WO2021208787A1 - Polypeptide cible utilisé pour le criblage de médicaments et procédé de criblage associé - Google Patents

Polypeptide cible utilisé pour le criblage de médicaments et procédé de criblage associé Download PDF

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WO2021208787A1
WO2021208787A1 PCT/CN2021/085836 CN2021085836W WO2021208787A1 WO 2021208787 A1 WO2021208787 A1 WO 2021208787A1 CN 2021085836 W CN2021085836 W CN 2021085836W WO 2021208787 A1 WO2021208787 A1 WO 2021208787A1
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amino acid
myc
target polypeptide
wbc100
protein
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PCT/CN2021/085836
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Chinese (zh)
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徐荣臻
干小仙
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徐荣臻
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4418—Non condensed pyridines; Hydrogenated derivatives thereof having a carbocyclic group directly attached to the heterocyclic ring, e.g. cyproheptadine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/22—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains four or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the present invention relates to a target polypeptide for screening drugs for treating and/or preventing cancer and polynucleotides encoding the same, and also relates to a method for screening drugs for treating and/or preventing cancer by using the target polypeptide of the present invention.
  • MYC oncogene is the "main driver" of human cancer. It is used in many hematological malignancies (including leukemia (2-4), lymphoma (5, 6) and multiple myeloma (7, 8)) and various Types of solid tumors (e.g. pancreatic ductal adenocarcinoma (PDAC) (9,10), brain cancer (11-13), non-small cell lung cancer (NSCLC) (14,15) and small cell lung cancer (SCLC) (16,17) , Liver cancer (18-20), prostate cancer (21, 22), etc.).
  • PDAC pancreatic ductal adenocarcinoma
  • MYC molecular targeted therapy
  • overactivation of MYC is usually associated with aggressiveness and poor clinical outcomes (23-25).
  • genetic studies have shown that inhibition of c-MYC activation can rapidly regress tumors by inhibiting cell proliferation, inducing senescence and apoptosis, and reshaping the tumor microenvironment in animal models (26).
  • depletion of MYC transcriptional activity can lead to the eradication of mouse lung adenocarcinoma driven by oncogenic K-RAS without serious side effects (27).
  • targeting MYC represents an important milestone in the era of molecular targeted therapy of human cancer.
  • MYC inhibitors with similar drugs have not been developed for more than 30 years.
  • Previous studies have reported several MYC inhibitors, including 10074-G5, 10058-F4 and 10074-A4 (28,29).
  • MYCi361 and MYCi975 have been reported (30), but they are all related to "undruggable" "The bHLHZip domain (including the LZ domain and the HLH domain) binds and does not show significant anti-tumor activity in vivo (28-30).
  • MYC protein is "non-drugable" due to its helix-loop-helix topology lacks druggable domains and nuclear localization in cancer cells, which makes it difficult to be directly targeted by traditional small molecule inhibitors (31-34 ).
  • the art needs new screening methods to obtain drugs that can effectively target MYC protein and have anti-tumor activity.
  • the present invention satisfies the above-mentioned needs by providing target polypeptides for screening drugs for treating and/or preventing cancer and methods for screening drugs for treating and/or preventing cancer using the target polypeptides of the present invention.
  • the present invention provides a target polypeptide for screening drugs for the treatment and/or prevention of cancer, which is an isolated polypeptide with a length of ⁇ 200 amino acids and contains a nuclear localization signal domain with a mammalian MYC protein.
  • An amino acid sequence with about 70% to 100% identity.
  • the target polypeptide comprises an amino acid sequence having about 70% to 100% identity with the multiple nuclear localization signal domain of the mammalian MYC protein, the multiple nuclear localization signal domain from the N-terminus to the The C-terminus is the first nuclear localization signal domain of the MYC protein, the domain between the first nuclear localization signal domain of the MYC protein and the second nuclear localization signal domain of the MYC protein, Optionally, the second nuclear localization signal domain of the MYC protein.
  • the present invention provides a fusion protein comprising the target polypeptide of the present invention.
  • the present invention provides a surface display system comprising the target polypeptide or fusion protein of the present invention, wherein the target polypeptide or the fusion protein is positioned on the surface of the surface display system.
  • the present invention provides an isolated polynucleotide encoding the target polypeptide or fusion protein of the present invention.
  • the present invention provides an expression vector comprising the polynucleotide of the present invention.
  • the present invention provides a host cell comprising the polynucleotide or expression vector of the present invention.
  • the present invention provides a method for producing the target polypeptide or fusion protein of the present invention, which comprises culturing the target polypeptide or fusion protein of the present invention under conditions suitable for expression of the target polypeptide or the fusion protein. Host cell, and the expressed target polypeptide or the fusion protein recovered from the culture medium.
  • the present invention provides a kit comprising the target polypeptide, fusion protein, or surface display system of the present invention.
  • the present invention provides a method for screening drugs for the treatment and/or prevention of cancer using the target polypeptide, fusion protein, or surface display system of the present invention, which includes screening for the target polypeptide, the fusion The protein or the candidate compound to which the surface display system binds.
  • the method for screening drugs for the treatment and/or prevention of cancer further comprises determining the binding affinity of the candidate compound to the target polypeptide, the fusion protein or the surface display system; and The affinity further screens the candidate compound to obtain the selected compound.
  • the WBC100 compound (green) simultaneously contacts the NLS2 and NLS1 motifs in the docking model.
  • the NLS1 and NLS2 motifs are colored light blue and blue, respectively. Except for the NLS motif, the potential ligand binding region is colored yellow.
  • Figure 5 WBC100 selectively induces MYC protein degradation by recruiting ubiquitin E3 ligase CHIP.
  • MOLM-13 cells were pretreated with WBC100 (2.0 ⁇ mol/L) or DMSO for 6 hours, and then treated with cycloheximide (CHX; 25 ⁇ mol/L). The cells were harvested at the indicated time points, and the MYC level was determined by Western blotting.
  • H9 cells were pretreated with WBC100 (2.0 ⁇ mol/L) or DMSO for 6 hours, and then treated with cycloheximide (CHX; 25 ⁇ mol/L). The cells were harvested at the indicated time points, and the MYC level was determined by Western blotting.
  • WBC100 induces ubiquitin-mediated degradation of MYC protein.
  • the 293T cells expressing HA-MYC were treated with WBC100 (2 ⁇ M) for designated hours, and then HA-MYC protein was pulled down by immunoprecipitation (IP) with HA antibody magnetic beads.
  • IP immunoprecipitation
  • Ubiquitin-specific monoclonal antibodies were used to determine ubiquitinated HA-MYC by Western blotting.
  • MOLM-13 cells were treated with WBC100 (2 ⁇ M) or DMSO for 4 hours. The cells were collected for coIP with MYC antibody and Western blot analysis of MYC, CHIP, Hsp70 and Hsp90 proteins.
  • WBC100 CHIP-mediated MYC degradation model by WBC100.
  • WBC100 binds to the MYC NLS domain, and recruits CHIP and Hsp70 to MYC protein for 26S proteasome-mediated degradation.
  • Figure 6 WBC100 cures MYC-positive AML in vivo and effectively regresses tumors in the PDX mouse model.
  • Figure 8 WBC100 effectively regressed MYC-positive human non-small cell lung cancer (NSCLC) in NOD/SCID mice.
  • NSCLC non-small cell lung cancer
  • WBC100-FITC and RFP-MYC protein co-localize in the nucleus and induce apoptosis of RFP-MYC positive cells.
  • Blank Incubate WBC100-FITC (0.25mg/ml) and biotin (0.25mg/ml) in PBS buffer at 4°C overnight, and then mix with streptavidin magnetic beads (Invitrogen) at 4 Incubate overnight at °C.
  • Negative control Incubate biotin-MYC NLS (1mg/ml) and FITC (0.25mg/ml) in PBS buffer at 4°C overnight, and then mix with streptavidin magnetic beads (Invitrogen) in Incubate overnight at 4°C.
  • Figure 16 WBC100 reduced the levels of Ki-67 and c-Myc, and was accompanied by an increase in apoptotic cells in mouse tumor xenografts.
  • Figure 18 WBC100 effectively regressed MYC-positive human NSCLC in vivo.
  • Figure 20 The effect of WBC100 on blood cell count and liver enzyme ALT in healthy mice.
  • ALT a
  • WBC white blood cells
  • PLT platelets
  • HGB hemoglobin
  • the present invention provides a target polypeptide for screening drugs for the treatment and/or prevention of cancer.
  • MYC protein such as MYCi361 and MYCi975
  • the present invention finds for the first time that the nuclear localization signal domain of MYC can be used as a target to effectively target MYC protein. It overcomes the problem of MYC that is unsolvable for decades, and “converts” MYC protein from a non-drugable target to Targets that can be made into medicines.
  • the present invention also provides a method for screening drugs for the treatment and/or prevention of cancer using the target polypeptides described herein, making it possible to screen for drugs with excellent binding affinity to MYC protein (such as small molecule drugs or macromolecular drugs) , And make it possible to screen for drugs that can effectively target MYC protein and have anti-tumor activity.
  • MYC protein such as small molecule drugs or macromolecular drugs
  • the NLS domain of MYC is the first druggable pocket used to target the otherwise "undruggable” MYC protein.
  • the 14-D-valine-triptolide (WBC100) small molecule inhibitor that targets MYC protein by binding to the nuclear localization signal domain of MYC can cause the degradation of MYC protein, which in turn leads to the apoptosis of MYC-positive cells. Death.
  • WBC100 combined with the nuclear localization signal domain of MYC has better anti-tumor effects, especially for deadly cancer cells and highly aggressive cancer cells. Tumor effect.
  • the present invention provides a target polypeptide for screening drugs for the treatment and/or prevention of cancer, which is an isolated polypeptide with a length of ⁇ 200 amino acids and contains a nuclear localization signal domain with a mammalian MYC protein.
  • An amino acid sequence with about 70% to 100% identity.
  • the target polypeptide comprises an amino acid sequence having about 70% to 100% identity with the multiple nuclear localization signal domain of the mammalian MYC protein, the multiple nuclear localization signal domain from the N-terminus to the The C-terminus is the first nuclear localization signal domain of the MYC protein, the domain between the first nuclear localization signal domain of the MYC protein and the second nuclear localization signal domain of the MYC protein, Optionally, the second nuclear localization signal domain of the MYC protein.
  • the mammalian MYC protein includes human C-MYC, human N-MYC, and human L-MYC.
  • the target polypeptide comprises an amino acid sequence having about 70% to 100% identity with the amino acid sequence shown in SEQ ID NO:1, wherein the second amino acid A in SEQ ID NO:1 remains unchanged. Change.
  • the target polypeptide comprises an amino acid sequence having about 70% to 100% identity with the amino acid sequence shown in SEQ ID NO: 2, wherein the amino acid A at position 2 and the amino acid sequence at position 2 in SEQ ID NO: 2 Amino acid L at position 14 remains unchanged.
  • the target polypeptide comprises an amino acid sequence having about 70% to 100% identity with the amino acid sequence shown in SEQ ID NO: 3, wherein the second amino acid A and the first amino acid sequence in SEQ ID NO: 3 Amino acid L at position 14 and amino acid R at position 27 remain unchanged.
  • the target polypeptide comprises an amino acid sequence having about 70% to 100% identity with the amino acid sequence shown in SEQ ID NO: 4, wherein the second amino acid A and the first amino acid sequence in SEQ ID NO: 4 The 14th amino acid L, the 27th amino acid R and the 32nd amino acid E remain unchanged.
  • the target polypeptide comprises an amino acid sequence having about 70% to 100% identity with the amino acid sequence shown in SEQ ID NO: 5, wherein the second amino acid A and the first amino acid sequence in SEQ ID NO: 5
  • the 14th amino acid L, the 27th amino acid R, the 32nd amino acid E, and the 46th amino acid Q remain unchanged.
  • the target polypeptide comprises an amino acid sequence having about 70% to 100% identity with the amino acid sequence shown in SEQ ID NO: 6, wherein the second amino acid A and the first amino acid sequence in SEQ ID NO: 6 The 14th amino acid L, the 27th amino acid R, the 32nd amino acid E, and the 46th amino acid Q remain unchanged.
  • the target polypeptide comprises an amino acid that is about 70% to 100% identical to the nuclear localization signal domain or multiple nuclear localization signal domain of the human N-MYC amino acid sequence shown in SEQ ID NO: 8. sequence.
  • the target polypeptide comprises an amino acid having about 70% to 100% identity with the nuclear localization signal domain or multiple nuclear localization signal domain of the human L-MYC amino acid sequence shown in SEQ ID NO: 9 sequence.
  • the target polypeptide is an amino acid sequence that is about 70% to 100% identical to a truncated mammalian MYC protein, which is truncated at the N-terminus and at the C The ends are truncated, are truncated at both the N-terminus and the C-terminus, or are truncated inside the MYC protein.
  • the target polypeptide is ⁇ 150, 100, 90, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9 in length. , 8, 7, or 6 amino acid polypeptides.
  • identity is at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100%.
  • the amino acid sequence of the target polypeptide is PAAKRVKLD (SEQ ID NO: 1).
  • the amino acid sequence of the target polypeptide is PAAKRVKLDSVRVL (SEQ ID NO: 2).
  • the amino acid sequence of the target polypeptide is PAAKRVKLDSVRVLRQISNNRKCTSPR (SEQ ID NO: 3).
  • the amino acid sequence of the target polypeptide is PAAKRVKLDSVRVLRQISNNRKCTSPRSSDTE (SEQ ID NO: 4).
  • the amino acid sequence of the target polypeptide is PAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEENVKRRTHNVLERQ (SEQ ID NO: 5).
  • amino acid sequence of the target polypeptide is PAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEENVKRRTHNVLERQRRNELKRSF (SEQ ID NO: 6).
  • the target polypeptide does not comprise the LZ domain, HLH domain, or both of the mammalian MYC protein.
  • the present invention provides a fusion protein comprising the target polypeptide of the present invention.
  • the fusion protein further includes a tag.
  • the tag includes HA, Flag, GST, His tag, biotin or streptavidin, or any combination thereof.
  • the target polypeptide or the fusion protein is a synthetic polypeptide or a synthetic protein.
  • the target polypeptide or the fusion protein is a polypeptide or protein expressed through genetic engineering.
  • the present invention provides a surface display system, which comprises the target polypeptide of the present invention or the fusion protein of the present invention, wherein the target polypeptide or the fusion protein is located on the surface of the surface display system. surface.
  • the surface display system is a yeast surface display system or a baculovirus surface display system.
  • the present invention provides an isolated polynucleotide encoding the target polypeptide or fusion protein of the present invention.
  • the present invention provides an expression vector comprising the polynucleotide of the present invention.
  • the present invention provides a host cell comprising the polynucleotide or expression vector of the present invention.
  • the present invention provides a method for producing the target polypeptide or fusion protein of the present invention, which comprises culturing the target polypeptide or fusion protein of the present invention under conditions suitable for expression of the target polypeptide or the fusion protein. Host cell, and the expressed target polypeptide or the fusion protein recovered from the culture medium.
  • the present invention provides a kit comprising the target polypeptide, fusion protein, or surface display system of the present invention.
  • the present invention provides a method for screening drugs for the treatment and/or prevention of cancer using the target polypeptide, fusion protein, or surface display system of the present invention, which includes screening for the target polypeptide, the fusion The protein or the candidate compound to which the surface display system binds.
  • the method for screening drugs for the treatment and/or prevention of cancer further comprises determining the binding affinity of the candidate compound to the target polypeptide, the fusion protein or the surface display system; and The affinity further screens the candidate compound to obtain the selected compound.
  • the method for screening drugs for the treatment and/or prevention of cancer further comprises screening for a compound to be determined that binds to the target polypeptide, the fusion protein, or the surface display system having a point mutation.
  • the mutation is selected from one or more of the following: SEQ ID NO: 6 amino acid A, 14 amino acid L, 27 amino acid R, 32 amino acid E, 46 amino acid Q, and any of them Combining; and comparing the candidate compound with the to-be-determined compound to obtain a selected compound.
  • the method for screening drugs for the treatment and/or prevention of cancer further comprises determining whether the selected compound causes the degradation of mammalian MYC protein.
  • the method for screening drugs for treating and/or preventing cancer further includes determining the killing effect of the selected compound on cancer cells.
  • MYC oncogene is the "main driver" of human cancer, and is abnormally activated in many hematological malignancies and various solid tumors.
  • MYC protein is "non-drugable” due to its helix-loop-helix topology lacking druggable domains and nuclear localization in cancer cells, which makes it difficult to be directly targeted by traditional small molecule inhibitors.
  • small molecules that can bind to MYC protein such as MYCi361 and MYCi975
  • the art needs new screening methods to obtain drugs that can effectively target MYC and have anti-tumor activity.
  • MYC genes have been found to include C-MYC, N-MYC, L-MYC, etc., which are located on chromosome 8, chromosome 2 and chromosome 1, respectively.
  • the human C-MYC protein has 439 amino acids, as shown in SEQ ID NO: 7, which includes two nuclear localization signal domains separated from each other.
  • the human N-MYC protein has 464 amino acids, as shown in SEQ ID NO: 8, which includes two nuclear localization signal domains separated from each other.
  • the human L-MYC protein has 367 amino acids, as shown in SEQ ID NO: 9, which includes two nuclear localization signal domains separated from each other.
  • the present invention provides a target polypeptide for screening drugs for the treatment and/or prevention of cancer.
  • the target polypeptide is an isolated polypeptide with a length of ⁇ 200 amino acids and contains a nuclear localization signal with a mammalian MYC protein.
  • the domain has an amino acid sequence that is about 70% to 100% identical.
  • the identity is at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5%. In one embodiment, the identity is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%. %, 91%, 92%, 93%, 94%, 95%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5% or 99.9%.
  • the length of the target polypeptide is ⁇ 190, 180, 170, 160, 150, 140, 130, 120, 110, or 100 amino acids, more preferably, the length of the target polypeptide is ⁇ 100, 99 , 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74 , 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49 , 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24 , 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7
  • the nuclear localization signal domain of the mammalian MYC protein is the nuclear localization signal domain of C-MYC, N-MYC, L-MYC or other MYC gene family members or any combination thereof.
  • the mammalian MYC protein is a human MYC protein.
  • the target polypeptide comprises one or two or more (e.g., three, four, five or more) nuclear localization signal domains of mammalian MYC protein, or is combined with the one or
  • the amino acid sequences of the nuclear localization signal domains of two or more mammalian MYC proteins have about 70% to 100% identical amino acid sequences, specifically, at least about 70%, 80%, 90%, 95%, Amino acid sequences that are 96%, 97%, 98%, 99%, or 99.5% identical, more specifically, have at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85% , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99 %, 99.5%, or 99.9% identical amino acid sequence.
  • the nuclear localization signal domains of the one or more MYC may be the same or different.
  • the target polypeptide comprises an amino acid sequence having about 70% to 100% identity with the multiple nuclear localization signal domain of a mammalian MYC protein, specifically, at least about 70%, 80%, 90%. , 95%, 96%, 97%, 98%, 99%, or 99.5% identical amino acid sequences, more specifically, at least about 70%, 75%, 80%, 81%, 82%, 83%, 84 %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96.5%, 97%, 97.5%, 98%, Amino acid sequence with 98.5%, 99%, 99.5% or 99.9% identity.
  • the multiple nuclear localization signal domains of the MYC protein are the first nuclear localization signal domain of the MYC protein, the first nuclear localization signal domain of the MYC protein, and the second nuclear localization of the MYC protein from the N-terminus to the C-terminus.
  • the domain between the signal domains optionally, the second nuclear localization signal domain of the MYC protein.
  • the multiple nuclear localization signal domains may include one or more nuclear localization signal domains and domains between the nuclear localization signal domains.
  • the multiple nuclear localization domain comprises the first nuclear localization signal domain of MYC protein from N-terminus to C-terminus, and the first nuclear localization signal domain of the MYC protein and the first nuclear localization signal domain of the MYC protein The domain between the two nuclear localization signal domains.
  • the multiple nuclear localization domain comprises the first nuclear localization signal domain of MYC protein from N-terminus to C-terminus, and the first nuclear localization signal domain of the MYC protein and the first nuclear localization signal domain of the MYC protein The domain between the two nuclear localization signal domains, and the second nuclear localization signal domain of the MYC protein.
  • the target polypeptide comprises an amino acid sequence having about 70% to 100% identity with the amino acid sequence shown in SEQ ID NO:1, specifically, having at least about 70%, 80%, 90%, Amino acid sequences that are 95%, 96%, 97%, 98%, 99%, or 99.5% identical, more specifically, have at least about 70%, 75%, 80%, 81%, 82%, 83%, 84% , 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5% %, 99%, 99.5%, or 99.9% identical amino acid sequence, wherein the second amino acid A in SEQ ID NO:1 remains unchanged.
  • the target polypeptide comprises an amino acid sequence having about 70% to 100% identity with the amino acid sequence shown in SEQ ID NO: 2, specifically, at least about 70%, 80%, 90%, Amino acid sequences that are 95%, 96%, 97%, 98%, 99%, or 99.5% identical, more specifically, have at least about 70%, 75%, 80%, 81%, 82%, 83%, 84% , 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5% %, 99%, 99.5%, or 99.9% identical amino acid sequence, wherein the second amino acid A and 14th amino acid L in SEQ ID NO: 2 remain unchanged
  • the target polypeptide comprises an amino acid sequence having about 70% to 100% identity with the amino acid sequence shown in SEQ ID NO: 3, specifically, having at least about 70%, 80%, 90%, Amino acid sequences that are 95%, 96%, 97%, 98%, 99%, or 99.5% identical, more specifically, have at least about 70%, 75%, 80%, 81%, 82%, 83%, 84% , 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5% %, 99%, 99.5%, or 99.9% identical amino acid sequence, wherein the second amino acid A, the 14th amino acid L, and the 27th amino acid R in SEQ ID NO: 3 remain unchanged
  • the target polypeptide comprises an amino acid sequence having about 70% to 100% identity with the amino acid sequence shown in SEQ ID NO: 4, specifically, at least about 70%, 80%, 90%, Amino acid sequences that are 95%, 96%, 97%, 98%, 99%, or 99.5% identical, more specifically, have at least about 70%, 75%, 80%, 81%, 82%, 83%, 84% , 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5% %, 99%, 99.5%, or 99.9% identical amino acid sequence, wherein the amino acid at position 2 A, amino acid L at position 14, amino acid R at position 27, and amino acid E at position 32 in SEQ ID NO: 4 remain unchanged .
  • the target polypeptide comprises an amino acid sequence having about 70% to 100% identity with the amino acid sequence shown in SEQ ID NO: 5, specifically, at least about 70%, 80%, 90%, Amino acid sequences that are 95%, 96%, 97%, 98%, 99%, or 99.5% identical, more specifically, have at least about 70%, 75%, 80%, 81%, 82%, 83%, 84% , 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5% %, 99%, 99.5%, or 99.9% identical amino acid sequence, in which SEQ ID NO: 5 has amino acid A at position 2, amino acid L at position 14, amino acid R at position 27, amino acid E at position 32, and amino acid at position 46. The amino acid Q remains unchanged.
  • the target polypeptide comprises an amino acid sequence having about 70% to 100% identity with the amino acid sequence shown in SEQ ID NO: 6, specifically, at least about 70%, 80%, 90%, Amino acid sequences that are 95%, 96%, 97%, 98%, 99%, or 99.5% identical, more specifically, have at least about 70%, 75%, 80%, 81%, 82%, 83%, 84% , 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5% %, 99%, 99.5%, or 99.9% identical amino acid sequence, in which SEQ ID NO: 6 has amino acid A at position 2, amino acid L at position 14, amino acid R at position 27, amino acid E at position 32, and amino acid at position 46. The amino acid Q remains unchanged.
  • the target polypeptide comprises an amino acid that is about 70% to 100% identical to the nuclear localization signal domain or multiple nuclear localization signal domain of the human N-MYC amino acid sequence shown in SEQ ID NO: 8.
  • a sequence specifically, an amino acid sequence having at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity, more specifically, at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 99.9% identical amino acid sequence.
  • the target polypeptide comprises an amino acid having about 70% to 100% identity with the nuclear localization signal domain or multiple nuclear localization signal domain of the human L-MYC amino acid sequence shown in SEQ ID NO: 9 A sequence, specifically, an amino acid sequence having at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity, more specifically, at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 99.9% identical amino acid sequence.
  • the target polypeptide is an amino acid sequence with about 70% to 100% identity with the truncated MYC, specifically, at least about 70%, 80%, 90%, 95%, 96%, An amino acid sequence of 97%, 98%, 99%, or 99.5% identity, more specifically, at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86% , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5% % Or 99.9% identical amino acid sequence, the truncated MYC is truncated at the N-terminus, truncated at the C-terminus, or truncated at both the N-terminus and the C-terminus, or within the MYC protein Be truncated.
  • the present invention provides a fusion protein comprising the target polypeptide described herein.
  • the fusion protein further includes a tag.
  • the tag is an affinity tag, including but not limited to influenza hemagglutinin (HA) tag, FLAG tag, glutathione-S-transferase (GST), histidine (His) tag , Biotin/Streptavidin or any combination thereof.
  • HA hemagglutinin
  • FLAG tag glutathione-S-transferase
  • His histidine
  • Biotin/Streptavidin Biotin/Streptavidin or any combination thereof.
  • the tag is located at the N-terminus of the polypeptide.
  • the tag is located at the C-terminus of the polypeptide.
  • the tag is directly located at the N-terminus or C-terminus of the polypeptide.
  • the tag is located at the N-terminus or C-terminus of the polypeptide via a linker.
  • the tag and/or linker can be separated from the polypeptide, for example, by means of site-specific enzyme cleavage.
  • the target polypeptide or fusion protein described herein is a synthetic polypeptide or a synthetic protein.
  • Methods for obtaining synthetic polypeptides or synthetic proteins include, but are not limited to, solid-phase synthesis technology or liquid-phase synthesis technology.
  • the target polypeptide or fusion protein described herein is a polypeptide or protein expressed through genetic engineering.
  • the present invention provides a method for producing the target polypeptide or fusion protein described herein, which comprises culturing the host cell described herein under conditions suitable for the expression of the target polypeptide or fusion protein, and from culturing The expressed target polypeptide or fusion protein is recovered from the base.
  • the host cell is a prokaryotic cell, such as E. coli.
  • the host cell is an insect cell, such as Sf9 cell, Sf21 cell, etc.
  • the host cell is a mammalian cell, such as a human cell.
  • the present invention provides a method for screening drugs for the treatment and/or prevention of cancer using the target polypeptide, fusion protein or surface display system described herein, which includes screening for the target polypeptide, fusion protein or surface display system.
  • Candidate compounds for system binding the method further includes determining the binding affinity of the candidate compound to the target polypeptide, fusion protein or surface display system and further screening the candidate compound based on the affinity to obtain the selected compound.
  • the method further includes screening for a compound to be determined that binds to a target polypeptide, fusion protein, or surface display system having a point mutation, the point mutation being selected from one or more of the following: SEQ ID NO: 6 Amino acid A at position 2, amino acid L at position 14, amino acid R at position 27, amino acid E at position 32, amino acid Q at position 46, and any combination thereof; and comparing the candidate compound with the compound to be determined to obtain Selected compound.
  • the point mutation target comprises amino acid A at position 2, amino acid L at position 14, amino acid R at position 27, amino acid E at position 32, and amino acid Q at position 46 selected from SEQ ID NO: 6. One, two, three, four or five of them.
  • the candidate compound that binds to the target polypeptide, fusion protein or surface display system is compared with the candidate compound, and the candidate compound is removed from the candidate compound to obtain the selected compound.
  • the method further includes determining whether the selected compound causes degradation of MYC protein. In one embodiment, whether the selected compound causes degradation of MYC protein is determined in a cell.
  • the cell may be, for example, a cell that overexpresses MYC by transfection of the MYC gene, or a cancer cell that highly expresses MYC, as described herein.
  • whether the selected compound causes degradation of MYC protein is determined in a cell lysate.
  • the method further includes determining the killing effect of the selected compound on cancer cells, such as cancer cells that highly express MYC, such as the cancer cells detailed in the Examples herein.
  • WBC100 14-D-valine-triptolide
  • WBC100 is the first selective small molecule degradation agent for MYC protein that is effective both in vitro and in vivo.
  • WBC100 directly targets the MYC nuclear localization signal (NLS) domain (an undiscovered pocket that targets MYC protein), and has shown a variety of effects in mouse models of various hematological and solid malignancies in vitro and in vivo.
  • NLS MYC nuclear localization signal
  • mice tolerate WBC100 at a dose that effectively kills cancer cells, which provides a broad therapeutic window for the treatment of MYC-positive cancers.
  • MYC has been considered "non-drugable” due to its helix-loop-helix topology lacks druggable domains and nuclear localization in cancer cells, which makes it difficult to be directly inhibited by traditional small molecules.
  • Agent targeting (31-34).
  • the present disclosure has determined that the NLS domain is the first druggable pocket for targeting the "non-druggable" MYC protein.
  • WBC100 is an effective orally effective small molecule inhibitor, which directly targets MYC protein by binding to the MYC NLS domain to undergo CHIP-mediated degradation of the ubiquitin E3 ligase, thereby leading to the apoptosis of MYC-positive cells.
  • Oral administration of WBC100 showed complete tumor regression in a variety of refractory blood and solid tumor mouse models without serious side effects.
  • the present disclosure provides the first evidence that MYC protein is a druggable target by targeting its NLS domain, and the direct pharmacological inhibition of MYC protein with WBC100 is tolerable and effective against a variety of MYC-positive cancers.
  • the present disclosure shows that WBC100 can enter the nucleus and directly target the "undruggable" MYC protein by binding to the NLS domain for E3 ligase CHIP-mediated degradation.
  • XPB is considered to be a candidate target of triptolide (38,39), the parent compound of WBC100), Rpb1 and STAT3 at an effective anti-tumor dose, which indicates that WBC100 selectively targets MYC protein to the nucleus.
  • WBC100 can be completely tested at a safe dose. Eliminate MYC-positive AML MOLM-13 cells and T-cell lymphoma H9 cells while preserving normal tissues. AML is a common fatal human leukemia, with a 5-year overall survival (OS) rate of less than 40% using chemotherapy, which highlights the need for new targets and drugs. Treatment with WBC100 at a dose of 0.2-0.4 mg kg -1 can completely eliminate AML-MOLM-13 in the body, and all mice survived disease-free.
  • OS overall survival
  • Peripheral T-cell lymphoma is a highly aggressive tumor that is usually resistant to conventional chemotherapy (53,54), and because most patients relapse within 2 years, the prognosis is still poor. Therefore, improved front-line treatment strategies are needed.
  • the present disclosure shows that WBC100 can even completely eradicate MYC-positive T-cell lymphoma at a low dose (0.1-0.2 mg/kg) in a xenograft mouse model.
  • WBC100 can effectively regress MYC-positive pancreatic ductal adenocarcinoma (PDAC), non-small cell lung cancer (NSCLC) and gastric cancer.
  • PDAC pancreatic ductal adenocarcinoma
  • NSCLC non-small cell lung cancer
  • MYC is an important driver in PDAC (55).
  • Treatment with WBC100 at doses of 0.1 mg/kg, 0.2 mg/kg and 0.4 mg/kg produced tumor growth inhibition (TGI) of 71.94%, 87.63% and 96.14%, respectively, while the standard care drug gemcitabine only accounted for 31.83% of TGI.
  • TGI tumor growth inhibition
  • Non-small cell lung cancer is the most common tumor and the leading cause of cancer death worldwide.
  • treatment with WBC100 at a dose of 0.4 mg/kg showed 93.24% tumor growth inhibition in mouse MYC-positive H1975 xenografts.
  • the present disclosure demonstrates that treatment with WBC100 at a dose of 0.4 mg/kg is sufficient to inhibit the tumor growth of MYC-positive gastric cancer, a common gastrointestinal (GI) tumor.
  • GI gastrointestinal
  • MYC-positive cancer cells carrying mutant oncogenes such as MOLM13 with FLT3-ITD mutant, MIA-paca2 with K-ras mutant, and EGFR (T790M) mutation
  • MOLM13 with FLT3-ITD mutant a mutant oncogenes
  • MIA-paca2 with K-ras mutant a mutant oncogenes
  • EGFR (T790M) mutation The body H1975 is also very sensitive to WBC100. This suggests that WBC100 may be effective in situations where standard chemotherapy drugs are difficult to treat.
  • MYC is an ideal predictive biomarker for distinguishing sensitive tumors from drug-resistant tumors, and can be used to guide the WBC100 treatment of individual patients.
  • isolated polypeptide or “isolated polynucleotide” is a polypeptide or polynucleotide that is separated and/or recovered from a component of its natural environment, and does not require a specific level of purification.
  • the isolated polypeptide can be taken from its natural or natural environment.
  • artificially synthesized polypeptides or polypeptides recombinantly expressed in host cells by genetic engineering are considered to be isolated.
  • a polypeptide or polynucleotide isolated and/or recovered from its naturally-occurring polypeptide form or polynucleotide form is considered to be isolated, such as a polypeptide isolated from a naturally-occurring mammalian MYC protein, including but It is not limited to truncated parts of mammalian MYC protein, or a combination of different truncated parts, or variants or derivatives thereof.
  • nuclear localization signal domain is an amino acid sequence that can guide a protein or polypeptide into the nucleus, and it can be located in any part of the protein or polypeptide.
  • sequence identity between two polypeptides or nucleic acid sequences means the number of identical residues between the sequences as a percentage of the total number of residues, and the calculation of the total number of residues is determined based on the type of mutation.
  • Types of mutations include insertions (extensions) at either or both ends of the sequence, deletions (truncation) at either or both ends of the sequence, substitutions/substitutions of one or more amino acids/nucleotides, insertions within the sequence, and insertions in the sequence.
  • the mutation type is one or more of the following: substitution/substitution of one or more amino acids/nucleotides, insertions within the sequence, and deletions within the sequence .
  • the total number of residues is calculated with the larger of the compared molecules. If the mutation type also includes insertion (extension) at either end or both ends of the sequence or deletion (truncation) at either end or both ends of the sequence, the number of amino acids inserted or deleted at either end or both ends (not exceeding 10 of the full length) %) is not included in the total number of residues.
  • the sequences being compared are aligned in a way that produces the largest match between the sequences, and the gaps in the alignment (if any) are resolved by a specific algorithm.
  • the number of sequences extending at both ends of the sequence does not exceed 10% of the full length, such as 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% Or 1%.
  • variants of the amino acid sequence of the polypeptides described in the present disclosure are encompassed.
  • Amino acid sequence variants of the polypeptide can be prepared by introducing suitable modifications into the nucleotide sequence encoding the polypeptide, or by peptide synthesis. Such modifications include, for example, deletions, and/or insertions and/or substitutions of residues within the amino acid sequence of the polypeptide. Any combination of deletion, insertion, and substitution can be performed to obtain the final polypeptide.
  • amino acids can be grouped as follows:
  • Non-conservative substitutions would require replacing members of one of these categories with members of another category.
  • Non-critical amino acids can be substituted conservatively and/or non-conservatively without affecting the normal function of the polypeptide.
  • truncated means by removing one or more amino acids from the N and/or C-terminus of the polypeptide or deleting one or more amino acids within the polypeptide.
  • genetic engineering refers to the complex technology of manipulating genes at the molecular level.
  • genetic engineering includes, but is not limited to, introducing exogenous genes or fragments or derivatives thereof into host cells through in vitro recombination, so that the exogenous genes or fragments or derivatives are replicated and transcribed in the host cell. And/or the operation of translating expressions.
  • surface display system refers to the use of cell surface display technology to immobilize foreign proteins or polypeptides on the cell surface.
  • Surface display systems include, for example, prokaryotic surface display systems and eukaryotic surface display systems.
  • the eukaryotic surface display system includes a yeast surface display system or a baculovirus surface display system.
  • the eukaryotic surface display system can be used to display proteins or polypeptides that require post-translational modifications such as phosphorylation, glycosylation, methylation, acetylation, hydroxylation, and disulfide bond isomerization.
  • the surface display system can be used in antigen/antibody library construction, high-affinity antibodies, drug screening, vaccines, detection, affinity purification, cancer diagnosis and other fields.
  • vector means a nucleic acid molecule capable of multiplying another nucleic acid to which it is linked.
  • the term includes a vector as a self-replicating nucleic acid structure and as a vector integrated into the genome of a host cell into which the vector has been introduced.
  • Expression vectors can guide the expression of nucleic acids that are operably linked by such vectors. Suitable expression vectors include (but are not limited to) plasmids and viral vectors, including adenovirus, adeno-associated virus, retrovirus, cosmid and the expression vector disclosed in PCT Publication No. WO 87/04462.
  • Vector components may generally include (but are not limited to) one or more of the following: a signal sequence; an origin of replication; one or more marker genes; suitable transcription control components (such as promoters, enhancers, and terminator).
  • suitable transcription control components such as promoters, enhancers, and terminator.
  • transcription control components such as ribosome binding sites, translation initiation sites, and termination codons, are also usually required.
  • the vector containing the polynucleotide of interest and/or the polynucleotide itself can be introduced into the host cell by any of a number of appropriate methods, including electroporation, the use of calcium chloride, rubidium chloride, calcium phosphate, Transfection of DEAE-dextran or other substances; microprojectile bombardment; lipofection; and infection.
  • electroporation the use of calcium chloride, rubidium chloride, calcium phosphate, Transfection of DEAE-dextran or other substances; microprojectile bombardment; lipofection; and infection.
  • the choice of introducing a vector or polynucleotide will generally depend on the characteristics of the host cell.
  • host cell means a cell into which exogenous nucleic acid has been introduced, as well as the progeny of such a cell.
  • Host cells include “transformants” (or “transformed cells”), “transfectants” (or “transfected cells”), or “infectants” (or “infected cells”), each of which includes primary transformation, transfection, or Infected cells and descendants derived from them.
  • Such offspring may not be exactly the same as the parent cell in nucleic acid content, and may contain mutations.
  • the polypeptides described in the present disclosure can be recombinantly produced using suitable host cells.
  • the nucleic acid encoding the polypeptide can be cloned into an expression vector, which can then be introduced into host cells such as E. coli cells, yeast cells, insect cells, ape COS cells, Chinese hamster ovary (CHO) cells.
  • polypeptide of the present invention can be recovered and purified from recombinant cell culture by known methods, including but not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction Action chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. High performance liquid chromatography (“HPLC”) can also be used for purification.
  • HPLC high performance liquid chromatography
  • affinity refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule and its binding ligand. Unless otherwise specified, as used herein, “affinity” refers to the inherent binding that reflects the 1:1 interaction between the members of the binding pair (for example, polypeptides and small molecule compounds, or polypeptides and macromolecular drugs such as antibodies, etc.) Affinity.
  • the affinity of molecule X to its ligand Y can be expressed by the dissociation equilibrium constant (K D ).
  • kon refers to the rate constant of binding.
  • k off refers to the rate constant of dissociation.
  • K D association rate constant k on "and “dissociation rate constant k off” are usually used to describe the affinity between a molecule (such as a polypeptide) and its binding ligand (such as a small molecule compound or a large molecule drug), that is , How tightly the ligand binds to a specific polypeptide.
  • a higher value indicates a smaller K D affinity interaction, while the larger K D value indicates lower affinity interactions.
  • Binding affinity is affected by interactions between non-covalent molecules, such as hydrogen bonds, electrostatic interactions, hydrophobicity between two molecules and van der Waals forces. Affinity can be analyzed by conventional methods known in the art. For example, surface plasmon resonance (SPR) technology (e.g., ) Or biofilm interferometry (for example, ).
  • SPR surface plasmon resonance
  • biofilm interferometry for example, ).
  • Determining whether the selected compound causes degradation of MYC protein can be determined by commonly used methods for determining protein stability, including but not limited to protein half-life determination (such as pulse tracking or cycloheximide tracking, etc.), degradation-related post-translational modifications Measurement (such as protein ubiquitination modification, etc.).
  • protein half-life determination such as pulse tracking or cycloheximide tracking, etc.
  • degradation-related post-translational modifications Measurement such as protein ubiquitination modification, etc.
  • Apoptosis assay include, but are not limited to: morphological testing (such as the formation of apoptotic bodies), DNA fragmentation testing, TUNEL testing, cytochrome c localization testing, Caspase-3 and PARP activity testing, mitochondrial membrane potential testing Detection, expression and cell localization of apoptosis-related protein TFAR19, EELISA method (such as detection of nucleosome fragments in cell lysate), flow cytometry (such as Annexin V/PI method), changes in acetylcholinesterase, etc.
  • morphological testing such as the formation of apoptotic bodies
  • DNA fragmentation testing such as the formation of apoptotic bodies
  • TUNEL testing cytochrome c localization testing
  • Caspase-3 and PARP activity testing mitochondrial membrane potential testing Detection, expression and cell localization of apoptosis-related protein TFAR19
  • EELISA method such as detection of nucleosome fragments in cell ly
  • Human hematological malignant tumor cell lines MOLM-13, KG1-a, Kasumi-1, HL-60, NB4, THP-1, CEM, Jurkat, MOLT-4, Nalm6, Raji, Namalwa, Jeko-1, OCI-Ly3, OCI-Ly10, U2932, SU-DHL16, Pfeiffer, H9, RPMI-8226, U266 and KM3 were cultured in RPMI-1640 medium.
  • SEM and Supb15 were cultured in IMDM medium.
  • Human solid tumor cell lines A549, H1975, H82, Mia-Paca2, PANC-1, ASPC-1, HepG2, Huh7 and MGC-803 are retained in DMEM.
  • SW620, SW480 and HT29 are stored in L-15 medium. Routinely test all cell lines for Mycoplasma. All cells were cultured at 37°C in a humidified atmosphere containing 5% CO 2.
  • WBC100 powder is provided by Weben Pharmaceuticals (WBP), and its purity is 99.5%.
  • Thiazolyl blue tetrazolium bromide (MTT A600799) was purchased from Shenggong Bioengineering. Magnetic beads binding FITC antibody were purchased from BioMag Scientific Inc. M-PER TM mammalian protein extraction reagent (78505), protease and phosphatase inhibitor (78443) were purchased from (Thermo science).
  • RPB1 (F-12) antibody was purchased from Santa Cruz Biotechnology.
  • MYC (32072), XPB (190698) antibodies were purchased from Abcam.
  • STAT3 antibody was from Cell Signaling Technology.
  • Anti-GAPDH (60004-1) was obtained from protech.
  • Antibodies against PARP (9532) and Cleaved-Caspase3 (9664) were purchased from Cell Signaling Technology (CST); Caspase3 (ET1602-39) and horseradish peroxidase (HRP) conjugated secondary antibodies (goat anti-rabbit and goat) Anti-mouse) was purchased from Huaan Biotechnology Co., Ltd.
  • the content of WBC100-FITC and apoptotic cells were detected by FCM.
  • the cells were cultured in triplicate at 200,000 cells/ml (6 wells), and the cells were treated with 50 nM FITC-labeled WBC100 (WBC100-FITC) or DMSO (control) for 24 hours.
  • the cells were collected and washed twice with PBS, suspended in binding buffer, and then stained with Annexin V-APC and 7-AAD or Annexin V-FITC and PI for 15 minutes in the dark according to the manufacturer's instructions.
  • the apoptosis rate and absorption rate of WBC100 were performed on Canto-II (BD Biosciences).
  • IC50 is defined as the concentration of the drug that causes a 50% reduction in vitality.
  • the membrane was probed with a horseradish peroxidase-labeled secondary antibody at room temperature for 1 h, and the signal was detected by chemiluminescence, and imaging and quantification were performed by imageJ software ( https://imagej .nih.gov/ij/ ).
  • the cellular MYC protein competition pull-down assay was performed with WBC100.
  • the U2932 cell protein lysate was incubated with WBC100-FITC (0.1 ⁇ g/ml) with or without excess unlabeled WBC100 (10 ⁇ g/ml) at 4°C overnight.
  • the mixture was added to 25 ⁇ l of anti-FITC antibody magnetic beads (Biomag), and rotated and mixed overnight at 4°C.
  • MYC mutant and WBC100 binding assay 8 MYC mutants were constructed using site-directed mutagenesis: L297A, A321 ⁇ , L333A, R346A, S347A, E351A, V361A and Q365A. These MYC mutant plasmids were used to transfect 293T cells for 48 hours, and then cell proteins were extracted for the binding assay of WBC100 and these MYC mutants. To prevent non-specific binding, use free WBC100 as a binding competitor for competitive pull-down analysis.
  • MYC mutant cell protein was pre-incubated with free WBC100 (the dose of WBC100 is 100 times more than WBC100-FITC) at 4°C overnight, and then incubated with WBC100-FITC at 4°C overnight.
  • the complex of MYC mutant and WBC100-FITC was precipitated with FITC antibody-bound magnetic beads for Western blot analysis.
  • the MYC gene copy was detected by fluorescence in situ hybridization (FISH).
  • FISH fluorescence in situ hybridization
  • the surface plasmon resonance (SPR) experiment was performed by Orizymes Biotechnologies (Shanghai) Co., Ltd using a Biacore T200 (GE Healthcare) instrument.
  • a Biacore T200 GE Healthcare
  • the sample flowed through the surface at 30 ⁇ l/min for 120s binding time and 180s dissociation time. Analyze the data obtained through Biacore T200 evaluation software 3.0 (GE Healthcare).
  • the binding assay is carried out in the following three groups:
  • the binding model of WBC100 and MYC protein complex was studied on the computer by docking method.
  • the homology model of the monomer c-MYC in the range of 289-439 aa is constructed by merging two homology models, that is, by using eIF3c (PDB ID 4u1c) as a template in the 289-378 aa region on the SwissModel server And the 350-439 aa area model using the OmoMyc structure (PDB ID 5i50).
  • AML MOLM-13 in situ model and Xenogen imaging were established in situ model.
  • human AML MOLM-13 cells were stably transduced with lentiviral firefly luciferase.
  • Cells (1 ⁇ 10 6 ) were injected into NSG (NOD/SCID/IL2R ⁇ -/-) mice through the tail vein. After the obvious tumor signal was detected (day 7 after cell injection), the mice were randomly divided into four groups to receive vehicle or different doses of WBC100 (0.1mg/kg, 0.2mg/ kg, 0.4mg/kg).
  • An in vivo IVIS 100 bioluminescence/optical imaging system (Xenogen, Alameda, CA) was used to perform bioluminescence imaging of mice at different time points. Briefly, 10 minutes before measuring the luminescence signal, mice were injected intraperitoneally with d-Luferin (Promega, Madison, WI) dissolved in PBS (2.0 mg per mouse). Perform luminescence imaging once a week.
  • AML xenograft model in NSG mice In short, MOLM13 cells (1 ⁇ 10 7 ) were subcutaneously inoculated into the ventral subcutaneous (7 weeks) of NSG (NOD/SCID/IL2R ⁇ -/-) female mice. After the xenograft tumor reached 500-800mm 3 , the mice were randomly divided into four groups to receive vehicle or different doses of WBC100 (0.1mg/kg, 0.2mg/kg, 0.4mg/kg) by oral administration twice a day for 7 days. ), and then euthanized to analyze tumor weight, H&E staining, WB and IHC.
  • WBC100 0.1mg/kg, 0.2mg/kg, 0.4mg/kg
  • PDX patient-derived xenograft tumor
  • MYC MYC positive cells
  • a solid tumor xenograft model in NOD/SCID mice 1 ⁇ 10 7 solid tumor cells (A549, H1975, PANC-1, MGC-803) were subcutaneously inoculated on the ventral side of NOD/SCID female mice. After the xenograft tumor reached 200-300mm 3 , the mice were randomly divided into three or four groups to receive the vehicle or different doses of WBC100 (0.1mg/kg, 0.2mg/kg, 0.4 mg/kg). The tumor volume and mouse body weight were measured at different time points. At the end of the experiment, all mice were euthanized to analyze body weight, tumor weight, and survival rate.
  • PDAC tumor xenograft model in nude mice 1 ⁇ 10 7 Mia-Paca2 tumor cells were inoculated subcutaneously on the ventral side of female nude mice. After the xenograft tumor reached about 100 mm 3 , the mice were randomly divided into five groups to receive vehicle or different doses of WBC100 (0.1 mg/kg, 0.2 mg/kg, 0.4 mg/kg) by oral administration twice a day for 28 consecutive days. The positive drug gemcitabine was injected intraperitoneally at a dose of 100 mg/kg twice a week (BIW) for 4 weeks. The tumor volume and mouse body weight were measured at different time points. At the end of the experiment, all mice were euthanized to analyze body weight, tumor weight, and survival rate.
  • WBC100 0.1 mg/kg, 0.2 mg/kg, 0.4 mg/kg
  • Tumor specimens are prepared and analyzed by a pathologist certified by the committee.
  • the tumor was fixed in 4% PBS buffered formalin; dehydrated and embedded in paraffin; sectioned and stained with hematoxylin and eosin (H&E). These sections were deparaffinized; heated to recover the antigen; and incubated with the primary antibodies Ki-67 and c-Myc.
  • the polyclonal anti-rabbit immunoglobulin (Dako) was combined with streptavidin/horseradish peroxidase for biotinylation of the secondary antibody. Gently counterstain the sections with hematoxylin. Visualize the glass slide on a Zeiss confocal microscope.
  • TUNEL terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling
  • mice were used to evaluate the toxicity of WBC100.
  • WBC100 was administered to mice by oral gavage twice a day for 14 consecutive days.
  • Toxicity assessment is based on mortality, clinical observation, body weight, food consumption, clinical and anatomical pathology, blood counts (WBC, PLT, and HGB) and blood liver enzyme alanine aminotransferase (ALT) at specified time points.
  • Step 1 Dissolve triptolide (TPL) (500mg, 1.39mmol, 1.0eq.) and dextrorotatory (N-Boc)-valine (1500mg, 6.91mmol, 5eq.) in dichloromethane (20mL) Medium and cool to 0°C. At 0°C, add N,N'-diisopropylcarbodiimide (4mL, 13mmol, 9.35eq.) and 4-dimethylaminopyridine (130mg, 1.07mmol, 0.8eq.) to the mixed solution And react at 25°C for 24 hours.
  • TPL Triptolide
  • N-Boc dextrorotatory
  • the reaction solution was diluted with ethyl acetate, washed with water, then washed with saturated ammonium chloride, dried and concentrated to obtain a crude product.
  • the crude product was purified on a preparative silica gel column to produce D-Boc-valine-TPL (14-D-Boc-valine-triptolide) (250 mg) as a white solid, and the yield was 32.2%.
  • Step 2 Dissolve the product D-Boc-valine-TPL (14-D-Boc-valine-triptolide) (200mg) in the previous step in dichloromethane (15mL), and then 3 mL of trifluoroacetic acid was added dropwise. After the addition, the reaction solution was allowed to react at 25°C for 3.5 hours. The reaction solution was diluted with dichloromethane and washed with an aqueous sodium hydrogen carbonate solution, then dried and concentrated to obtain a crude product. The crude product was purified by preparative high performance liquid chromatography to produce 14-L-Valine-triptolide (WBC100) (120 mg) as a white solid with a yield of 73%.
  • WBC100 14-L-Valine-triptolide
  • triptolide is a natural small molecule that has a strong cytotoxic effect on some human cancer cells (35-37), but it is not yet known whether triptolide directly targets MYC protein .
  • Triptolide is considered to be a promising anti-cancer drug, but its clinical application is greatly restricted due to poor water solubility, dose-limited toxicity, and uncertainty of its anti-tumor target.
  • WBC100 The synthesis and characterization of WBC100 can be obtained in the patent application (US10238623).
  • the synthesis of WBC100 is as follows: Combine triptolide with Boc-D-Val-OH2, N,N-diisopropylcarbodiimide (DIC) and 4-dimethylaminopyridine (DMAP) ) Reacted in dichloromethane (DCM) at 25°C for 24 hours to produce 14-D-Boc-valine triptolide as a white powder. Subsequent 14-D-Boc-valine triptolide reacts with trifluoroacetic acid (TFA) to form a white powdery 14-D-valine triptolide TFA salt ( Figure 9a).
  • TFA trifluoroacetic acid
  • Example 2 WBC100 effectively kills MYC positive cancer cells in vitro
  • MYC-positive human hematological tumor cell lines CEM, Jurkat, Molt-4 and Sup-B15 and MYC-negative normal peripheral blood cell samples from healthy adult donors were treated with different concentrations of WBC100 (Figure 9b-c) and lasted 72 hours. It is found that WBC100 selectively kills MYC-positive cancer cells, but retains MYC-negative normal blood cells ( Figure 9b,c), and the average therapeutic index is 9.96 (from 3.64 to 18.58), indicating that the small molecule preferentially kills MYC-positive cells, and MYC protein may be crucial to its cancer-killing activity.
  • MYC is the main target of WBC100's anti-tumor activity
  • a large number of various tumor cell lines were used to detect and analyze the correlation between MYC levels and the anti-tumor activity of WBC100.
  • Western blotting was used to analyze the MYC protein levels of a group of different hematological malignant cell lines (including 11 leukemias, 9 lymphomas, 3 multiple myeloma cell lines) and 12 various solid tumor cell lines.
  • 3 cell lines with high MYC protein levels are highly sensitive to WBC100 (IC50 ⁇ 100nM), and 3 cell lines with medium MYC protein levels are moderately sensitive to WBC100 (100nM ⁇ IC50 ⁇ 200nM) , And 6 kinds of tumor cells with low MYC levels are low sensitivity (IC50>200nM) ( Figure 1b and Table 2).
  • MYC knockdown was performed in Mia-paca2 cells with high levels of MYC, and then the effect of WBC100 on the survival of Mia-paca2 cells was determined.
  • MYC KD reduced the sensitivity to WBC100 by 1.78 times (IC50: 78.87 ⁇ 7.748nM and 44.33 ⁇ 4.997nM) (Figure 11g-i).
  • Example 3 WBC100 accumulates in the nucleus and induces apoptosis of MYC positive cancer cells
  • WBC100-FITC FITC-labeled WBC100
  • Example 4 WBC100 directly binds to the nuclear localization signal (NLS) domain of MYC protein
  • FITC-conjugated WBC100 WBC100-FITC
  • FITC antibody-bound magnetic beads FITC antibody-bound magnetic beads
  • MYC positive cancer cell protein MYC positive cancer cell protein
  • Unlabeled WBC100 was used as a binding competitor.
  • the MYC-positive cancer cell protein was incubated with WBC100-FITC, and then the complex was precipitated with magnetic beads bound to FITC antibody for Western blot analysis.
  • WBC100 strongly interacts with mutant MYC1-328, but not with other deletion mutants: MYC1-140 (transcription activation domain, TAD), MYC1-320 (MYC-nick) structure Domain), MYC329-439 (DNA binding domain) and MYC355-439 (bHLHzip domain) ( Figure 3e).
  • MYC1-140 transcription activation domain, TAD
  • MYC1-320 MYC-nick) structure Domain
  • MYC329-439 DNA binding domain
  • MYC355-439 bHLHzip domain
  • the binding model of WBC100 in c-MYC protein complex was studied on the computer by docking method.
  • the homology model of the monomer c-MYC in the range of 289-439a.a. is constructed by merging two homology models, that is, by using eIF3c (PDB ID 4u1c) as a template on the SwissModel server (40) the 289- 378 aa area model and 350-439 aa area model using OmoMyc structure (PDB ID 5i50).
  • AAD omni-directional docking
  • WBC100 preferentially combines the NLS1 and NLS2 regions, as shown in Figure 4a.
  • WBC100 can form hydrogen bonds, hydrophobic interactions and cation- ⁇ interactions with MYC protein.
  • WBC100 forms two hydrogen bonds with S347/E351 (shown as blue dots), forms two cation- ⁇ interactions with R346 and Q365 (yellow arrows), and forms hydrophobic interactions with L297/L333/V361.
  • Function network (gray arrow).
  • the NLS1 and NLS2 motifs are colored light blue and blue, respectively.
  • 8 MYC mutants were constructed using site-directed mutagenesis: L297A, A321 ⁇ , L333A, R346A, S347A, E351A, V361A and Q365A.
  • 293T cells were transfected with these mutant MYC for 48 hours, and then the cell protein was extracted for the binding assay of WBC100 and these MYC mutants.
  • a competitive pull-down assay was performed using free WBC100 as a specific binding competitor.
  • MYC mutant cell protein was pre-incubated with free WBC100 overnight at 4°C, and then incubated with WBC100-FITC overnight at 4°C.
  • the complex of MYC mutant and WBC100-FITC was precipitated with FITC-Ab magnetic beads for Western blot analysis.
  • Example 5 WBC100 selectively induces MYC protein degradation by recruiting ubiquitin E3 ligase CHIP
  • MYC protein The stability of MYC protein is regulated by a variety of mechanisms.
  • the main way of MYC degradation in cells is through the ubiquitin-proteasome system (UPS) (48,49).
  • UPS ubiquitin-proteasome system
  • Example 6 Oral administration of WBC100 can cure MYC-positive refractory acute myeloid leukemia and T-cell lymphoma
  • NSG mice with refractory MOLM-13 luciferase cells with high MYC levels were then used to establish an orthotopic human AML model. Consistent with the in vitro results, oral administration of WBC100 twice a day for 20 days had a dose-dependent anti-tumor activity on human MYC-positive AML in NSG mice ( Figure 6a). Treatment with higher (0.4 mg kg -1 body weight) or medium dose (0.2 mg kg -1 body weight) of WBC100 completely eliminated AML-MOLM-13 from the body ( Figure 6a), and all mice were disease-free on day 35 Survive.
  • WBC100 also significantly inhibited tumor growth in leukemia mice and prolonged their survival ( Figure 6a-b).
  • in situ NSG mice with MOLM-13 luciferase cells were used to compare the anti-tumor activity of WBC100 with MYC transcription inhibitors (+)-JQ1 (BET inhibitor) (51) was compared with idarubicin (IDA), the standard care drug for human refractory AML.
  • IDA idarubicin
  • mice were randomly divided into four groups, and they received vehicle or different doses of WBC100 (0.1, 0.2 and 0.4 mg kg -1 ) orally twice a day for 7 days, and then euthanized .
  • WBC100 0.1, 0.2 and 0.4 mg kg -1
  • H&E staining of tumor tissues immunostaining of Ki-67 and MYC, TUNEL staining for apoptosis, and Western staining of MYC protein were performed.
  • Example 7 WBC100 makes the mouse model of AML patient-derived xenotransplantation (PDX) regression
  • a patient-derived xenograft (PDX) mouse model was established using primary AML cells from patients and NSG mice, and the effect of WBC100 on MYC-positive cells was tested. Inhibition.
  • Example 8 Activity of WBC100 in MYC-positive solid tumors in vivo
  • Example 9 Normal mice have a good tolerance to WBC100 at an effective dose
  • mice administered with WBC100 at the highest dose survived well, except for the moderately reversible increase in liver enzymes in the blood and slight weight loss. The weight returned to normal afterwards ( Figure 21).
  • Triptolide is an inhibitor of RNA polymerase I and II-dependent transcription leading predominantly to down-regulation of short-lived mRNA.Mol Cancer Ther. 8, 2780 (2009).

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Abstract

L'invention concerne un polypeptide cible utilisé pour le criblage de médicaments destiné au traitement et/ou à la prévention de cancers, le polypeptide cible étant un polypeptide d'une longueur inférieure ou égale à 200 acides aminés, et contient une séquence d'acides aminés qui est approximativement de 70 % à 100 % identique au domaine de signal de localisation nucléaire d'une protéine MYC de mammifère. L'invention concerne également une protéine de fusion contenant le polypeptide cible, un polynucléotide isolé codant pour le polypeptide cible, et un procédé de criblage de médicaments pour le traitement et/ou la prévention de cancers grâce à l'utilisation du polypeptide cible ou de la protéine de fusion.
PCT/CN2021/085836 2020-04-15 2021-04-07 Polypeptide cible utilisé pour le criblage de médicaments et procédé de criblage associé WO2021208787A1 (fr)

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WO2023143330A1 (fr) * 2022-01-25 2023-08-03 杭州卫本医药科技有限公司 Époxyde diterpène de tripterygium wilfordii ayant une fonction de destruction de cellule positive myc, son procédé de préparation et son utilisation
CN117186178A (zh) * 2022-09-09 2023-12-08 湖南大学 一种多肽及其制备方法

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