CN117659002A - PROTAC compound for inducing BCR-ABL degradation based on N-terminal rule and application thereof - Google Patents

PROTAC compound for inducing BCR-ABL degradation based on N-terminal rule and application thereof Download PDF

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CN117659002A
CN117659002A CN202311653111.8A CN202311653111A CN117659002A CN 117659002 A CN117659002 A CN 117659002A CN 202311653111 A CN202311653111 A CN 202311653111A CN 117659002 A CN117659002 A CN 117659002A
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bcr
abl
dasa
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arg
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饶海
张建超
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Southwest University of Science and Technology
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Abstract

The invention relates to a PROTAC compound for inducing BCR-ABL degradation based on an N-terminal rule and application thereof, belonging to the technical field of medicines. The molecular structure of the compound comprises single amino acid and ligand Dasatinib, and the two are connected by PEG linker. The invention designs novel PROTAC small molecules by utilizing the minimum degradation signal-single amino acid, the shortest PEG linker and replacing different amino acids, thereby improving the cell permeability and achieving better curative effect. Meanwhile, the level of BCR-ABL protein is better controlled and the growth of tumor is inhibited through unique adjustable degradation capability, so that a new choice is provided for future tumor targeted therapy.

Description

PROTAC compound for inducing BCR-ABL degradation based on N-terminal rule and application thereof
Technical Field
The invention relates to the technical field of medicines, in particular to a PROTAC compound or pharmaceutically acceptable salt thereof for inducing BCR-ABL degradation based on an N-terminal rule and application thereof.
Background
Protoc (proteolysis targeting chimeras, proteolytically targeted chimera) is a drug development technology that utilizes ubiquitin-proteasome systems to degrade target Proteins (POI). The PROTAC compound is based on protein degradation technology caused by ubiquitin-proteinase system (figure 1), PROTAC is a bifunctional small molecule, one end of which is a ligand binding to E3 ligase, the other end of which is a ligand binding to intracellular protein, and the two ligands are connected by a linker. The ubiquitin-linked enzyme E3 and the target protein are respectively recruited at two ends of the PROTAC to trigger ubiquitination of the target protein, and then the target protein is degraded by a proteasome. Linker is an important structure linking two active groups of PROTAC drugs, and theoretically does not participate in pharmacodynamic process. With the continuous and intensive research on the PROTAC technology, alkyl chains and PEG chains can be classified according to the difference of Linker constitution, and alkynyl bipiperidine rings with higher rigidity, spiro rings containing nitrogen atoms, bridge rings or the like are also used as linkers to limit the flexibility and the degree of freedom of the PROTAC molecules. The length of a typical Linker is typically 4-15 carbon atoms (or heteroatoms). Researchers find that the length of the Linker structure and the molecular layer structure have very important influence on the activity of PROTAC drugs, and the influence of the length of the Linker on the degradation activity is different according to different action targets. However, no rule for the passing of Linker structural design exists at present. In addition, click chemistry is often applied to Linker of the PROTAC molecule for linking molecules at both ends due to milder reaction conditions and higher efficiency.
The E3 ligases reported in the literature for use in PROTAC are mainly CRBN, VHL, cIAP and MDM2. The E3 ligase with better effect and highest frequency of use is mainly two kinds of CRBN (60.1%) and VHL (30.1%). Wherein the ligand of CRBN is mainly lenalidomide, pomalidomide and analogues thereof, and the ligand of VHL is mainly VHL-L. In the development process of PROTAC, craig Crews doctor at the university of Yersinia is first to make a critical breakthrough. One end of the designed PROTAC small molecule can recognize an estrogen receptor, and the other end of the PROTAC small molecule acts with ubiquitin ligase VHL, so that the ubiquitination of the estrogen receptor is caused to be degraded, and the growth of breast tumors is prevented. Meanwhile, the Bradner group of the harvard medical institute also designed a PROTAC compound, which utilized CRBN ubiquitin ligase to degrade the transcription factor BRD4, which plays an important role in cancer. Both studies successfully inhibited tumors in a mouse model, overcoming a major obstacle to protein knockdown methods for treatment. From this PROTAC, which is an emerging technology, there is a great deal of attention in the biomedical world and industry, and especially two PROTAC compounds recently available from Arvinas company exhibit very good phase II clinical effects.
Although the protoc technology has made great progress in recent years, there are still some problems: (1) The PROTAC medicine has larger molecular weight and is difficult to penetrate cell membranes; (2) The length of the linker and the binding site both affect the degradation capacity of the procac; (3) As one of the constituents of PROTAC, E3 ubiquitin ligase plays a vital role. Although E3 ligases are a large family of proteins, it has been found that over 600E 3 ligases can function in human cells, there are only VHL, CRBN, IAPs, MDM 2E 3 ligase ligands that can now be truly used in PROTAC designs. The lack of available E3 ligases and ligands has limited the development of the PROTAC technology. (4) VHL, CRBN, IAPs, MDM2, although used on PROTAC to date, lack regulation of the rate of degradation of the target protein, as too fast or too slow degradation of the protein may be detrimental in some cases; (5) In addition, the two ubiquitin ligases VHL and CRBN most commonly used in PROTAC have some defects, such as their low activity in some cells, and may not be easily used for tumor treatment such as blood cancer, brain cancer, lung cancer and kidney cancer. It remains a critical and urgent issue how to make the procac more universally applicable.
Disclosure of Invention
The invention aims to provide a PROTAC compound or pharmaceutically acceptable salt thereof for inducing BCR-ABL degradation based on an N-terminal rule and application thereof, wherein the PROTAC compound is designed with novel PROTAC small molecules by utilizing a minimum degradation signal-single amino acid and a shortest PEG linker (PEG linker) and replacing different amino acids, so that the cell permeability is improved, better curative effect is achieved, and the level of BCR-ABL protein is better controlled by unique adjustable degradation capability.
The technical scheme of the invention is realized as follows:
the invention provides a PROTAC compound or pharmaceutically acceptable salt thereof for inducing BCR-ABL degradation based on an N-terminal rule, wherein the molecular structure of the compound comprises single amino acid and a ligand Dasatinib, and the two are connected by a PEG linker.
As a further development of the invention, the monoamino acid can be selected from Gly, ala, val, leu, ile, phe, pro, trp, ser, tyr, cys, met, asp, asn, gln, glu, thr, lys, arg or His, preferably Arg, lys, leu or Phe.
The present invention provides a protoc compound or a pharmaceutically acceptable salt thereof that induces BCR-ABL degradation based on the N-terminal rule, including organic or inorganic salts, including but not limited to: sodium, potassium, cesium, calcium, magnesium, triethylamine, pyridine, picoline, ethanolamine, triethanolamine, dicyclohexylamine, N-dibenzylethylenediamine, hydrochloride, hydrobromide, sulfate, nitrate, phosphate, formate, acetate, trifluoroacetate, pantothenate, succinate, citrate, tartrate, fumarate, maleate, gluconate, glucuronate, gluconate, benzoate, lactate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, arginate, aspartate, glutamate, pantothenate, ascorbate, and combinations of the foregoing.
The present invention also provides pharmaceutical compositions comprising a PROTAC compound or a pharmaceutically acceptable salt thereof that induces degradation of BCR-ABL based on the N-terminal rule, and a pharmaceutically acceptable carrier or excipient thereof.
As a further improvement of the present invention, the ligand Dasatinib has a structure as shown in formula I:
wherein, one of x= F, cl, br, I; r is R 1 、R 2 ＝CH 3 、C 2 H 5 、C 3 H 7 、C 4 H 9 、C 5 H 11 One of them.
As a further improvement of the present invention, the PEG linker includes, but is not limited to, diethylene glycol, triethylene glycol, tetraethylene glycol, pentaethylene glycol.
As a further improvement of the present invention, the compound comprises Arg-PEG1-Dasa:
in the present invention, the pharmaceutically acceptable composition will comprise from about 1 to about 99% by weight of the conjugate of the invention, and from 99 to 1% by weight of a suitable carrier or pharmaceutically acceptable excipient, depending on the mode of administration desired. Preferably the composition comprises from about 5 to 75% by weight of the conjugate of the invention, the remainder being a suitable carrier or pharmaceutically acceptable excipient. More preferably, the composition comprises about 10 to 50% by weight of the conjugate of the invention, the remainder being a suitable carrier or pharmaceutically acceptable excipient.
In embodiments of the present invention, the pharmaceutical compositions of the present invention may also contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, antioxidants and the like, for example: citric acid, sorbitan monolaurate, triethanolamine oleate, butylated hydroxytoluene, and the like.
In the embodiment of the invention, the pharmaceutical composition is in the forms of tablets, capsules, pills, granules, powder, suppositories, injections, solutions, suspensions, pastes, patches, lotions, drops, liniments, sprays and the like.
In embodiments of the invention, the conjugates of the invention may be administered in the form of a pure compound or in the form of a suitable pharmaceutical composition, using any acceptable mode of administration or agent for similar use. Thus, the mode of administration employed may be selected from oral, intranasal, parenteral, topical, transdermal or rectal modes of administration in the form of solid, semi-solid or liquid pharmaceutical forms, for example, tablets, suppositories, pills, soft and hard gelatin capsules, powders, solutions, suspensions, injections and the like, preferably in unit dosage forms suitable for simple administration of precise doses.
Pharmaceutical compositions which can be administered in liquid form can be prepared, for example, by dissolving, dispersing or the like the conjugate of the invention (about 0.5 to about 20%) and optionally pharmaceutically acceptable adjuvants in a carrier, examples of which are water, saline, aqueous dextrose, glycerol, ethanol or the like, to form a solution or suspension.
The invention further provides application of the PROTAC compound or pharmaceutically acceptable salt thereof for inducing the degradation of BCR-ABL based on the N-terminal rule in preparation of medicines for degrading the protein level of BCR-ABL in a body.
The invention further protects an application of the PROTAC compound or the pharmaceutically acceptable salt thereof for preparing the antitumor drug, wherein the PROTAC compound is used for inducing the degradation of BCR-ABL based on the N-terminal rule.
As a further improvement of the present invention, the tumors include, but are not limited to, blood cancer, brain cancer, lung cancer and kidney cancer.
Protoc (proteolysis targeting chimeras, proteolytically targeted chimera) is a drug development technology that utilizes ubiquitin-proteasome systems to degrade target Proteins (POI). In this case, the invention designs a novel and unique PROTAC small molecule which utilizes the degradation way of the cell to degrade the BCR-ABL protein kinase, namely the PROTAC small molecule synthesized by the invention takes single amino acids such as Arg, lys, leu and Phe as ligands of E3 ligase UBR, and the other end is combined with the target protein BCR-ABL through a ligand Dasatinib (Dasa), and the two ligands are connected by PEG linker with different lengths. Such chemical molecules link the UBR to the BCR-ABL, which is ubiquitinated and proteolytically degraded. The PROTAC designed by the invention has the advantages that the target protein can be degraded by a small degradation signal (single amino acid), so that the size of the PROTAC molecule is obviously reduced, and the permeability and the effectiveness of cells are improved; secondly, by replacing different amino acids (such as Lys, leu, phe, etc.), the degradation speed of the target protein can be changed by the small molecules with different N-terminal only due to different affinities of ubiquitin ligase UBR, so that the regulation of protein stability can be optimized. The Arg-PEG1-Dasa, arg-PEG2-Dasa, arg-PEG3-Dasa, arg-PEG4-Dasa, lys-PEG1-Dasa, leu-PEG1-Dasa and Phe-PEG1-Dasa which are designed and synthesized are proved to be high-efficiency BCR-ABL degradation agents in-vivo and in-vitro experiments, and the degradation speed of the BCR-ABL can be regulated and controlled. The design of the invention not only can provide a new treatment strategy for leukemia, but also can be easily transplanted to other target medicines, and has wide application feasibility.
Compared with the prior art, the invention has the following beneficial effects:
1. previous studies by the inventors found that BCR-ABL is relatively stable in leukemia and is not easily degraded, thereby promoting uncontrolled cell growth. Therefore, degradation of BCR-ABL with PROTAC would be an effective approach. The PROTAC small molecule synthesized by the invention starts from single amino acid Arg (or Lys, leu, phe and the like) and is fused with Dasatinib through PEG linker with different lengths. By this Arg (Lys, leu, phe) -PEG (1-4) -Dasa small molecule intermediate bridge linking the UBR with BCR-ABL such that BCR-ABL is ubiquitinated and significantly reduced in level, inhibiting leukemia growth. One major advantage of protein degradation is that, unlike typical enzyme-based approaches, it requires only transient drug target interactions, and does not require direct inhibition of kinase activity.
2. The invention improves the existing common PROTAC: (1) A small degradation signal (e.g., single amino acid Arg, lys, leu, phe) significantly reduces the size of the PROTAC compound to increase cell permeability and effectiveness; (2) By comparing PEG markers with different lengths, the target protein is degraded to different degrees, so that the shortest marker is found to achieve the best degradation effect; (3) The UBR adopted is widely expressed and has strong activity of E3 ubiquitin ligase; (4) By replacing different amino acids (e.g. Arg, lys, leu, phe, etc.), these small molecules with only a different N-terminal can change the degradation rate due to different affinities for ubiquitin ligase UBR, thus optimizing the regulation of protein stability.
3. The invention establishes and verifies a novel PROTAC double-function small molecule for identifying E3 ubiquitin ligase by utilizing a minimum degradation signal-single amino acid based on an N-terminal rule, degrades target protein, and provides a new technical choice for realizing target attack; secondly, the invention selects single amino acid as a degradation signal, on one hand, the application range of E3 is further expanded, and on the other hand, the molecular weight of the compound is greatly reduced, so that the compound is easier to enter cells to exert effects; the third invention can achieve the best degradation effect by using the shortest PEG linker (one PEG), further reduces the molecular weight of the compound and improves the penetrability; finally, the Arg-PEG1-Dasa PROTAC bifunctional molecular degradation agent with high activity, which is designed by the invention, proves the capability of reducing the level of BCR-ABL protein in vivo and inhibits the growth of tumors, thereby providing a new strategy for future tumor targeted treatment.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.
FIG. 1 is a schematic diagram of PROTAC technology;
FIG. 2 is a high resolution mass spectrum of Arg-PEG 1-Dasa;
FIG. 3 is a high resolution mass spectrum of Arg-PEG 2-Dasa;
FIG. 4 is a high resolution mass spectrum of Arg-PEG 3-Dasa;
FIG. 5 is a high resolution mass spectrum of Arg-PEG 4-Dasa;
FIG. 6 shows in vitro degradation of BCR-ABL by Arg-PEG (1-4) -Dasa;
FIG. 7 is NH 2 -HRMS profile of PEG 1-Dasa;
FIG. 8 shows that different amino acids PROTAC have different degradation effects and degradation speeds on BCR-ABL;
FIG. 9 shows Arg-PEG1-Dasa degrades BCR-ABL and inhibits tumor growth in vivo.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The preparation methods of Arg-PEG1-Dasa, arg-PEG2-Dasa, arg-PEG3-Dasa, arg-PEG4-Dasa, lys-PEG1-Dasa, leu-PEG1-Dasa and Phe-PEG1-Dasa serial PROTAC compounds are proved to be practical through experiments, and the experimental conditions are repeatable and the yield is stable.
1. Preparation method of Arg-PEGn-Dasa
Step one Compound Fmoc-PEG n Synthesis of Dasa
To the dried reaction flask were added Dasatinib (Dasatinib) (1.0 eq) and Fmoc-PEG n -(CH 2 ) 2 -COOH (1.5 eq) was dissolved by adding an appropriate amount of anhydrous DMF followed by an equal volume of an anhydrous dichloromethane solution of DCC (1.5 eq) and DMAP (0.2 eq). The mixed reaction solution was stirred at room temperature for 4 hours, then the solvent was removed by rotary evaporator under reduced pressure, and the residue was purified by semi-preparative RP-HPLC and lyophilized to give Fmoc-PEG as a white powder n -Dasa。
By the method of step one, the following compounds can be synthesized:
the compound Fmoc-PEG1-Dasa.
The compound Fmoc-PEG2-Dasa.
The compound Fmoc-PEG3-Dasa.
The compound Fmoc-PEG4-Dasa.
Step two compound Arg-PEG n Synthesis of Dasa:
Fmoc-PEG n Dasa (1.0 eq) was dissolved in DMF solution containing 20% piperidine, and the mixture was stirred at room temperature for 1 hour and monitored by HPLC. After the reaction was completed, the solvent was removed by rotary evaporator under reduced pressure and high vacuum. The residue was dissolved in DMF and Boc-Arg (Pbf) -OH (1.2 eq), DIPEA (3.0 eq), HATU (1.2 eq) were added in sequence, the mixture was stirred at room temperature for 2 hours and purified by semi-preparative RP-HPLC, after which the Boc-Arg (Pbf) -PEG was obtained as a white powder after lyophilization n Dasa. The above product was dissolved in a dichloromethane solution containing 95% TFA, the reaction solution was stirred at room temperature for 0.5 hours, the protecting group was removed, and the solvent was removed by a rotary evaporator under reduced pressure, and the residue was purified by semi-preparative RP-HPLC to give a precipitate after lyophilizationArg-PEG in the form of white powder n -Dasa。
By the method of step two, the following compounds can be synthesized:
compound Arg-PEG1-Dasa, 73% yield. HRMS (ESI) m/z C 35 H 51 ClN 12 O 6 S[M+H] + Calculated values: 759.3274, found: 759.32794. the high resolution mass spectrum of Arg-PEG1-Dasa is shown in FIG. 2.
The compound Arg-PEG2-Dasa was 53% yield. HRMS (ESI) m/z C 35 H 51 ClN 12 O 6 S[M+H] + Calculated values: 803.3537, found: 803.35297. the high resolution mass spectrum of Arg-PEG1-Dasa is shown in FIG. 3.
The compound Arg-PEG3-Dasa was produced in 53% yield. HRMS (ESI) m/z C 37 H 55 ClN 12 O 7 S[M+H] + Calculated values: 847.3799, found: 847.37909. the high resolution mass spectrum of Arg-PEG1-Dasa is shown in FIG. 4.
The compound Arg-PEG4-Dasa was produced in 44% yield. HRMS (ESI) m/z C 39 H 59 ClN 12 O 8 S[M+H] + Calculated values: 891.4061, found: 891.40521. the high resolution mass spectrum of Arg-PEG1-Dasa is shown in FIG. 5.
EXAMPLE 1 verification of Arg-PEG (1-4) In vitro degradation of BCR-ABL by Dasa
ImmunoprintingTrace experiments revealed whether Arg-PEG (1-4) -Dasa reduced BCR-ABL protein levels: k562 cells expressing endogenous BCR-ABL gene were plated in 6-well plates, 1X 10 6 Cells were treated with Arg-PEG-Dasa PROTAC small molecules Arg-PEG1-Dasa, arg-PEG2-Dasa, arg-PEG3-Dasa, arg-PEG4-Dasa of different lengths of PEG linker per well. Each PROTAC was used at 5 different concentrations of 0, 0.1, 1, 10 2 、10 3 48 hours after nM treatment of cells, centrifugation at 5000rpm for 2mins, supernatant was discarded, cells were lysed with RIPA for 20mins, BCA protein was quantified, and immunoblotting experiments were performed with 50. Mu.g protein samples at each concentration to detect the protein levels of BCR-ABL at different concentrations of drug.
CCK-8 cell proliferation experiments confirm whether Arg-PEG (1-4) -Dasa inhibited K562 cell viability: k562 cells expressing the endogenous BCR-ABL gene were plated in 96-well plates, 5000/well, and cells were treated with Arg-PEG-Dasa PROTAC small molecules Arg-PEG1-Dasa, arg-PEG2-Dasa, arg-PEG3-Dasa, arg-PEG4-Dasa of different lengths of PEG linker. After cells were treated with 8 different concentrations of 0, 0.01, 0.1, 0.25, 0.5, 0.75, 1, 2.5nM for 48 hours for each PROTAC, 10. Mu.L of CCK-8 was added to each well and incubated at 37℃for 2 hours, OD was measured with a multifunctional microplate reader 450 Absorbance values were then calculated for different ProTAC versus K562 cells 50 。
Immunoblotting experiments revealed the effect of four PROTACArg-PEG (1-4) -Dasa on reducing BCR-ABL protein levels: k562 cells expressing endogenous BCR-ABL gene were plated in 6-well plates, 1X 10 6 After cells were treated with Arg-PEG-Dasa PROTAC small molecules Arg-PEG1-Dasa, arg-PEG2-Dasa, arg-PEG3-Dasa, arg-PEG4-Dasa of different lengths of PEG linker at the same concentration for 48 hours, centrifuged at 5000rpm for 2 minutes, the supernatant was discarded, the cells were lysed for 20 minutes with RIPA, BCA protein was quantified, and immunoblotting experiments were performed for 50. Mu.g protein samples at each concentration to examine the effect of the above four compounds on reducing BCR-ABL protein levels.
Evaluation of Arg-PEG1-Dasa whether BCR-ABL was degraded by proteasome: k562 cells expressing endogenous BCR-ABL gene were plated in 6-well plates, 1X 10 6 Cells were treated with DMSO (3 wells) and Arg-PEG1-Dasa (6 wells) for 36h, respectively, followed by DMSO (3 wells) and two Arg-PEG1-Dasa (3 wells) treatmentsGroups were treated with 50MG/kg CHX for 0, 3, 6h, wherein 10. Mu.M MG1326 h was added to a group of Arg-PEG1-Dasa treated cells (3 wells), centrifuged at 5000rpm for 2mins, the supernatant was discarded, cells were lysed with RIPA for 20mins, BCA protein was quantified, and immunoblotting experiments were performed at 50. Mu.g protein samples for each concentration to detect the protein level of BCR-ABL.
Verification of Arg-PEG (1-4) In vitro degradation of BCR-ABL by Dasa (fig. 6A): 0, 0.1, 1, 10 2 、10 3 nM five different concentrations of Arg-PEG1-Dasa, arg-PEG2-Dasa, arg-PEG3-Dasa, arg-PEG4-Dasa treated K562 cells for 48h, immunoblots showed that the four compounds all significantly reduced the protein level of BCR-ABL at 1nM (FIG. 6B). After cells were treated with 8 different concentrations of 0, 0.01, 0.1, 0.25, 0.5, 0.75, 1, 2.5nM for each PROTAC for 48 hours, CCK-8 cell proliferation experiments found that each of the four compounds significantly reduced cell activity, with Arg-PEG1-Dasa IC50:0.3595nM, arg-PEG2-Dasa IC50:0.5304nM, arg-PEG3-Dasa IC50:0.4803nM, arg-PEG4-Dasa IC50:0.5245nM (FIG. 6C). K562 cells were treated with 1nM Arg-PEG1-Dasa, arg-PEG2-Dasa, arg-PEG3-Dasa, arg-PEG4-Dasa for 48h and immunoblots showed the best inhibition of BCR-ABL by Arg-PEG1-Dasa at 1nM (FIG. 6D). According to Arg-PEG1-Dasa, the inhibition effect on BCR-ABL is best and the IC50 is lowest, so that further analysis on DC50 of Arg-PEG1-Dasa is found<1nM (FIG. 6E). Finally, by treating Arg-PEG1-Dasa for 36h and then CHX for 0, 3 and 6h and MG123 for 6h, the protein level of BCR-ABL was gradually reduced with the increase of the CHX addition time, and the reduction of BCR-ABL was completely inhibited after MG132 was added, which proves that Arg-PEG1-Dasa added promoted the degradation of BCR-ABL by proteasome (FIG. 6F).
Example 2 comparison of the degradation Effect and degradation Rate of different amino acids PROTAC on BCR-ABL
Immunoblotting experiments analyzed degradation concentrations of different amino acids procac to BCR-ABL: k562 cells expressing endogenous BCR-ABL gene were plated in 6-well plates, 1X 10 6 Cells were treated with PROTAC small molecules Arg-PEG1-Dasa, lys-PEG1-Dasa, leu-PEG1-Dasa, phe-PEG1-Dasa of different amino acids per well. Each PROTAC with 5 nothingAfter 48 hours of treatment of cells at the same concentrations 0, 0.1, 1, 2.5, 5nM, centrifugation at 5000rpm for 2mins, supernatant was discarded, cells were lysed with RIPA for 20mins, BCA protein was quantified, and immunoblotting experiments were performed with 50. Mu.g protein samples at each concentration to detect the protein levels of BCR-ABL at different concentrations of drug.
Immunoblotting experiments analyze the degradation effect of different amino acids PROTAC on BCR-ABL: k562 cells expressing endogenous BCR-ABL gene were plated in 6-well plates, 1X 10 6 Holes with minimum concentration of DMSO, arg-PEG1-Dasa, lys-PEG1-Dasa, leu-PEG1-Dasa, phe-PEG1-Dasa and NH capable of inhibiting BCR-ABL 2 After 48 hours of treatment of the cells with PEG1-Dasa intermediate (whose HRMS profile is shown in FIG. 7), centrifugation at 5000rpm for 2mins, the supernatant was discarded, cells were lysed with RIPA for 20mins, BCA protein was quantified, and immunoblotting experiments were performed with 50. Mu.g protein samples to detect the protein levels of BCR-ABL at the different compounds.
CCK-8 cell proliferation experiments compare the effect of different amino acids PROTAC on K562 cell viability: k562 cells expressing the endogenous BCR-ABL gene were plated in 96-well plates, 5000/well, and treated with PROTAC small molecules Lys-PEG1-Dasa, leu-PEG1-Dasa, phe-PEG1-Dasa of different amino acids. After cells were treated with 8 different concentrations of 0, 0.01, 0.1, 0.25, 0.5, 0.75, 1, 2.5nM for 48 hours for each PROTAC, 10. Mu.L of CCK-8 was added to each well, incubated at 37℃for 2 hours in an incubator, OD450 absorbance was measured with a multifunctional microplate reader, and then the IC of the different PROTACs against K562 cells was calculated 50 。
Immunoblotting experiments analyzed degradation rates of different amino acids PROTAC on BCR-ABL: k562 cells expressing endogenous BCR-ABL gene were plated in 6-well plates, 1X 10 6 Cells were treated with Arg-PEG1-Dasa, lys-PEG1-Dasa, leu-PEG1-Dasa, phe-PEG1-Dasa at the lowest concentration capable of inhibiting BCR-ABL for various times 0, 12, 24, 36, 48h,5000rpm respectively, centrifuged for 2mins, the supernatant was discarded, cells were lysed for 20mins with RIPA, BCA protein was quantified, and immunoblotting experiments were performed with 50. Mu.g protein samples to detect protein levels of BCR-ABL at various compounds. The cells were treated with Arg-PEG1-Dasa, lys-PEG1-Dasa, leu-PEG1-Dasa, phe-PEG1-Dasa at high concentrations capable of inhibiting the BCR-ABL protein level by 95% for different times 0, 12, respectivelyCentrifugation at 5000rpm for 2mins at 24, 36, 48h, supernatant was discarded, cells were lysed for 20mins with RIPA, BCA protein was quantified and immunoblotted with 50. Mu.g protein samples to detect protein levels of BCR-ABL at different compounds.
The degradation effect and degradation speed of different amino acids PROTAC on BCR-ABL are compared: by exchanging Arg for Lys, leu, phe we designed and synthesized the Lys-PEG1-Dasa, leu-PEG1-Dasa, phe-PEG1-Dasa series PROTAC compounds (FIG. 8A). Treatment of K562 cells with five different concentrations of Arg-PEG1-Dasa, lys-PEG1-Dasa, leu-PEG1-Dasa, phe-PEG1-Dasa at 0, 0.5, 1, 2.5, 5nM for 48h showed that the immunoblots significantly reduced the protein levels of BCR-ABL in each of the four compounds described above at 1nM (FIG. 8B). After 8 different concentrations of 0, 0.01, 0.1, 0.25, 0.5, 0.75, 1, 2.5nM of three PROTACs, lys-PEG1-Dasa, leu-PEG1-Dasa, phe-PEG1-Dasa, were used to treat cells for 48 hours, CCK-8 cell proliferation experiments were found to significantly reduce cell activity in all four of the above compounds, with Lys-PEG1-Dasa IC50:0.4569nM, leu-PEG1-Dasa IC50:0.2202nM, phe-PEG1-Dasa IC50:0.2939nM (FIG. 8C). With 1nM Arg-PEG1-Dasa, lys-PEG1-Dasa, leu-PEG1-Dasa, phe-PEG1-Dasa and intermediate NH 2 PEG1-Dasa treated K562 cells for 48h and immunoblots showed the best inhibition of BCR-ABL by Leu-PEG1-Dasa at 1nM (FIG. 8D). We treated K562 cells with the above four PROTACs at a minimum concentration of 1nM for reducing BCR-ABL for no time of 0, 12, 24, 36, 48h, found that four PROTACs were able to degrade BCR-ABL 50% at 1nM for Arg-PEG1-Dasa, respectively: 48h, lys-PEG1-Dasa: 48h, leu-PEG1-Dasa: about 12h, phe-PEG1-Dasa: 36h (fig. 8E). When we treated K562 cells with 10nM of the four PROTACs described above for no time 0, 12, 24, 36, 48 hours, four PROTACs were found to degrade BCR-ABL 50% of the time under 10nM conditions were Arg-PEG1-Dasa, respectively: 48h, lys-PEG1-Dasa: about 36h, leu-PEG1-Dasa: about 12h, phe-PEG1-Dasa: 24h (FIG. 8F).
Example 3 evaluation of Arg-PEG1-Dasa ability to degrade BCR-ABL level in vivo and anti-tumor Activity
Female nude mice of 4 weeks of age were modeled for tumor allograft. K562 cells (1X 10) 7 )/100μLPBS+Into 100 μl matrigel, subcutaneously injected into 4-week-old female nude mice. In case of tumor with size of 150mm 3 (0.5×L×W 2 ) After that, animals were randomly divided into two groups of 3 animals each. Control group Vehicle and administration group Arg-PEG1-Dasa (10 mg/kg) were injected intraperitoneally once every two days for a total of 10 times. Tumor size and mouse body weight were measured prior to each injection. Mice were sacrificed 27 days after injection of K562 cells and tumor specimens were collected for further analysis. Tumor growth curves and nude mice body weight change curves were plotted using mean ± SEM (n=3), using P ×<0.05、**P<0.01、***P<The statistical significance was assessed by the 0.001 method. All animal experiments were conducted in accordance with the relevant guidelines and regulations and were approved by the animal care and use committee of the experimental animal center at the university of south technology.
Evaluation of Arg-PEG1-Dasa ability to degrade BCR-ABL levels in vivo and anti-tumor Activity: k562 cells (1X 10) 7 ) 100 μl PBS+100 μl Matrigel was subcutaneously injected into 4 week old female nude mice. In case of tumor with size of 150mm 3 (0.5×L×W 2 ) After that, animals were randomly divided into two groups of 3 animals each. Control group Vehicle and administration group Arg-PEG1-Dasa (10 mg/kg) were injected intraperitoneally once every two days for a total of 10 times. The results show that the body weight of mice in the drug administration group and the control group is not obviously changed; the tumor volume of the administration group is slowly increased, and the tumor volume of the control group is in a steady-state increasing trend; after the experiment, mice were sacrificed, the stripped tumor tissues were weighed, the proteins were lysed and extracted, and immunoblots showed a significant decrease in BCR-ABL protein levels compared to the control drug group tumors, indicating that Arg-PEG1-Dasa was able to significantly degrade BCR-ABL protein levels in vivo and effectively inhibit tumor growth with fewer toxic and side effects (fig. 9).
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Claims (9)

1.A PROTAC compound or pharmaceutically acceptable salt thereof for inducing BCR-ABL degradation based on N-terminal rule, which is characterized in that the molecular structure of the compound comprises single amino acid and ligand Dasatinib, and the two are connected by PEG linker.
2. The protoc compound or pharmaceutically acceptable salt thereof inducing BCR-ABL degradation based on the N-terminal rule according to claim 1 wherein said single amino acid is selected from Gly, ala, val, leu, ile, phe, pro, trp, ser, tyr, cys, met, asp, asn, gln, glu, thr, lys, arg or His, preferably Arg, lys, leu or Phe.
3. The protoc compound or pharmaceutically acceptable salt thereof that induces BCR-ABL degradation based on N-terminal law according to claim 1, wherein the ligand Dasatinib has the structure shown in formula I:
wherein, one of x= F, cl, br, I; r is R 1 、R 2 ＝CH 3 、C 2 H 5 、C 3 H 7 、C 4 H 9 、C 5 H 11 One of them.
4. The protoc compound or pharmaceutically acceptable salt thereof that induces BCR-ABL degradation based on N-terminal law according to claim 1, wherein the PEG linker includes, but is not limited to, diethylene glycol, triethylene glycol, tetraethylene glycol, pentaethylene glycol.
5. The protoc compound or pharmaceutically acceptable salt thereof that induces BCR-ABL degradation based on N-terminal law according to claim 1, wherein said compound is selected from the group consisting of:
6. a pharmaceutical composition comprising a PROTAC compound or a pharmaceutically acceptable salt thereof that induces degradation of BCR-ABL based on the N-terminal law as claimed in any one of claims 1-5 and a pharmaceutically acceptable carrier or excipient.
7. Use of a protoc compound or a pharmaceutically acceptable salt thereof for inducing degradation of BCR-ABL according to any of claims 1-5 based on N-terminal law for the manufacture of a medicament for degrading the protein level of BCR-ABL in a body.
8. Use of a protoc compound or a pharmaceutically acceptable salt thereof according to any of claims 1-5 for the manufacture of an antitumor drug based on N-terminal law induced degradation of BCR-ABL.
9. The use according to claim 7, wherein the tumour is a blood cancer, brain cancer, lung cancer or kidney cancer.
CN202311653111.8A 2023-04-14 2023-12-03 PROTAC compound for inducing BCR-ABL degradation based on N-terminal rule and application thereof Pending CN117659002A (en)

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