S, amido, piperazinyl, NR is¹²、S(O)、S(O)₂、-S(O)₂O、-OS(O)₂、OS(O)₂O、

Wherein E¹Is O, S, CHR¹²Or NR¹²，R¹²Is H or C1-C3 alkyl optionally substituted with one or two hydroxy groups;

In a more preferred embodiment, the TBM is a group having a structure represented by formula (2), or a group further modified by a substituent group at the position of (c), (c,

wherein R is¹⁴Is C1-C4 alkylene (e.g. methylene, ethylene, propylene or butylene), R¹⁵And R¹⁶Each C1-C4 alkyl (such as methyl, ethyl, propyl, n-butyl or isobutyl), R¹⁷Is a bond, H, C1-C4 alkyl (such as methyl, ethyl, propyl or butyl) or-R¹⁸-O-, wherein R¹⁸Is C1-C4 alkylene (such as methylene, ethylene, propylene or butylene).

Wherein, the substituent group at the position of (r), (C), (d), (C) or (C) in the group of the modified structure shown in formula (2) can be halogen (such as fluorine or chlorine), alkyl (such as methyl, ethyl, propyl or butyl) of C1-C4, alkoxy (such as methoxy, ethoxy, propoxy or butoxy) of C1-C4, carboxyl, amino, aryl (such as phenyl) of C6-C18 or benzyl.

Further preferably, in the structure of formula (2), TBM is attached to the linker group L through the position of the fifth in formula (2), and R is¹⁴Is ethylene, R¹⁵And R¹⁶Each is methyl, R¹⁷Is a bond, methylene or-CH₂CH₂-O-。

In the invention, by constructing the autophagy targeting chimera compound 1 and compound 2 aiming at tau protein, the autophagy targeting chimera of the invention can be verified to degrade target protein by utilizing autophagy specificity, and the structures of the compound 1 and the compound 2 are as follows.

Compound 1

Compound 2

The autophagy targeting chimera compound 1 and compound 2 are named as C090019 and C080019 respectively, and can be prepared according to the following process routes:

(1) intermediate substances compound a and compound B synthetic route:

synthetic route of compound a:

synthetic route of compound B:

(2) synthetic route of compound 1:

(3) synthetic route of compound 2:

the specific preparation process of the intermediate compound A and the intermediate compound B comprises the following steps:

(1) preparation of Compound A

(1.1) preparation of Compound 3-2

Dissolving compound 3-1 and Triethylamine (TEA) in Tetrahydrofuran (THF) at 0-5 deg.C, adding 1- (1, 4-diazepan-1-yl) ethanone, stirring the mixture at 0-5 deg.C for 1-1.5h, and stirring at room temperature for 1-1.5 h. After completion of the reaction, the reaction mixture was concentrated and purified by column chromatography to obtain compound 3-2 (white solid).

(1.2) preparation of Compound A

Mixing the compound 3-2, 2- (thiophene-2-yl) ethylamine and K at 70-90 deg.C₂CO₃Soluble in methyl nitrile (CH)₃CN), stirring overnight. After the reaction is complete, K is filtered off₂CO₃And the organic layer was concentrated and purified by column chromatography to give compound a (white solid).

(2) Preparation of Compound B

At 70-90 deg.C, compound 3-3, 2- (chloromethyl)Ethylene oxide and K₂CO₃Soluble in methyl nitrile (CH)₃CN), stirring overnight. After the reaction is complete, K is filtered off₂CO₃And the organic layer was concentrated and purified by column chromatography to give compound B (colorless oil).

The specific preparation process of the compound 1 comprises the following steps:

(1) preparation of Compound 4-2

Dissolving compound 4-1 and Triethylamine (TEA) in Dichloromethane (DCM) at 0-5 deg.C, adding MsCl, stirring the resulting mixture at 0-5 deg.C for 10-15min, and heating to room temperature for 1-1.5 h. After completion of the reaction, the resulting mixture was concentrated, and the resulting residue was purified by column chromatography using petroleum ether/ethyl acetate (PE/EA) to give compound 4-2 (colorless oil).

(2) Preparation of Compounds 4-3

Compound 4-2 and KI were dissolved in acetone at 70-90 ℃ and stirred overnight. After completion of the reaction, the reaction mixture was concentrated, and the resulting residue was purified by column chromatography using petroleum ether/ethyl acetate (PE/EA) to give compound 4-3 (colorless oil).

(3) Preparation of Compound 4-4

Dissolving compound 4-3 in Tetrahydrofuran (THF), adding NaH, stirring at 0-5 deg.C for 20-40min, adding compound C, and stirring the resulting mixture at room temperature overnight. After the reaction is finished, the solution is treated with H₂Quench O and extract with Ethyl Acetate (EA). The organic layer was dried and concentrated in vacuo, and the residue was purified by silica gel chromatography using 5-10% methanol/dichloromethane (MeOH/DCM) to give compound 4-4 (yellow solid).

(4) Preparation of Compounds 4-5

Dissolving compound 4-4 in methanol (MeOH), adding Pd/C, and mixing the obtained mixture at 20-30 deg.C and H₂Stirring for 6-10h under protection. After the reaction was complete, the resulting mixture was filtered, the filtrate was concentrated in vacuo, and the residue was purified by silica gel chromatography using 5-20% methanol/dichloromethane (MeOH/DCM) to give compounds 4-5 (colorless oil).

(5) Preparation of Compounds 4-6

At 70-90 deg.C, mixing compound 4-5, compound A and compound K₂CO₃Dissolved in CH₃CN (methylnitrile), stirred overnight. After the reaction is complete, K is filtered off₂CO₃And the organic layer was concentrated and purified by prep-TLC using methanol/dichloromethane (MeOH/DCM) to give compounds 4-6 (white solids).

(6) Preparation of Compounds 4-7

The compound 4-6 is dissolved in dioxane, HCl/dioxane is added, and the mixture is stirred at room temperature for 3-5 h. After completion of the reaction, the solution obtained was concentrated in vacuo and the residue was taken up with Na₂CO₃(aq), extracted with Ethyl Acetate (EA), the organic layer dried and concentrated in vacuo, and the residue purified by silica gel chromatography using 5-10% methanol/dichloromethane (MeOH/DCM) to give compounds 4-7 (yellow solids).

(7) Preparation of Compound 1 (target Compound "C090019")

Dissolving compound 4-7 and compound B in CH at 60-70 deg.C₃OH, stirring overnight. After the reaction was complete, the solvent was removed and the residue was purified by prep-TLC and prep-HPLC to give compound 1 (brown oil).

The specific preparation process of the compound 2 comprises the following steps:

(1) preparation of Compound 5-2

At 20-30 deg.C, mixing compound 5-1 and carbon tetrabromide (CBr)₄) And triphenylphosphine (PPh3) was dissolved in Tetrahydrofuran (THF) and stirred overnight. After completion of the reaction, the solution was concentrated and purified by column chromatography using petroleum ether/ethyl acetate (PE/EA) to give compound 5-2 (yellow oil).

(2) Preparation of Compounds 5-3

Compound 5-2 was dissolved in Tetrahydrofuran (THF), NaH was added, stirring was carried out at 0-5 ℃ for 20-40min, then compound C was added, and the resulting mixture was stirred at room temperature overnight. After the reaction is completed, the solution is treated with H₂Quench O and extract with Ethyl Acetate (EA), dry the organic layer and concentrate in vacuo, purify the residue by silica gel chromatography using 5-10% methanol/dichloromethane (MeOH/DCM) to give compound 5-3 (yellow oil).

(3) Preparation of Compounds 5-4

Dissolving compound 5-3 in methanol (MeOH), adding Pd/C, and mixing the obtained mixture at 20-30 deg.C and H₂Stirring for 6-10h under protection. After the reaction was complete, the resulting mixture was filtered through silica, the filtrate was concentrated in vacuo, and the residue was purified by silica gel chromatography using 5-15% methanol/dichloromethane (MeOH/DCM) to give compound 5-4 (a colorless oil).

(4) Preparation of Compounds 5-5

At 70-90 deg.C, mixing compound 5-4, compound A and compound K₂CO₃Soluble in methyl nitrile (CH)₃CN), stirring overnight. After the reaction is complete, K is filtered off₂CO₃The organic layer was concentrated and purified by column chromatography using methanol/dichloromethane (MeOH/DCM) to give compound 5-5 (white solid).

(5) Preparation of Compounds 5-6

The compound 5-5 is dissolved in dioxane, HCl/dioxane is added, and the mixture is stirred at room temperature for 3-5 h. After completion of the reaction, the resulting mixed solution was concentrated in vacuo, and the residue was taken up with Na₂CO₃(aq), extracted with Ethyl Acetate (EA), the organic layer dried and concentrated in vacuo, and the residue purified by silica gel chromatography using 5-10% methanol/dichloromethane (MeOH/DCM) to give compound 5-6 (yellow solid).

(6) Preparation of Compound 2 (target Compound "C080019")

Dissolve compound 5-6 and compound C in methanol (MeOH), stir the resulting solution at room temperature for 3-5h, then add NaBH₄The resulting mixture was stirred at room temperature for 1-1.5 h. After the reaction is complete, the mixture is washed with H₂Decomposing O and removing the solvent, and reacting the residue with H₂Diluted O, extracted with Ethyl Acetate (EA), the organic phase dried and concentrated. The residue was purified by prep-TLC and prep-HPLC to give compound 2 (colorless oil).

The invention also provides a targeted protein degradation method for degrading a target protein by autophagy, which comprises the following steps: the degradation of the target protein by autophagy is mediated by an autophagy targeting chimera, which is the autophagy targeting chimera described above.

The present invention also provides a method of degrading tau protein in a patient in need thereof, comprising administering to said patient an effective amount of compound 1 or compound 2 as provided herein above.

In the above method, the compound 1 or compound 2 is administered to the patient by at least one means selected from the group consisting of: nasal, inhalation, topical, oral, intramuscular, subcutaneous, transdermal, intraperitoneal, epidural, intrathecal and intravenous routes.

The invention also provides application of the compound 1 or the compound 2 in preparing a medicament for treating or preventing tau protein related diseases. The disease may be at least one of alzheimer's disease, frontotemporal dementia with parkinson's disease linked to chromosome 17, pick's disease, progressive supranuclear palsy, corticobasal degeneration, primary age-related tauopathies, silvery particle disease, aging-related tau astrocytosis, chronic traumatic encephalopathy, globoid tauopathy, parkinson's disease, huntington's disease, stroke, and epilepsy.

The present invention will be described in detail below by way of examples.

Example 1

Preparation of intermediate substances compound a and compound B:

(1) preparation of Compound A

(1.1) preparation of Compound 3-2

Compound 3-1(1.20g, 6.55mmol) and Triethylamine (TEA) (1.10g, 9.82mmol) were dissolved in Tetrahydrofuran (THF) (60mL) at 0 deg.C, then 1- (1, 4-diazepan-1-yl) ethanone (0.93g, 6.55mmol) was added and the resulting mixture was stirred at 0 deg.C for 1h and at room temperature for 1 h. Completion of the reaction was confirmed by TLC (EA/PE ═ 1: 4) showing complete consumption of compound 3-1. The reaction mixture was concentrated and purified by column chromatography to give 900mg of compound 3-2 as a white solid.

(1.2) preparation of Compound A

Compounds 3-2(900mg, 3.1mmol), 2- (thien-2-yl) ethylamine (590g, 4.6mmol) and K were added at 80 deg.C₂CO₃(635mg，46mmol) in methyl nitrile (CH)₃CN) (60mL) and stirred overnight. Completion of the reaction was confirmed by TLC (EA/PE ═ 1: 1) showing complete consumption of compound 3-2. Filtration of K₂CO₃And the organic layer was concentrated and purified by column chromatography to give 600mg of compound a as a white solid.

(2) Preparation of Compound B

Compound 3-3(1.0g, 3.26mmol), 2- (chloromethyl) oxirane (359mg, 3.91mmol) and K were added at 80 deg.C₂CO₃(674mg, 4.89mmol) in methyl nitrile (CH)₃CN) (60mL) and stirred overnight. Completion of the reaction was confirmed by TLC (EA/PE ═ 1: 9) showing complete consumption of compound 3-3. Filtration of K₂CO₃And the organic layer was concentrated and purified by column chromatography to give 400mg of compound B as a colorless oil.

Preparation of compound 1:

(1) preparation of Compound 4-2

Compound 4-1(1.50g, 5.92mmol) and Triethylamine (TEA) (1.20g, 11.85mmol) were dissolved in Dichloromethane (DCM) (100mL) at 0 deg.C, MsCl (0.89g, 7.12mmol) was added, and the resulting mixture was stirred at 0 deg.C for 10min and heated to room temperature for 1 h. Completion of the reaction was confirmed by TLC (EA/PE ═ 3: 7) showing complete consumption of compound 4-1. The resulting mixture was concentrated, and the resulting residue was purified by column chromatography using Petroleum Ether (PE) to give 1.2g of compound 4-2 as a colorless oil.

(2) Preparation of Compounds 4-3

Compound 4-2(1.2g, 3.62mmol) and KI (1.8g, 10.87mmol) were dissolved in acetone (100mL) at 80 ℃ and stirred overnight. Completion of the reaction was confirmed by TLC (EA/PE ═ 1: 1) showing complete consumption of compound 4-2. The reaction mixture was concentrated, and the resulting residue was purified by column chromatography using Petroleum Ether (PE) to give 650mg of compound 4-3 as a colorless oil.

(3) Preparation of Compound 4-4

Compound 4-3(650mg, 1.79mmol) was dissolved in Tetrahydrofuran (THF) (50mL), NaH (86mg, 3.58mmol) was added, stirring was performed at 0 ℃ for 30min, then Compound C (469mg, 2.68mmol) was added, and the resulting mixed solution was stirred at room temperature overnight.Completion of the reaction was confirmed by TLC (MeOH/DCM ═ 1: 9) showing complete consumption of compounds 4-3. Subjecting the solution to H₂O (30mL) was quenched and extracted with Ethyl Acetate (EA) (40 mL. times.3). The organic layer was dried and concentrated in vacuo, and the residue was purified by silica gel chromatography using 8 wt% methanol (MeOH) to give 120mg of compound 4-4 as a yellow solid.

(4) Preparation of Compounds 4-5

Compound 4-4(120mg, 0.1mmol) was dissolved in methanol (MeOH) (5mL), Pd/C (50mg) was added, and the resulting mixture was heated at 25 deg.C under H₂Stirring for 8h under protection. Completion of the reaction was confirmed by TLC (MeOH/DCM ═ 1: 9) showing complete consumption of compounds 4-4. The resulting mixture was filtered, and the filtrate was concentrated in vacuo, and the residue was purified by silica gel chromatography using 8 wt% methanol (MeOH) to give 80mg of compounds 4-5 as a colorless oil.

(5) Preparation of Compounds 4-6

Compounds 4-5(80mg, 0.25mmol), Compound A (142mg, 0.37mmol) and K were added at 80 deg.C₂CO₃(69mg, 0.50mmol) in CH₃CN (methyl nitrile) (20mL) was stirred overnight. Completion of the reaction was confirmed by TLC (MeOH/DCM ═ 1: 19) showing complete consumption of compounds 4-5. Filtration of K₂CO₃And the organic layer was concentrated and purified by prep-TLC using methanol (MeOH) to give 100mg of compounds 4-6 as white solids.

(6) Preparation of Compounds 4-7

Compound 4-6(100mg, 0.15mmol) was dissolved in dioxane (10mL), HCl (3mL, 3.0mmol) was added, and the resulting mixture was stirred at room temperature for 4 h. Complete reaction was confirmed by LCMS (MeOH/DCM ═ 1: 4) showing complete consumption of compounds 4-6. The reaction solution was concentrated in vacuo and the residue was taken up with Na₂CO₃(aq), extracted with Ethyl Acetate (EA) (10mL × 3), the organic layer dried and concentrated in vacuo, and the residue purified by silica gel chromatography using 8 wt% methanol (MeOH) to give 62mg of compound 4-7 as a yellow solid.

(7) Preparation of Compound 1 (target Compound "C090019")

Compounds 4-7(62mg, 0.1mmol), Compound B (39mg, 0.1mmol) were dissolved in CH at 65 deg.C₃OH (10mL), stir overnight. Completion of the reaction was confirmed by TLC (MeOH/DCM ═ 1: 4) showing complete consumption of compounds 4-7. The solvent was removed and the residue was purified by prep-TLC and prep-HPLC to give 22mg of Compound 1 as a brown oil. The nuclear magnetic resonance spectrum of the compound is shown in figure 2.

Example 2

Preparation of intermediate compound a:

(1) preparation of Compound 3-2

(2) Preparation of Compound A

Compounds 3-2(900mg, 3.1mmol), 2- (thien-2-yl) ethylamine (590g, 4.6mmol) and K were added at 80 deg.C₂CO₃(635mg, 4.6mmol) was dissolved in methylnitrile (CH)₃CN) (60mL) and stirred overnight. Completion of the reaction was confirmed by TLC (EA/PE ═ 1: 1) showing complete consumption of compound 3-2. Filtration of K₂CO₃And the organic layer was concentrated and purified by column chromatography to give 600mg of compound a as a white solid.

Preparation of compound 2:

(1) preparation of Compound 5-2

At 25 deg.C, compound 5-1(1.0g, 3.36mmol), carbon tetrabromide (CBr)₄) (1.0g, 3.36mmol) and triphenylphosphine (PPh3) (1.0g, 4.04mmol) were dissolved in Tetrahydrofuran (THF) (60mL) and stirred overnight. Completion of the reaction was confirmed by TLC (EA/PE ═ 1: 19) showing complete consumption of compound 5-1. The solution was concentrated and purified by column chromatography using Petroleum Ether (PE) to give 850mg of compound 5-2 as a yellow oil.

(2) Preparation of Compounds 5-3

Compound 5-2(850mg, 2.36mmol) was dissolved in tetrahydrofuranPyran (THF) (50mL), NaH (113mg, 4.72mmol) was added, stirring was continued at 0 deg.C for 30min, followed by the addition of Compound C (569mg, 3.54mmol), and the resulting mixture was stirred at room temperature overnight. Completion of the reaction was confirmed by TLC (EA/PE ═ 1: 4) showing complete consumption of compound 5-2. Subjecting the solution to H₂O (30mL) was quenched and extracted with Ethyl Acetate (EA) (30mL × 3), the organic layer was dried and concentrated in vacuo, and the residue was purified by silica gel chromatography using 8 wt% methanol (MeOH) to give 200mg of compound 5-3 as a yellow oil.

(3) Preparation of Compounds 5-4

Compound 5-3(120mg, 0.27mmol) was dissolved in methanol (MeOH) (15mL), Pd/C (50mg) was added, and the resulting mixture was heated at 25 deg.C under H₂Stirring for 8h under protection. Completion of the reaction was confirmed by TLC (MeOH/DCM ═ 1: 9) showing complete consumption of compounds 5-3. The resulting mixture was filtered through silica, the filtrate was concentrated in vacuo, and the residue was purified by silica gel chromatography using 8 wt% methanol (MeOH) to give 75mg of compound 5-4 as a colorless oil.

(4) Preparation of Compounds 5-5

Compounds 5-4(75mg, 0.21mmol), Compound A (97mg, 0.25mmol) and K were added at 80 deg.C₂CO₃(69mg, 0.50mmol) in methyl nitrile (CH)₃CN) (20mL), stirred overnight. Completion of the reaction was confirmed by TLC (MeOH/DCM ═ 1: 19) showing complete consumption of compounds 5-4. Filtration of K₂CO₃The organic layer was concentrated and purified by column chromatography using methanol (MeOH) to give 80mg of compound 5-5 as a white solid.

(5) Preparation of Compounds 5-6

Compound 5-5(80mg, 0.12mmol) was dissolved in dioxane (10mL), HCl (3.6mL, 3.6mmol) was added, and the resulting mixture was stirred at room temperature for 4 h. Complete reaction was confirmed by LCMS (MeOH/DCM ═ 1: 4) showing complete consumption of compounds 5-5. The resulting mixed solution was concentrated in vacuo, and the residue was taken up with Na₂CO₃(aq), extracted with Ethyl Acetate (EA) (10mL × 3), the organic layer dried and concentrated in vacuo, and the residue purified by silica gel chromatography using 8 wt% methanol (MeOH) to give 50mg of compound 5-6 as a yellow solid.

(6) Preparation of Compound 2 (target Compound "C080019")

Compounds 5-6(50mg, 0.08mmol) and Compound C (26mg, 0.08mmol) were dissolved in methanol (MeOH) (10mL), the resulting solution was stirred at room temperature for 4h, then NaBH was added₄(12mg, 0.32mmol) and the resulting mixture was stirred at room temperature for 1 h. Subjecting the mixture to hydrogenation with H₂Decomposing O and removing the solvent, and reacting the residue with H₂O diluted, extracted with Ethyl Acetate (EA) (10mL × 3), the organic phase dried and concentrated. The residue was purified by prep-TLC and prep-HPLC to give 23mg of Compound 2 as a colorless oil. The nuclear magnetic resonance spectrum of the compound is shown in figure 3.

Test example

First, preparation of animal sample

1. Raising of animals

C57BL/6 mice were purchased from Wafukang (Beijing Corp.). The experimental animals were strictly managed according to the regulations on the management of laboratory animals in China, the temperature was controlled at 25 ℃, the circadian rhythm was reversed for 12 hours, and all animal experiments were approved by the ethical committee of the college of Tongji medical college of Huazhong university of science and technology.

2. Pharmaceutical treatment of animals

Mice were administered by subcutaneous injection. Firstly, preparing a cosolvent (20% hydroxypropyl-beta-cyclodextrin (HP-beta-CD)), dissolving 2g of powder in 10mL of physiological saline, and shaking and uniformly mixing. Weighing a mouse, calculating the dosage (15mg/kg) required by subcutaneous injection, respectively adding equal volumes of a drug (experimental group) and DMSO (control group) into a cosolvent with the volume 10 times that of the drug, shaking and uniformly mixing, sucking a proper amount of the drug by using a 1mL injector, fixing the head and the neck of the mouse, inserting a needle along the loose part of the neck skin, slowly injecting the drug, sensing that the drug forms a skin dome, and observing whether the drug leaks or not after the needle is taken out. If there was a leak, the mouse was discarded and replaced by another injection.

3. Preparation of samples

The instruments and consumables used for homogenization of mouse brain tissue are first prepared, fresh homogenate (50mM Tris-HCl, pH 7.4-7.5,100mM NaCl, 1% Triton,5mM EDTA,1mM PMSF (Sigma, P-7626), 1 Xprotease inhibitor cocktail (Protease inhibitors cocktail, Sigma, P8340)) is prepared and the homogenate is placed on ice for precooling. After anesthetizing a mouse with 6% chloral hydrate, the mouse is decapitated, the whole brain tissue is taken out, the whole brain tissue is placed on a glass plate placed on ice, the cerebellum is cut off rapidly, the left brain and the right brain are separated, one half of the brain tissue is placed in paraformaldehyde solution for fixation, the other half of the brain tissue is separated into a cortex and a hippocampus, and the cortex is taken as 1/3 close to frontotemporal lobe and is respectively placed in a precooled EP tube with the volume of 1.5 mL. Weighing brain tissue, putting the brain tissue into a homogenate tube, adding the homogenate at a ratio of 1:10 (brain tissue mass: homogenate volume: 1:10), opening the homogenate machine, homogenizing under 50 times for forward and reverse, collecting the homogenate liquid into an EP tube, standing on ice for 30 minutes, blowing and beating the liquid in the tube uniformly every 10 minutes, centrifuging at 12000rpm multiplied by 20 minutes in a centrifuge precooled to 4 ℃, and dividing the supernatant into two parts. The first portion was taken in appropriate volume, added to 4 x SDS loading buffer at 3:1, and placed in a fume hood at 10: 1, adding 250mM beta-mercaptoethanol, uniformly mixing, heating for 10 minutes at 95 ℃ in an iron bath, cooling, shaking, uniformly mixing, centrifuging, and storing in a refrigerator at-80 ℃. The other part is kept about 10 mu L, the protein concentration is measured, and the rest is put into liquid nitrogen for quick freezing and then transferred to a refrigerator at the temperature of 80 ℃ below zero for storage. Before immunoblotting hybridization loading, diluting the sample by 4-5 times with a proper amount of 1 xSDS loading buffer solution, reheating for 10 minutes, cooling, centrifuging for a short time, and mixing the samples uniformly.

Secondly, preparation of cell sample

1. Cell culture

Cells were incubated at 37 ℃ with 5% CO₂Culturing in a cell culture box. The medium for HEK293tau cells (stably expressing wild type full length tau protein) was DMEM/HIGH GLUCOSE (hyclone, 1234), 10% Fetal Bovine Serum FBS (Fetal Bovine Serum, FBS) (Biological Industries, 04-001-1ACS), 0.2mg/ml G418. After cell inoculation, the medium was changed every 2-3 days, and the state and growth of cells were observed under an inverted microscope. When the cell coverage in the culture flask reached 80% -90%, passaging or plating.

2. Drug treatment

Cells were seeded one day in 24-well plates approximately 2.5 × 105 cells per well. After 24 hours of cell culture, the original culture medium was removed and replaced with fresh medium, and then different doses of the small molecule compounds (0. mu.M, 0.01. mu.M, 0.1. mu.M, 1. mu.M, 5. mu.M) prepared in the above examples were added, and the culture was continued for 24 hours, and finally cell samples were collected.

3. Preparation of samples

(1) 1 XPBS and cell lysates (50mM Tris-HCl, pH 7.4-7.5,100mM NaCl, 1% Triton,5mM EDTA,1mM PMSF (Sigma, P-7626), 1 XProtease inhibitor cocktail (Sigma, P8340)) were chilled on ice.

(2) Taking out a six-hole cell culture plate from a constant-temperature cell culture box at 37 ℃, placing the six-hole cell culture plate on ice, sucking a culture medium to be discarded, slowly adding precooled PBS along the hole wall, slightly shaking the six-hole cell culture plate to be discarded, then adding fresh precooled PBS, washing the six-hole cell culture plate twice, completely sucking the PBS for the last time, adding a proper amount of prepared cell lysate, slightly shaking the cell culture plate to uniformly cover the cell, standing the cell culture plate on ice for 10 minutes, scraping the cell along the bottom of a hole plate by using a clean cell scraper, collecting the cell lysate in a 1.5mL EP tube, standing the cell lysate on ice for 30 minutes, and uniformly blowing liquid in the tube every 10 minutes.

(3) The samples were centrifuged at 12000rpm for 20 minutes in a centrifuge precooled to 4 ℃ and the supernatant was split into two portions, the first of which was taken in appropriate volume and added to 4 xSDS loading buffer (4 xSDS loading buffer: 0.2M Tris-HCl pH 6.8, 2% SDS, 40% glycerol) at 3:1 in a fume hood as 10: 1, adding 250mM beta-mercaptoethanol, heating for 10 minutes at 95 ℃ in an iron bath, shaking, mixing uniformly, centrifuging, and storing in a refrigerator at-80 ℃. The other fraction was left at about 10. mu.L and the protein concentration was measured.

Third, determination of protein content of sample (BCA method)

1. Shaking a protein sample, then appropriately diluting (5 mu l of each sample is mixed and diluted with 45 mu l of double distilled water, and 2 parallel samples are arranged respectively), and centrifuging and shaking;

2. setting six standard tubes, respectively taking 0 μ l, 10 μ l, 20 μ l, 30 μ l, 40 μ l and 50 μ l of 20mg/ml BSA (100mg BSA dissolved in 5ml double distilled water), respectively adding 1000 μ l, 990 μ l, 980 μ l, 970 μ l, 960 μ l and 950 μ l double distilled water to prepare standard proteins of 0 μ g/μ l, 0.2 μ g/μ l, 0.4 μ g/μ l, 0.6 μ g/μ l, 0.8 μ g/μ l and 1.0 μ g/μ l;

3. the diluted protein sample and the diluted standard protein were added to a 96-well plate (5. mu.l/well, one tip was changed for each well at the junction between the wall and the bottom of the PCR gun) with 3 parallel wells each.

4. The working solution is prepared from solution A and solution B in the kit according to the proportion of 50: 1. Adding the working solution into a 96-well plate, quickly suspending and adding 95 mu l of the working solution into each well, covering a cover after adding, quickly attaching the cover to the bottom, vibrating along the same direction, keeping hands from touching the bottom of the 96-well plate, and incubating for 30 minutes at 37 ℃ by using a plastic box;

5. de-bubbling with a 1ml syringe needle, turning on a BioTek switch, turning on Gen5, pointing a left arrow icon, clicking OK, and exporting Excel;

6. replicate standard protein OD values, frame select OD values and standard protein concentrations and insert scatter plots. Data points are selected to add trend lines, formulas are displayed, R square values (at least 2 should be found after the decimal point is 9) are displayed, and outliers are removed. Copy the protein OD value of the sample, input the corresponding sample group number above, remove the abnormal value.

Fourth, tau protein assay (immunoblotting)

1. Building frame (two glass plates, three bottles, five reagents, filter paper, toilet paper, garbage can, gun head, comb teeth)

(1) Cleaning the tabletop and the underframe, cleaning the comb teeth, the glass plate, the distilled water bottle, the upper rubber bottle and the lower rubber bottle, drying the upper rubber bottle and the lower rubber bottle, taking out the reagent for preparing the electrophoresis gel, and returning to the room temperature;

(2) the higher glass plates are folded inwards together, the upper part is pressed to enable the lower part to be tightly attached to the desktop to enable the upper part to be flush with the desktop, the clamp is turned outwards to clamp the upper part, and the upper part is placed on the underframe and is buckled by the clamp.

(3) And injecting double distilled water to check whether leakage exists, and detecting leakage after reloading if leakage exists.

2. Preparation of an electrophoretic gel

TABLE 1

(1) Sequentially adding 20% Arc/Bis, Tris buffer solution, TEMED and 10% APS, and blowing and uniformly mixing by using a pipette, wherein bubbles are prevented from being mixed in the mixed solution in the whole process;

(2) respectively and slowly injecting separation glue into the glue film along two corners (the separation glue penetrates under the liquid surface during suction and is gently blown and uniformly mixed, a small amount of liquid is reserved at a gun head each time to prevent bubbles from being generated), the use amount of each piece of glue is 3 multiplied by 900 mu l, and after the glue is observed to be not leaked, double distilled water is respectively used for filling gaps of the glue film along the two corners (the oxygen is prevented from inhibiting polymerization, the glue at the lower part is kept horizontal, and the glue can be put for a period of time);

(3) waiting for about 30 minutes for the gel to separate, pouring back to remove double distilled water, completely absorbing the residual water by using filter paper, and marking the upper edge of the lower part of the gel by using a marker pen;

(4) the concentrated gel is respectively and slowly injected into the gel film along two corners, the comb teeth with the required specification (the small comb teeth are used for loading the sample amount less than 20 mu l, the large comb teeth are used for loading the sample amount more than 20 mu l) are obliquely inserted from left to right, the gel is supplemented between lanes to avoid the gel shrinkage, and the gel is waited (about 50 minutes is needed).

3. And (3) carrying out electrophoretic separation on the sample and the protein (a sample needle, a sample, a socket, a Marker, an electrophoretic fluid, an electrophoretic tank and a distilled water bottle).

The conductive wire under the electrophoresis frame is cleaned, the conductive wire is transferred to the electrophoresis frame, the lanes and the numbers are marked by a marking pen, the comb teeth are slowly and vertically pulled out, the gel tank is filled with electrophoresis liquid, and a sample is taken by a microsyringe and is added into each lane (Marker adds 1 mul to lane 1). After the sample is loaded, the electrophoresis rack is transferred to an electrophoresis tank, the electrophoresis tank is added with electrophoresis liquid, then a cover is covered to ensure that the red color is red, the black color is black, after the sample is loaded, the electrophoresis is firstly carried out for about 30 minutes by using a constant current of 10 mA/piece of gel (starting according to two times), when the bromophenol blue indicator electrophoresis is carried out until the junction of the concentrated gel and the separation gel is linear, the electrophoresis is changed into constant voltage of 100V (if the constant voltage can be adjusted to high current) electrophoresis for about 60 minutes until the bromophenol blue reaches the bottom of the gel and the Marker strips are completely.

4. Transferring membrane (marking NC membrane, transferring membrane liquid, filter paper, ice box, basin, dish, transferring membrane groove, plastic plate, cleaning tweezers)

(1) And (3) marking the NC membrane by using a marker pen, then soaking the NC membrane in the recovered membrane transferring liquid for 10-20 minutes (which is beneficial to fixing protein, balancing gel and removing SDS), pressing bayonets at two sides to take off the gel tank, prying the middle parts at the right sides of the glass plate and the white porcelain plate by using a small plate, and keeping electrophoresis of the residual gel in the process.

(2) Slightly inclining a glass plate vertically according to the molecular weight range to be displayed and slightly cutting glue back and forth left and right once, sticking three layers of filter paper soaked in the membrane transferring liquid on the glue by using a pair of tweezers, carefully prying up the glue by using a small plate and placing the glue on a sponge (the filter paper faces downwards), sticking an inverted NC membrane on the other side, immersing the glue and the NC membrane into the membrane transferring liquid (the glue is on the upper side), removing bubbles by using a glass rod, carefully clamping the glue by using the pair of tweezers and placing the glue on the hand (the glue is on the upper side), sticking the three layers of filter paper soaked in the membrane transferring liquid on the glue by using the pair of tweezers, inversely placing the glue on the sponge, and. From bottom to top, a black plastic plate → a layer of sponge → three layers of filter paper → glue → NC film → three layers of filter paper → a layer of sponge → a transparent plastic plate, if not tight, can be fixed by a rubber band.

(3) After the electrodes are correctly arranged, the membrane transferring groove is placed in an ice bath (the glue is not required to be soaked in the membrane transferring liquid for a long time before the electrification so as to avoid the diffusion and the decomposition of the protein), the transferring current is constant current 276mA, the voltage is generally 140V (the methanol can be properly supplemented to increase the voltage), the specific transferring time is determined according to the molecular weight of the protein to be transferred, the time when the molecular weight of the transferred protein is less than 100kDa is 1h, and the time when the molecular weight is more than 100kDa is 1.5 h.

5. Immunoblotting color development (cleaning tweezers, box with double distilled water, milk, fresh-keeping bag, toilet paper, primary antibody, ice box, plate, transparent glue, TBST, black plastic bag, secondary antibody)

(1) And (3) sealing: after the completion of the membrane transfer, the NC membrane was carefully blocked with 5% skimmed milk powder in TBS blocking solution at room temperature for 1h or overnight at 4 ℃ to recover the filter paper not in contact with the gel.

(2) Primary antibody incubation: taking out the NC film, rinsing residual milk stain on the surface of the film by using 1 × TBS, clamping the NC film by using tweezers, standing on toilet paper to remove excessive water, placing the NC film Marker side outwards in a freshness protection package, and draining and exhausting by using the toilet paper. Add primary antibody (0.1% Tween 20 can be added to reduce background) and seal on plate (with Marker and protein side up), clear tape is not pressed on target strip, and incubate overnight at 4 deg.C.

(3) And (3) secondary antibody incubation: the next day the NC membrane was removed from the incubation bag and primary antibody was recovered, rinsed with TBST buffer for 3X 5 minutes, rinsed with 1X TBS for removing residual salt ions on the membrane surface, set up on toilet paper with tweezers to remove excess water, placed in a freshness-keeping bag and vented with toilet paper. Adding horseradish peroxide labeled goat anti-rabbit or goat anti-mouse Odyssey secondary antibody (0.1% Tween 20(Tween 20) can be added to reduce background) in a dark place, sealing and pasting the plate (the side with Marker and protein faces upwards), placing a transparent adhesive tape on a target strip, incubating for about 1 hour with slow shaking at room temperature, taking out the NC membrane from the incubation bag and recovering the secondary antibody, and rinsing for 3 multiplied by 5 minutes by TBST buffer solution. After rinsing, the residual salt ions on the membrane surface were removed by rinsing with 1 × TBS.

(4) Color development: the glass plate is firstly wiped clean by using a piece of lens wiping paper dipped with absolute ethyl alcohol. The NC film Marker was placed on a glass plate side up, incubated for about 1 minute with an ECL chemiluminescent substrate (BeyoECL Star, P0018AM) configured as described in the specification, and then photographed with an ECL developing apparatus (Shanghai volkong scientific instruments Co., Ltd., ChemiScope 3300 Mini).

(5) Semi-quantitative analysis: the obtained Image was quantified for gray scale using Image J software.

(6) Statistical analysis: statistical analysis was done using GraphPad Prism software.

FIG. 4 shows the results of immunoblot hybridization (a) and semi-quantitative analysis (b) of the degradation of intracellular tau protein using different concentrations of compound C090019 provided by the present invention. The results show that: 0.1. mu.M, 0.5. mu.M, 1. mu.M and 5. mu.M of the compound significantly reduced the amount of cellular tau protein. FIG. 5 shows the results of immunoblot hybridization (a) and semi-quantitative analysis (b) of the degradation of intracellular tau protein using different concentrations of compound C080019 provided by the present invention. The results show that: 0.5. mu.M, 1. mu.M and 5. mu.M of the compound significantly reduced the amount of cellular tau protein. FIG. 6 shows the results of immunoblot hybridization (a, C) and semi-quantitative analysis (b, d) of the effect of subcutaneously injecting the compound C090019 provided by the present invention on tau protein content in cerebral cortex and hippocampus of mice. The results show that: the small molecule compound C090019 can obviously reduce the content of tau protein in cerebral cortex and hippocampus of a mouse.

Therefore, the autophagy targeting chimera compound C090019 or compound C080019 for tau protein constructed by the invention can reduce the content of tau protein in cells. Therefore, the target protein degradation method utilizing autophagy to degrade the target protein can specifically degrade the target protein.

Since tau protein is abnormally accumulated in cells and is involved in more than 20 kinds of neurodegenerative diseases, the accumulation amount thereof is positively correlated with neurodegeneration and memory impairment of these degenerative diseases. Thus, the tau protein degradation can achieve the purpose of preventing or/and treating tau-related neurodegenerative diseases, such as Alzheimer disease, frontotemporal dementia with Parkinson's disease linked to chromosome 17, pick's disease, progressive supranuclear palsy, corticobasal degeneration, primary age-related tauopathies, silvery granulosis, aging-related tau astrocytosis, chronic traumatic encephalopathy, globulo-like gliosis, Parkinson's disease, Huntington's disease, stroke and epilepsy.

The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.

Sequence listing

<110> Shanghai Qiangri Biotechnology Limited

<120> autophagy targeting protein degradation technology and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 36

<212> PRT

<213> Human P62 protein ZZ domain sequence

<400> 1

Cys Asp Gly Cys Asn Gly Pro Val Val Gly Thr Arg Tyr Lys Cys Ser

1 5 10 15

Val Cys Pro Asp Tyr Asp Leu Cys Ser Val Cys Glu Gly Lys Gly Leu

20 25 30

His Arg Gly His

35

Claims

1. A targeted protein degradation method for degrading a target protein by autophagy, which is characterized in that the degradation of the target protein by autophagy is mediated by an autophagy targeting chimera, wherein the autophagy targeting chimera is a bifunctional molecule having the chemical structure TBM-L-ABM or a pharmaceutically acceptable salt, enantiomer, stereoisomer, solvate, polymorph or N-oxide thereof, wherein TBM is a target protein binding moiety, L is a linker group, ABM is an autophagy receptor binding moiety, and the target protein binding moiety is connected with the autophagy receptor binding moiety through the linker group.

2. An autophagy-targeting chimera having the chemical structure TBM-L-ABM or a pharmaceutically acceptable salt, enantiomer, stereoisomer, solvate, polymorph or N-oxide thereof, wherein TBM is a target protein binding moiety, L is a linker group, ABM is an autophagy receptor binding moiety, and the target protein binding moiety and the autophagy receptor binding moiety are linked by a linker group.

3. The autophagy-targeting chimera of claim 2, wherein the target protein to which the TBM is capable of binding is tau protein, alpha-synuclein, polyglutamine protein including huntingtin protein, copper/zinc superoxide dismutase, TDP-43, C9orf72, FUS, or a polymer of one or more thereof.

4. The autophagy-targeting chimera of claim 2, wherein the ABM is capable of binding to an autophagy receptorIs P62, NBR1, OPTN, CALCOCO2/NDP₅₂TAX1BP1, NIX, BNIP3, FUNDC1, Bcl2L13 or FKBP 8;

5. The autophagy targeting chimera of claim 2 or 4, wherein ABM is a group having a structure shown in formula (1),

wherein R is¹And R²Is H or C1-C4 alkyl;

6. The autophagy-targeting chimera of claim 2, wherein L is a group-X-Y-Z-, X is linked to TBM, Z is linked to ABM,

wherein X is a bond, alkylene of C1-C4, or-NH-;

7. The autophagy targeting chimera according to claim 2 or 3, characterized in that TBM is a group with a structure shown in formula (2), or a group with a structure shown in formula (2), wherein the group is further modified by a substituent group at the position of (i), (ii), (iii), (iv), (c), (,

8. The autophagy-targeting chimera of claim 2, wherein the autophagy-targeting chimera compound 1 and compound 2 have the structure:

9. a targeted protein degradation method for degrading a target protein using autophagy, the method comprising: mediating degradation of a target protein by autophagy using an autophagy-targeting chimera according to any one of claims 3-8.

10. A method of degrading tau protein in a patient in need thereof, comprising administering to the patient an effective amount of the autophagy-targeting chimera of any one of claims 2-8;

preferably, the autophagy-targeting chimera is administered to the patient by at least one means selected from the group consisting of: nasal, inhalation, topical, oral, intramuscular, subcutaneous, transdermal, intraperitoneal, epidural, intrathecal and intravenous routes.

11. Use of an autophagy-targeting chimera according to any one of claims 2-8 in the preparation of a medicament for treating or preventing a tau protein associated disease;

wherein the disease is at least one of Alzheimer's disease, frontotemporal dementia with Parkinson's disease linked to chromosome 17, pick's disease, progressive supranuclear palsy, corticobasal degeneration, primary age-related tauopathies, silvery particle disease, aging-related tau astrocytosis, chronic traumatic encephalopathy, globoid tauopathy, Parkinson's disease, Huntington's disease, stroke and epilepsy.