Specific covalent inhibitors targeting the catalytic active center of mll1

Abstract

This application relates to compounds, compositions, and methods for inhibiting mixed lineage leukemia 1 (MLL1), wherein the compounds target the catalytic active site of MLL1 to inhibit the activity of MLL1.

Description

Invention Field

[0001] This application relates to compounds, compositions, and methods for inhibiting mixed lineage leukemia 1 (MLL1). Background Technology

[0002] MLL1 (Mixed Lineage Leukemia 1) is a histone H3 lysine 4 (H3K4) methyltransferase, a member of the MLL family of proteins. It uses S-adenosylmethionine (SAM) as a methyl donor to catalyze the transfer of a methyl group to the ε-amino group of histone H3 lysine 4 (H3K4). Methylation at the H3K4 position serves as an important epigenetic marker, widely involved in crucial life processes such as gene transcription regulation and cell fate transition.

[0003] In mammals, the MLL family comprises three subfamilies with a total of six members: MLL1, MLL2, MLL3, MLL4, SET1A, and SET1B. These six members mediate monomethylation, dimethylation, and trimethylation of histone H3K4, respectively. MLL3 and MLL4 are responsible for monomethylation at the H3K4 site, MLL1 and MLL2 catalyze dimethylation, and SET1A and SET1B catalyze trimethylation. They play distinct functions in different biological processes. Unlike histone methyltransferases such as G9a and Dot1L, MLL family proteins themselves have low catalytic activity and require complexes with other regulatory subunits to efficiently perform their methyltransferase functions (Hu et al., 2013). Studies have shown that the methyltransferase activity of MLL family proteins is regulated by four common subunits: WDR5, RBBP5, ASH2L, and DPY30 (WRAD). The loss of any WRAD subunit leads to a decrease or even disappearance of methyltransferase activity in MLL family proteins. In addition to the activity-regulating subunits WDR5, RBBP5, ASH2L, and DPY30, the MLL1 complex can also interact with MENIN proteins, thereby being recruited to corresponding target gene sites to exert methyltransferase activity and establish the correct epigenetic landscape.

[0004] Mixed lineage leukemia (MLL leukemia) is a subtype of acute leukemia with poor response to chemotherapy and a high risk of relapse. Its main genetic variant is MLL1 gene rearrangement. MLL1 enzyme activity dysregulation is highly correlated with the occurrence and progression of malignant tumors such as MLL leukemia, and targeting and inhibiting MLL1 enzyme activity has become a potential treatment option for MLL leukemia. Current development of MLL1-specific inhibitors targets the interaction interface between MLL1 and its co-regulatory proteins, showing some therapeutic effect. However, due to the complexity of the protein-protein interaction network in vivo, these inhibitors inevitably have potential risks such as poor specificity, strong side effects, and complex mechanisms, which restricts the further development of MLL leukemia treatment methods. Furthermore, the catalytic domains of the six members of the MLL family are highly conserved, and their active pockets are highly similar in sequence and structure, which poses a challenge to the development of specific inhibitors targeting the MLL1 active site. Therefore, all current inhibitor development efforts have bypassed the active site target and instead focused on the regulatory subunit interaction interface; to date, there are no reports of specific inhibitors targeting the MLL1 active site. Therefore, it is still necessary to find new targets and develop effective inhibitor molecules to overcome the above-mentioned challenges. Summary of the Invention

[0005] To find a breakthrough in developing inhibitor molecules that target the active site of MLL1, we explored the structural differences of MLL family proteins, revealed the key factors and structural basis affecting the affinity and selectivity of inhibitors, and clarified the subtle structural differences and key amino acid residues between MLL1 and other members. Based on this, we designed and screened specific covalent inhibitors MI9-14 and MI9-14-TAT fusion peptides that target the substrate binding pocket of MLL1.

[0006] This application provides MI9-14 peptides that inhibit MLL1, MI9-14-TAT fusion peptides, and compositions and methods for treating or preventing diseases or conditions mediated by MLL1.

[0007] The specific technical solution of this application is as follows:

[0008] 1. A polypeptide whose amino acid sequence comprises CRX1{NLE}QX2X3RY or CRX1{NLE}QX2X3RY; wherein

[0009] X1 is L, M, V, or I; X1 is preferably L;

[0010] X2 is T, F, I, W, Y, L, M, or V; X2 is preferably T;

[0011] X3 is any amino acid selected from the group consisting of: A, W, F, L, M, I, P, V, Q, C, G, N, S, Y, T, D, E, H, R, and K; X3 is preferably A.

[0012] 2. A polypeptide, which is an MI9-14-TAT fusion polypeptide, that is, the C-terminus of MI9-14 fused with TAT;

[0013] The amino acid sequence of MI9-14 includes CRX1{NLE}QX2X3RY or CRX1{NLE}QX2X3RY; wherein X1 is L, M, V or I, preferably L; X2 is T, F, I, W, Y, L, M or V, preferably T; X3 is any amino acid selected from the group consisting of A, W, F, L, M, I, P, V, Q, C, G, N, S, Y, T, D, E, H, R and K, preferably A;

[0014] The amino acid sequence of the TAT is GRKKRRQRRRPQ (SEQ ID NO:2);

[0015] The preferred amino acid sequence of the MI9-14-TAT fusion polypeptide is CRL{NLE}QTARYGRKKRRQRRRPQ.

[0016] 3. The polypeptide described in item 1 or 2, which targets the catalytic active site of MLL1 to inhibit the activity of MLL1.

[0017] 4. A pharmaceutical composition comprising a polypeptide as described in item 1 or 2 and one or more pharmaceutically acceptable carriers.

[0018] 5. A combination comprising a polypeptide as described in item 1 or 2, and one or more therapeutic agents.

[0019] 6. The combination as described in item 5, wherein one or more therapeutic agents are anticancer agents, analgesics, or anti-inflammatory agents.

[0020] 7. A method for treating a disease or condition in which one benefits by inhibiting mixed lineage leukemia 1 (MLL1), comprising administering to a subject in need a therapeutically effective amount of the compound as described in item 1 or 2.

[0021] 8. The method of claim 7, wherein the disease or condition in which MLL1 is benefited by inhibition is chronic myeloid leukemia (CML), acute T-lymphoblastic leukemia (T-ALL), acute myeloid leukemia with MLL1 translocation rearrangement (AML), or acute lymphoblastic leukemia with MLL1 translocation rearrangement (ALL); wherein the disease or condition is preferably acute myeloid leukemia with MLL1 translocation rearrangement (AML) or acute lymphoblastic leukemia with MLL1 translocation rearrangement (ALL).

[0022] 9. Use of the compound described in item 1 or 2 in the preparation of a medicament for treating a disease or condition in which the patient benefits by inhibiting mixed lineage leukemia 1 (MLL1).

[0023] 10. The use as described in item 9, wherein the disease or condition in which MLL1 is benefited by inhibition is chronic myeloid leukemia (CML), acute T-lymphoblastic leukemia (T-ALL), acute myeloid leukemia with MLL1 translocation rearrangement (AML), or acute lymphoblastic leukemia with MLL1 translocation rearrangement (ALL); wherein the disease or condition is preferably acute myeloid leukemia with MLL1 translocation rearrangement (AML) or acute lymphoblastic leukemia with MLL1 translocation rearrangement (ALL).

[0024] 11. Use of the compound described in item 1 or 2 in combination with a second therapeutic agent in the preparation of a medicament for treating a disease or condition that can be treated by inhibiting mixed lineage leukemia 1 (MLL1).

[0025] Technical effect

[0026] Currently, there are no reported specific inhibitors targeting the substrate binding pocket of MLL1 histones. Only in 2020, an article in ACS Med. Chem. Lett. reported a series of small-molecule inhibitors of MLL1 obtained through AdoMet molecular modification (Chern TR, et al. Discovery of Potent Small-Molecule Inhibitors of MLL Methyltransferase. ACS Med Chem Lett. 2020 May 14; 11(6):1348-1352). However, these inhibitors did not occupy both the AdoMet binding pocket and the substrate H3 channel of MLL1 as designed; instead, they only occupied the AdoMet binding site, inhibiting MLL1 activity by locking it in a relatively open conformation. Furthermore, it is unclear whether these inhibitors have selective and specific inhibitory effects on MLL1, and their impact on the proliferation of MLL fusion-type leukemia cells has not been evaluated. In contrast, the MI9-14 molecule in this application can compete with histone H3 for substrate pockets, showing selective inhibition of MLL1, effectively inhibiting the proliferation of MLL fusion leukemia cells such as MV4 and 11 and exhibiting a pro-apoptotic effect.

[0027] In addition, there is another class of MLL1 inhibitors with a completely different mechanism, collectively known as WDR5 inhibitors. These inhibitors inhibit the enzyme activity of MLL1 by disrupting the interaction between the active regulatory subunit WDR5 and MLL1. Currently, several WDR5 inhibitors have been developed and have shown strong anti-leukemia activity in preclinical in vitro and in vivo models. For example, OICR-9429, the most widely studied WDR5 inhibitor, is a very potent MLL1 inhibitor with a Ki of 64 nM for inhibiting MLL1 enzyme activity (Yu X, et al. A selective WDR5 degrader inhibits acute myeloid leukemia inpatient-derived mouse models. Sci Transl Med. 2021 Sep 29; 13(613):eabj1578.). Our test results show that OICR-9429 can inhibit the proliferation of MLL fusion leukemia cell lines MV4;11, MOLM-13 and NOMO-1 cells, with IC50 values ​​of 8.7 μM, 25.2 μM and 10.2 μM, respectively. For example, the inhibitor MM-401, developed based on the MLL WIN motif, has an IC50 of 0.32 μM for inhibiting MLL1 enzyme activity and IC50s of 12.42 μM and 23.97 μM for inhibiting the proliferation of MLL fusion leukemia cell lines MV4;11 and MOLM-13, respectively (Cao F, et al. Targeting MLL1 H3K4methyltransferase activity in mixed-lineage leukemia. Mol Cell. 2014 Jan 23; 53(2):247-61). Compared with MM-401 and OICR-9429, the peptide inhibitor MI9-14-TAT provided in this application showed stronger inhibitory effects on proliferation in all four MLL fusion leukemia cell lines. In particular, the MLL-AF9 fusion leukemia cell line THP-1 was completely insensitive to the potent WDR5 inhibitor OICR-9429, but MI9-14 could effectively inhibit its cell proliferation with an IC50 of 10.8 μM.

[0028] In summary, this application provides the first specific inhibitor targeting the catalytic active site of MLL1. Compared with existing WDR5 inhibitors (such as OICR-9429 and MM-401), this inhibitor exhibits stronger efficacy in inhibiting the proliferation of MLL fusion-type leukemia cells, especially showing broad and superior proliferation-inhibiting activity against different fusion-type leukemia cell lines. These results demonstrate that MI9-14 and MI9-14-TAT in this application have unique advantages as specific inhibitors targeting the catalytic active site of MLL, providing new targets and molecular tools for the development and application of targeted drugs for MLL fusion-type leukemia. Attached Figure Description

[0029] Figure 1 The results show that MI9-14 covalently binds to cysteine ​​at the "door loop" of MLL1. Figure 1 A: Structural formula of the MI9-14 polypeptide molecule; Figure 1 B: MALDI-TOF detection of the covalent binding of MI9-14 with MLL1 and MLL3 after 6 hours of incubation; Figure 1 C: HCD detection of the covalent sites of MI9-14 and MLL1. The figure shows the HCDMS / MS spectrum of the [M+4H]4+ ion at m / z 491.2413. The predicted b-type and y-type ions are listed below. Figure 1 Above and below the peptide sequence shown in the upper right corner of C.

[0030] Figure 2 MI9-14 specifically binds to MLL1 and inhibits its methyltransferase activity. Figure 2 A: Fluorescence polarization detection of the affinity of MI9-14 for M1AR, K d It is 2.57 ± 0.4 μM; Figure 2 B: Enzyme activity inhibition assay was used to detect the inhibition efficiency of MI9-14 on M1WRAD without prior incubation. The IC50 was 12508±2023 nM. After 6 h of incubation, the IC50 of MI9-14 on M1WRAD enzyme activity inhibition was 1610±392.2 nM. After 12 h of incubation, the IC50 of MI9-14 on M1WRAD enzyme activity inhibition was 480.5±68.6 nM. Figure 2 C: Enzyme activity inhibition assays tested the effects of MI9-14 on the activities of various methyltransferases and demethylases, showing that the inhibitory effect on MLL1 methyltransferase activity has certain specificity and selectivity.

[0031] Figure 3 MI9-14-TAT showed that it inhibited the proliferation of leukemia cells. Figure 3A: Four MLL fusion leukemia cell lines (MV4;11, MOLM-13, NOMO-1, and THP-1), two MLL non-fusion leukemia cell lines (Jurkat E6-1 and K562), and human embryonic kidney cells HEK293T were treated with MI9-14 fused with a transmembrane peptide (i.e., forming MI9-14-TAT), and human embryonic kidney cells HEK293T were also treated. Cell proliferation was detected by CCK-8 assay after 4 days. Figure 3 B: Cell images of MOLM-13 cells after treatment with 40 μM MI9-14-TAT for 72 h. It can be seen that MI9-14-TAT significantly inhibited the proliferation of MOLM-13 cells and caused cell death. Figure 3 C: Flow cytometry results of Annexin V-FITC / PI staining after MI9-14-TAT treatment for 48 h showed that MV4;11 cells exhibited significant apoptosis after MI9-14-TAT treatment for 48 h, while no apoptosis was detected in Jurkat E6.1 cells. Figure 3 D: MV4 cells were treated with different concentrations of MI9-14-TAT; after 1148h, Annexin V-FITC / PI staining and flow cytometry were performed to detect the ratio of early apoptotic cells to late apoptotic cells. It was found that MI9-14-TAT-induced apoptosis of MV4 cells was concentration-dependent.

[0032] Figure 4 The binding and activity characterization of MI9-11 and MI9-17 are shown. Figure 4 A: MALDI-TOF detection of the covalent binding of MI9-11 and MI9-17 to MLL1 after 6 hours of incubation; Figure 4 B: Fluorescence polarization detection of the affinity of MI9-11 and MI9-17 for M1AR; Figure 4 C: Enzyme activity inhibition assay to detect the inhibition efficiency of MI9-11 and MI9-17 on M1WRAD after 12 h of incubation; Figure 4 D: Two MLL fusion leukemia cell lines (MOLM-13, NOMO-1) and one MLL non-fusion leukemia cell line (K562) were treated with MI9-17-TAT. Cell proliferation was detected by CCK-8 assay after 72 hours.

[0033] Figure 5 The results show the specific MI9-14 variant tested and its affinity for M1AR. Invention Details

[0034] This application provides MI9-14 peptides that inhibit MLL1, MI9-14-TAT fusion peptides, and compositions and methods for treating or preventing diseases or conditions mediated by MLL1.

[0035] This document describes various enumerated embodiments of this application. The features specified in each embodiment may be combined with other specified features to provide further embodiments of this application.

[0036] For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular form will also include the plural form, and vice versa.

[0037] In a first aspect, this application provides a polypeptide whose amino acid sequence comprises CRX1{NLE}QX2X3RY or CRX1{NLE}QX2X3RY; wherein

[0038] X1 is L, M, V, or I; X1 is preferably L;

[0039] X2 is T, F, I, W, Y, L, M, or V; X2 is preferably T;

[0040] X3 is any amino acid selected from the group consisting of: A, W, F, L, M, I, P, V, Q, C, G, N, S, Y, T, D, E, H, R, and K; X3 is preferably A.

[0041] In this article, "NLE" is an abbreviation for the non-natural amino acid ortholeucine, with the molecular formula C6H12O. 13 NO2, CAS number 327-57-1, structural formula:

[0042] In a second aspect, this application provides a polypeptide, which is an MI9-14-TAT fusion polypeptide, i.e., a C-terminus of MI9-14 fused with TAT; wherein the amino acid sequence of MI9-14 includes CRX1{NLE}QX2X3RY or CRX1{NLE}QX2X3RY; wherein X1 is L, M, V or I, preferably L; X2 is T, F, I, W, Y, L, M or V, preferably T; X3 is any amino acid selected from the group consisting of A, W, F, L, M, I, P, V, Q, C, G, N, S, Y, T, D, E, H, R and K, preferably A; the amino acid sequence of the TAT is GRKKRRQRRRPQ (SEQ ID NO:2);

[0043] The preferred amino acid sequence of the MI9-14-TAT fusion polypeptide is CRL{NLE}QTARYGRKKRRQRRRPQ.

[0044] MI9-14 in this article is a polypeptide with the amino acid sequence CRL{NLE}QTARY. Its structural formula is:

[0045]

[0046] MI9-14 has the following two important features: (1) it enhances the hydrophobic interaction between key residue sites and the catalytic active pocket of MLL1 by introducing the non-natural amino acid ortholeucine (NLE); (2) it can target key cysteine ​​residues on the “door loop” region of MLL1 and covalently bind to them.

[0047] MI9-11 in this application is also a polypeptide with the amino acid sequence CRTMQTARY. Compared with MI9-14, the third and fourth amino acids are less hydrophobic.

[0048] The MI9-17 of this application is also a polypeptide with the amino acid sequence ARL{NLE}QTARY. Compared with MI9-14, only the first amino acid is different. Due to the lack of cysteine, MI9-17 cannot covalently bind to the "door loop" region of MLL1.

[0049] In this article, "TAT" refers to TAT-penetrating peptide, with the amino acid sequence GRKKRRQRRRPQ. TAT-penetrating peptide is a short peptide that penetrates the cell membrane, enabling it to rapidly enter the cell via a non-specific pathway and deliver its attached substances (such as drugs and proteins) into the cell. This property makes TAT-penetrating peptide widely used in cell delivery and gene transfection research. TAT-penetrating peptide has multiple cationic amino acid residues, which allows it to interact with the negatively charged cell membrane and enter the cell via intracellular transport pathways.

[0050] The peptides described in the first and second aspects above target the catalytic active site of MLL1, thereby inhibiting the activity of MLL1.

[0051] As used herein, the term “inhibition” refers to the reduction or suppression of a given condition, symptom, disorder, or disease, or a significant decrease in baseline activity in a biological activity or process.

[0052] In a third aspect, this application provides a pharmaceutical composition comprising a polypeptide as described in the first or second aspect above and one or more pharmaceutically acceptable carriers.

[0053] As used herein, the term "pharmaceutical composition" means a compound of the present application, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier suitable for oral or parenteral administration.

[0054] As used herein, the term “pharmaceutically acceptable carrier” means a substance that can be used in the preparation or use of a pharmaceutical composition, and includes, for example, suitable diluents, solvents, dispersion media, surfactants, antioxidants, preservatives, isotonic agents, buffers, emulsifiers, absorption delay agents, salts, pharmaceutical stabilizers, binders, excipients, disintegrants, lubricants, wetting agents, sweeteners, flavoring agents, dyes, and combinations thereof.

[0055] In a fourth aspect, this application provides a combination comprising a polypeptide as described in the first or second aspect, and one or more therapeutic agents; wherein the one or more therapeutic agents are anticancer agents, analgesics, or anti-inflammatory agents.

[0056] As used in this article, the term "anticancer agent" or "antitumor agent" refers to a therapeutic agent used to treat or control the growth of cancer cells.

[0057] As used in this article, the term "anti-inflammatory agent" refers to a therapeutic agent that reduces inflammation (redness, swelling, and / or pain) in the body. Anti-inflammatory agents block certain substances in the body that cause inflammation.

[0058] In a fifth aspect, this application provides a method for treating a disease or condition that benefits from the inhibition of mixed lineage leukemia 1 (MLL1), comprising administering a therapeutically effective amount of the compound as described in the first or second aspect to a subject in need.

[0059] As used herein, the term "subject" refers to mammals, primates (e.g., humans (male or female)), dogs, rabbits, guinea pigs, pigs, rats, and mice. In some embodiments, the subject is a primate. In other embodiments, the subject is a human.

[0060] As used herein, the term “treatment” for any disease or disorder means relief or improvement of the disease or disorder (i.e., slowing or halting the development of the disease or at least one of its clinical symptoms); or relief or improvement of at least one physical parameter or biomarker associated with the disease or disorder, including those physical parameters or biomarkers that the patient may not be able to identify.

[0061] As used herein, the term "therapeuticly effective amount" of the compound of this application refers to the amount of the compound of this application that will elicit a biological or medical response in a subject (e.g., a reduction or inhibition of enzyme or protein activity, or improvement of symptoms, relief of symptoms, slowing or delaying disease progression, or prevention of disease, etc.).

[0062] As used in this article, a subject is considered "needing" treatment if the subject will benefit from the treatment biologically, medically, or in terms of quality of life.

[0063] In some respects, the diseases or conditions for which the aforementioned patients benefit from MLL1 inhibition are chronic myeloid leukemia (CML), acute T-lymphoblastic leukemia (T-ALL), acute myeloid leukemia with MLL1 translocation rearrangement (AML), or acute lymphoblastic leukemia with MLL1 translocation rearrangement (ALL); the diseases or conditions are preferably acute myeloid leukemia with MLL1 translocation rearrangement (AML) or acute lymphoblastic leukemia with MLL1 translocation rearrangement (ALL).

[0064] In a sixth aspect, this application provides the use of the compounds described in the first or second aspect in the preparation of a medicament for treating a disease or condition that benefits from the inhibition of mixed lineage leukemia 1 (MLL1).

[0065] In some respects, the diseases or conditions for which MLL1 is benefited are chronic myeloid leukemia (CML), acute T-lymphoblastic leukemia (T-ALL), acute myeloid leukemia with MLL1 translocation rearrangement (AML), or acute lymphoblastic leukemia with MLL1 translocation rearrangement (ALL); the diseases or conditions are preferably acute myeloid leukemia with MLL1 translocation rearrangement (AML) or acute lymphoblastic leukemia with MLL1 translocation rearrangement (ALL).

[0066] In a seventh aspect, this application provides the use of the compound as described in the first or second aspect in combination with a second therapeutic agent in the preparation of a medicament for treating a disease or condition that is benefited from or can be treated by inhibiting mixed lineage leukemia 1 (MLL1).

[0067] In this article, "M1AR" refers to the complex composed of three protein fragments: MLL1 (3814-3969N3861I / Q3867L), RBBP5 (AS-ABM domain), and ASH2L (SPRY domain), which is the smallest active unit of the MLL1 protein.

[0068] In this article, “M1WRAD” refers to a complex composed of five proteins: MLL1 (3754-3969) + WDR5 (full-length) + RBBP5 (full-length) + ASH2L (full-length) + DPY30 (full-length), which is the complete active unit of the MLL1 protein; here, “M1WRAD” is synonymous with “M1WRAD complex” in the Examples section.

[0069] In this article, "MWRAD" refers to a complex composed of five proteins: MLL, WDR5 (full-length), RBBP5 (full-length), ASH2L (full-length), and DPY30 (full-length). However, depending on the specific experimental context, M can be MLL1 (3754-3969), MLL2 (2508-2715), MLL3 (4700-4911), MLL4 (5335-5537), SET1A (1491-1707), or SET1B (1706-1923) (all members of the MLL family of proteins).

[0070] In this article, "H3 polypeptide" refers to amino acids 1-20 of human histone H3 with a Y added to the C-terminus for accurate quantification by UV 280 nm light absorption. The sequence is ARTKQTARKSTGGKAPRKQLY.

[0071] In this article, "AdoHcy" refers to S-adenosylhomocysteine. "AdoMet" refers to S-adenosylmethionine.

[0072] In this article, "TFA" refers to trifluoroacetic acid.

[0073] SUV39H1, SUV39H2, G9a, and GLP: All four are H3K9 methyltransferases.

[0074] DNMT1: DNA methyltransferase.

[0075] KDM5A and LSD1: Both are H3K4 demethylases.

[0076] In this article, "THP-1" refers to a type of human monocytic leukemia cell.

[0077] In this article, "CML" refers to chronic myeloid leukemia.

[0078] In this article, "T-ALL" refers to acute T-lymphoblastic leukemia.

[0079] In this article, "AML" refers to acute myeloid leukemia.

[0080] "CCK-8" refers to the Cell Counting Kit-8, a reagent kit developed by Dojindo Chemical Research Institute in Japan for detecting cell proliferation and cytotoxicity, and is an alternative to the MTT assay. The CCK-8 reagent contains WST-8: chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt. Under the action of the electron carrier 1-methoxy-5-methylphenazine sulfate dimethyl ester (1-Methoxy PMS), it is reduced by dehydrogenases in the cell mitochondria to a highly water-soluble yellow formazan product. The amount of formazan produced is directly proportional to the number of viable cells. The absorbance value is measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader, which indirectly reflects the number of viable cells. This method has been widely used for the detection of the activity of some bioactive factors, large-scale screening of antitumor drugs, cell proliferation assays, cytotoxicity assays, and drug sensitivity tests. The CCK-8 kit used in this application is the super-powerful CCK-8 kit (catalog number: MA0225-1) manufactured by Dalian Meilun Biotechnology Co., Ltd.

[0081] As used herein, the term “prevention” for any disease or disorder means preventive treatment of the disease or disorder; or delaying the onset or progression of the disease or disorder.

[0082] As used herein, the terms “a”, “the”, and similar terms used in the context of this application (especially in the context of the claims) shall be interpreted to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context.

[0083] This application is further illustrated by the following embodiments, but should not be construed as further limitation. All accompanying drawings and references, patents, and published patent applications cited in this application are expressly incorporated herein by reference. Example

[0084] Example 1: Development of MLL1-specific covalent inhibitor MI9-14

[0085] Based on the analysis of key factors of inhibitor affinity and selectivity, we designed the MI9-14 peptide molecule. MI9-14 has the following two important features: (1) it enhances the hydrophobic interaction between key residue sites and the catalytic active pocket of MLL1 by introducing the non-natural amino acid ortholeucine (NLE); (2) it can covalently bind to the key cysteine ​​residue on the "door loop" region of MLL1. The sequence of MI9-14 is CRL{NLE}QTARY; please refer to the molecular structure of MI9-14. Figure 1A.

[0086] 1.1 To characterize the covalent binding ability of MI9-14 with MLL1, we used MALDI-TOF MS / MS to detect the covalent relationship between MI9-14 and MLL1 and MLL3. The specific operation steps are as follows:

[0087] (1) Mix 100 μM MLL1 or MLL3 protein with 2 mM MI9-14 molecules in buffer (25 mM Tris-HCl, pH 8.0, 150 mM NaCl) until homogeneous. Take 10 μL of the solution and add 90 μL of 0.5% TFA (trifluoroacetic acid) as the sample before the reaction.

[0088] (2) After incubating the reaction system at 25°C for 6 hours, take 10 μL of the solution and add 90 μL of 0.5% TFA to dilute the system 10 times to terminate the binding reaction. This is recorded as the sample after the reaction.

[0089] (3) For both the pre-reaction and post-reaction samples, take 1 μL of each sample and mix it with 1 μL of SA matrix (10 mg / mL concentration dissolved in MALDI matrix buffer (70% acrylonitrile (v / v) + 30% 0.1% trifluoroacetic acid (v / v))). Spot the mixture onto a 384-well MALDI target plate and allow it to air dry at room temperature.

[0090] (4) Detection was performed using an AB Sciex 5800MALDI TOF / TOF mass spectrometer equipped with TOF / TOF Series Explorer Software. The theoretical molecular weight of the protein fragment used for MLL1 was 18211.15, the theoretical molecular weight of the protein fragment used for MLL3 was 18086.67, and the theoretical molecular weight of MI9-14 was 1123.33. Therefore, the covalent binding of MI9-14 to MLL1 could be determined by detecting the changes in signal intensity at molecular weights of 18211.15 and 19334.48; the covalent binding of MI9-14 to MLL3 could be determined by detecting the changes in signal intensity at molecular weights of 18086.67 and 19210.

[0091] like Figure 1 As shown in Figure B, after 6 hours of incubation, 67% of MLL1 molecules were covalently bound to MI9-14 (the molecular weight increased by 1123 Da), while the control group MLL3 did not covalently bind to MI9-14 (the molecular weight remained unchanged). This indicates that MI9-14 can specifically covalently bind to MLL1.

[0092] 1.2 To further verify whether MI9-14 covalently binds to the C3882 residue in the "door loop" region of MLL1, we performed high-energy collision dissociation mass spectrometry (HCD MS / MS). HCD MS / MS is commonly used to study post-translational modifications of proteins. By generating specific fragment patterns through HCD and then measuring the molecular weight of secondary fragment ions, the unique fingerprints of these fragment patterns can be used to infer the post-translational modification sites. We used HCD MS / MS to analyze the covalent binding site between MI9-14 and MLL1. The sample preparation procedure is as follows:

[0093] (1) Mix 100 μM MLL1 protein and 2 mM MI9-14 molecules in a buffer (25 mM Tris-HCl, pH 8.0, 150 mM NaCl) until homogeneous;

[0094] (2) Incubate the reaction system at 25°C for 6 hours, then add 10 μL of the solution to 90 μL of 0.5% TFA solution to obtain the mass spectrometry sample.

[0095] After preparing the mass spectrometry samples, HCD MS / MS and data analysis were performed using the mass spectrometry system of the National Protein Science Center (Shanghai). Figure 1 The spectrum in the upper right corner of C shows that MI9-14 achieves specific covalent binding by forming a disulfide bond with C3882 in MLL1 through the first cysteine ​​residue.

[0096] Example 2: MI9-14 specifically inhibits the methyltransferase activity of MLL1

[0097] 2.1 After clarifying the covalent binding of MI9-14 to MLL1, we characterized its affinity for MLL1 and its inhibitory activity against MLL1. We labeled MI9-14 with 5-FAM fluorescence and detected its binding ability to M1AR using fluorescence polarization experiments. The experimental procedure is as follows:

[0098] (1) The synthesized FAM fluorescently labeled MI9-14 peptide was diluted to a final concentration of 200 nM with FP buffer (20 mM HEPES pH 7.4, 100 mM NaCl, 0.5 mg / ml BSA, 5% glycerol). The purified M1AR complex was also diluted to a final concentration of 0.3 mM with FP buffer.

[0099] (2) The M1AR complex was diluted 2-fold, and a total of 16 gradients were set;

[0100] (3) Transfer the M1AR complex of each concentration to a black 384-well plate, and then add an equal volume of MI9-14-FAM peptide to each M1AR complex concentration and incubate for 10 min.

[0101] (4) Set up the Synergy NEO multifunctional microplate reader, select the FP485 / 528 filter group, set the average adjustment value to 10000 RFU, set the gain well to FP buffer+MI9-14-FAM peptide well, request polarization 20mp, and perform detection.

[0102] (5) Using Graphpad Prism8 to process the data, nonlinear fitting was performed on the logarithm of each polarization value against the protein concentration. The affinity constant K between MI9-14 and M1AR was obtained by fitting the Sigmoidal dose-response formula. d .

[0103] Analysis of the polarization results shows that the K-polarization of MI9-14 combined with M1AR d The affinity was 2.57 ± 0.4 μM, which is higher than the affinity of M1AR for binding to H3WT (K). d =49.2±1.4μM) increased by about 20 times ( Figure 2 A).

[0104] 2.2 Detection Experiment of the Inhibitory Efficiency of MI9-14 on MLL1 Activity

[0105] Subsequently, to clarify the inhibitory efficiency of MI9-14 on MLL1, we used 20 μM H3 peptide and 40 μM AdoMet (S-adenosylmethionine) as substrates in vitro, utilizing Promega's MTase-Glo TM The Methyltansferase Assay Kit (catalog number: V7602) was used to detect the IC50 of MI9-14 inhibition of 80 nM M1WRAD complex activity based on bioluminescence. MTase-Glo TM The Methyltansferase Assay Kit works by converting the methyltransferase product AdoHcy (S-adenosylhomocysteine) into a bioluminescent signal through a series of reactions, thereby monitoring the AdoHcy production rate. Therefore, it can be used to detect the catalytic activity and activity changes of various methyltransferases, including DNA, protein, RNA, and small molecule methyltransferases. The experimental steps for detecting the inhibitory efficiency of MI9-14 on MLL1 activity are as follows:

[0106] (1) Determination of AdoHcy (S-adenosylhomocysteine) standard curve

[0107] To quantitatively detect the rate of the methyl transfer reaction, it is first necessary to establish a linear relationship between the bioluminescent signal intensity and the molar amount of AdoHcy.

[0108] a. AdoHcy was diluted 2-fold with 1×MTase buffer (25mM HEPES, pH 7.5, 100mM NaCl, 0.1mg / mL BSA), starting with the highest concentration of 1μM, and diluted in 8 gradients (1μM, 500nM, 250nM, 125nM, 62.5nM, 31.25nM, 15.6nM, and 7.8nM) to 7.8nM;

[0109] b. Transfer the eight concentrations of AdoHcy and the blank control 0 μM AdoHcy to white 384-well plates, with three replicates for each concentration, and add 2 μL of 0.5% TFA to each well;

[0110] c. Add 2 μL of 6×MTase-Glo Reagent from the kit to each well and incubate at room temperature for 30 min;

[0111] d. Add 12 μL of MTase-Glo Detection Solution from the kit to each well and incubate at room temperature for 30 min;

[0112] e. Set the Luminescence detection scheme in the multi-functional microplate reader interface, set the blank control well as the sample well, select a fixed gain of 100, run the detection and save the data;

[0113] f. Using Graphpad Prism 8.0, the bioluminescence intensity was linearly fitted to the molar amount of AdoHcy to obtain the AdoHcy standard curve.

[0114] (2) IC50 detection of MI9-14's inhibition of MWRAD complex enzyme activity

[0115] a. Set up a 2x substrate system: 40 μM H3 peptide, 80 μM AdoMet (S-adenosylmethionine), 25 mM HEPES, pH 7.5, 100 mM NaCl, 0.1 mg / mL BSA;

[0116] b. Set up a 2x enzyme system: 160 nM MWRAD, different concentrations of MI9-14, 25 mM HEPES, pH 7.5, 100 mM NaCl, 0.1 mg / mL BSA; (MI9-14 concentration gradients were set as follows: 10.24, 5.12, 2.56, 1.28, 0.64, 0.32, 0.16, 0.08, 0 μM)

[0117] c. For the 2x enzyme system, incubate at 4℃ for 6h, 12h, or without incubation, respectively;

[0118] d. Add an equal volume of the 2x enzyme system to the 2x substrate system, so that the reaction system contains 80 nM MWRAD, 20 μM H3 peptide, 40 μM AdoMet, and different concentrations of MI9-14 (5.12, 2.56, 1.28, 0.64, 0.32, 0.16, 0.08, and 0 μM). Mix well at 30°C, and transfer 8 μL of the reaction solution to a white 384-well microplate containing 2 μL of 0.5% TFA at 0 min, 5 min, 10 min, 20 min, 30 min, 45 min, and 60 min to terminate the reaction.

[0119] e. After all sampling is completed, proceed with the reaction and detection according to steps (1)c-(1)e of this embodiment;

[0120] f. The detected bioluminescence intensity was converted into the molar amount of AdoHcy using the AdoHcy standard curve. Then, the amount of AdoHcy was plotted against the reaction time in Graphpad Prism 8.0. The slope was obtained by selecting the linear interval and performing linear fitting, thus obtaining the initial rate of the MWRAD catalytic reaction at different MI9-14 concentrations.

[0121] g. The initial reaction rate at each MI9-14 concentration was nonlinearly fitted to the logarithm of the inhibitor concentration, and the IC50 values ​​of MI9-14 inhibiting the activity of different proteins were obtained by fitting the Sigmoidal dose-response formula.

[0122] The results of the enzyme activity inhibition experiment showed that when MI9-14 was not pre-incubated with M1WRAD, the IC50 of MI9-14 inhibiting the enzyme activity of M1WRAD was 12508±2023 nM. Figure 2 B); When MI9-14 was incubated with M1WRAD for 6 hours in advance to achieve covalent binding, the IC50 of MI9-14 inhibiting the enzyme activity of M1WRAD decreased significantly to 1610±392.2 nM. Figure 2 B); after incubating MI9-14 with M1WRAD for 12 h, the activity of MI9-14 was further enhanced, and the IC50 for inhibiting the enzyme activity of M1WRAD was 480.5 ± 68.6 nM. Figure 2 B) indicates that covalent bonding significantly enhances the activity of the MI9-14 molecule.

[0123] To verify the specificity of MI9-14 in inhibiting MLL1 enzyme activity, we also used MTase-Glo TMThe Methyltansferase Assay Kit was used to test the effects of MI9-14 on the activities of other methyltransferases using the method described above. Figure 2 The results of C showed that MI9-14 had no significant effect on the activity of other histone methyltransferases, including G9a (amino acids 913-1193, i.e., the catalytic domain of G9a), GLP (amino acids 982-1266, i.e., the catalytic domain of GLP), SUV39H1 (amino acids 82-412, i.e., the catalytic domain of SUV39H1), SUV39H2 (amino acids 112-410, i.e., the catalytic domain of SUV39H2), NSD1 (amino acids 1852-2105, i.e., the catalytic domain of NSD1), NSD2 (amino acids 953-1240, i.e., the catalytic domain of NSD2), and DOT1L (amino acids 1-420, i.e., the catalytic domain of DOT1L). It also had no significant effect on the activity of DNA methyltransferase DNMT1, and did not inhibit the activity of H3K4 demethylases KDM5A and LSD1. For other members of the MLL family, MI9-14 also showed some inhibitory effect on MLL2, which belongs to the same subfamily as MLL1 and also plays a role in the occurrence and progression of leukemia, but its inhibitory effect on the four members of the other two subfamilies was weak. Figure 2 C).

[0124] Example 3: MI9-14-TAT effectively inhibits leukemia cell proliferation and induces apoptosis.

[0125] 3.1 First, the C-terminus of the MI9-14 molecule was fused with the cell-penetrating peptide TAT (GRKKRRQRRRPQ) to promote the effective transmembrane entry of MI9-14 into cells. Subsequently, various leukemia cell lines were treated with MI9-14-TAT (amino acid sequence CRL{NLE}QTARYGRKKRRQRRRPQ), including the MLL-AF9 fusion cell lines MOLM-13 and THP-1, the MLL-AF4 fusion cell line MV4;11 (MOLM-13, THP-1, and MV4;11 are all derived from AML), and the non-MLL fusion cell lines K562 (BCR-ABL, CML) and Jurkat E6-1 (T-ALL); and the 293T cell line was used as a control to test cytotoxicity. Four days after treatment, the cell viability of each group was measured and calculated using the Meilun Biotech Super CCK-8 kit (catalog number: MA0225-1) to characterize the effect of MI9-14-TAT on leukemia cell proliferation. The specific experimental procedures are as follows:

[0126] (1) Seven cell lines—MOLM-13, THP-1, MV4;11, NOMO-1, Jurkat, K562, and 293T—were seeded in 96-well plates. 100 μL of 1500 THP-1 cells were seeded per well, and 1000 cells of the other cell lines were seeded per well. Each cell line was seeded in 30 replicate wells. The culture medium for MOLM-13, THP-1, MV4;11, NOMO-1, and Jurkat was RPMI 1640 + 10% FBS; for K562, it was RPMI 1640 + 20% FBS; and for 293T, it was DMEM + 10% FBS.

[0127] (2) After pre-culturing at 37°C in a 5% CO2 incubator for 24 hours, 100 μL of MI9-14-TAT diluted with the corresponding culture medium and 0.2% DMSO (three replicates for each group) were added to each cell well to make the final concentrations of MI9-14-TAT 180, 90, 45, 33.75, 22.5, 11.25, 5.6, 2.8, 1 and 0.1 μM, respectively. The cells were then cultured at 37°C in a 5% CO2 incubator.

[0128] (3) After culturing for 4 days, discard the old culture medium, add 100 μL of new culture medium and 10 μL of CCK-8 solution to each well, and be careful not to generate bubbles. At the same time, set up 100 μL of new culture medium with 10 μL of CCK-8 added as a blank control. Place the culture plate in an incubator and continue to incubate at 37°C for 2 to 6 hours. Then, read the absorbance at 450 nm using an ELISA reader.

[0129] (4) Calculate cell viability: Cell viability = [(absorbance of experimental wells - absorbance of blank wells) / (absorbance of control wells - absorbance of blank wells)].

[0130] (5) The cell survival rate at each MI9-14 concentration was nonlinearly fitted to the logarithm of the MI9-14 concentration, and the IC50 value of MI9-14 inhibiting the proliferation of different cell lines was obtained by fitting the Sigmoidal dose-response formula.

[0131] like Figure 3As shown in Figure A, MI9-14-TAT treatment did not affect the proliferation of 293T cells, indicating that MI9-14-TAT did not exhibit significant cytotoxicity. However, MI9-14-TAT significantly inhibited the proliferation of both MLL-AF4 and MLL-AF9 MLL fusion leukemia cell lines, with IC50 values ​​of MV4, 11 (3.5±0.5μM), MOLM-13 (7.2±0.8μM), NOMO-1 (4.9±0.4μM), and THP-1 (10.8±1.8μM), respectively. Observation of MOLM-13 cells treated with 40μM MI9-14-TAT for 72h revealed significant cell death and fragmentation compared to solvent treatment, accompanied by the release of contents (see Figure A). Figure 3 B). In addition, we found that MI9-14-TAT can also inhibit the proliferation of MLL non-fusion leukemia cell lines K562 and Jurkat E6-1 to some extent. This means that the methyltransferase activity of MLL1 / MLL2 may also play a certain role in promoting the occurrence and progression of this type of CML or T-ALL, indicating that MLL1 / MLL2 may be a more broad-spectrum leukemia drug target.

[0132] 3.2 Leukemia cells often evade cell death and achieve unlimited proliferation by upregulating the expression of anti-apoptotic factors or mutating and inactivating pro-apoptotic factors. Although the mechanisms of action and targets of commonly used chemotherapy drugs and targeted drugs vary, most drugs ultimately kill leukemia cells by inducing apoptosis. Therefore, we treated MV4;11 cells with MI9-14-TAT for 48 h and used the Annexin V-FITC / PI apoptosis detection kit (Elabscience, catalog number: E-CK-A211) in combination with flow cytometry to investigate the pro-apoptotic effect of MI9-14-TAT, to explore its mechanism of action, and to evaluate its potential in inhibiting malignant proliferation and delaying disease progression. The Annexin V-FITC / PI apoptosis detection kit works by using FITC-labeled calcium-dependent phospholipid-binding protein Annexin V (FITC-Annexin V) to bind to phosphatidylserine (PS) residues that evert onto the cell membrane during apoptosis. Meanwhile, the DNA-binding dye propidium iodide (PI) specifically stains the double-stranded DNA of necrotic cells or cells that have lost their cell membrane integrity in late apoptosis. The combination of FITC-Annexin V and PI dye allows for the differentiation of cells at different stages of apoptosis. The specific experimental procedures are as follows:

[0133] (1) The MLL fusion leukemia cell line MV4;11 and the control MLL non-fusion leukemia cell line JurkatE6.1 were seeded in 12-well plates, with 1 mL of MV4;11 seeded per well (7×10⁻⁶ cells). 5 Jurkat E6.1 cells were seeded at 1 mL per well (6 × 10⁶ cells per well). 5 10 cells, each cell line seeded in 27 replicate wells;

[0134] (2) After pre-culturing at 37°C in a 5% CO2 incubator for 12 h, 100 μL of MI9-14-TAT diluted with the corresponding culture medium and 0.2% DMSO were added to each cell well (three replicates for each group) to make the final concentrations of MI9-14-TAT 50, 40, 30, 20, 15 and 10 μM respectively. The cells in 6 wells were left untreated and continued to be cultured at 37°C in a 5% CO2 incubator.

[0135] (3) After culturing for 48 h, 100 μL of camptothecin (CPT) diluted with the corresponding culture medium was added to three untreated wells of each cell type (apoptosis positive control, repeated three times). The final concentration of CPT for MV4;11 cells was 4 μM, and the final concentration of CPT for Jurkat cells was 6 μM. The cells were then cultured at 37 °C in a 5% CO2 incubator.

[0136] (4) Collect cells after culturing for 4 hours;

[0137] a. The total cell density, viable cell density, and cell viability of each group of treated cells were analyzed and recorded by trypan blue staining.

[0138] b. Transfer the cell culture medium completely to each well into a centrifuge tube, rinse the culture dish with 400 μL / well of pre-cooled PBS, combine the two, centrifuge at 300×g for 5 min, and discard the supernatant;

[0139] c. Gently resuspend cells in 1.2 mL / well of pre-cooled PBS, centrifuge at 300×g for 5 min, and discard the supernatant;

[0140] d. Gently resuspend cells in 1 mL / well of pre-chilled PBS;

[0141] e. Count by viable cell density, each sample according to (5 × 10⁻⁶) 5 Samples were aliquoted into 3 tubes (1 cell / tube) and then placed on ice.

[0142] (5) Streaming analysis;

[0143] a. Select the B525 / B690 fluorescence excitation channel;

[0144] b. Centrifuge the untreated cell group at 300×g for 5 min, discard the supernatant, add 500μL of 1×binding buffer (from Annexin V-FITC / PI cell apoptosis detection kit) to gently resuspend the cells, and adjust the voltage before instrumentation.

[0145] c. CPT treatment group: centrifuge at 300×g for 5 min, discard supernatant, gently resuspend cells in 100 μL of 1×binding buffer, add 2.5 μL of FITC for 10 min at room temperature in the dark (FITC single staining) or 2.5 μL of LPI for 5 min at room temperature in the dark (PI single staining), then add 400 μL of 1×binding buffer, mix well, and adjust compensation on the instrument.

[0146] Centrifuge sample group 300×g for 5 min, discard supernatant, gently resuspend cells in 100 μL of 1×binding buffer, add 2.5 μL of FITC and stain at room temperature in the dark for 5 min, then add 2.5 μL of PI and stain for 5 min (FITC+PI double staining), add 400 μL of 1×binding buffer, mix well, and test on the instrument.

[0147] (5) Data were processed using FlowJo 10.8.1 software. The distribution of single-stained cells in the two groups of CPT-treated groups was used to define the cross-shaped cell populations, where Q1 was the naked nucleus cell population, Q2 was the late apoptotic cell population, Q3 was the early apoptotic cell population, and Q4 was the normal cell population.

[0148] like Figure 3 As shown in Figure C: Compared with DMSO, treatment of MV4.11 cells with 20 μM MI9-14-TAT for 48 h significantly increased the number of early apoptotic cells (Q3, Annexin V-FITC+, PI-) and late apoptotic cells (Q2, Annexin V-FITC+, PI+), reaching 35.2% and 16.7%, respectively. Furthermore, the proportion of apoptotic cells gradually increased with increasing MI9-14-TAT concentration. Figure 3 D). These data indicate that MI9-14-TAT can induce apoptosis in MLL fusion-type leukemia cells MV4;11 in a dose-dependent manner. However, for non-MLL fusion-type T-ALL leukemia cells Jurkat E6.1, MI9-14-TAT treatment did not alter their cell state or induce apoptosis. These results confirm the specific pro-apoptotic effect of MI9-14-TAT on MLL fusion-type leukemia cell lines.

[0149] Example 4: Some variants of MI9-14 still maintain a considerable affinity for M1AR.

[0150] To investigate whether certain amino acids in MI9-14 could be substituted by other amino acids, we performed amino acid substitution mutations at certain sites in MI9-14 and tested their affinity for M1AR. The specific MI9-14 variants tested and their affinity test results for M1AR are shown in Table 1 and [Table data missing]. Figure 5 .

[0151] Table 1: MI9-14 variants whose affinity for M1AR was experimentally verified.

[0152]

[0153] From Table 1 and Figure 5 The results shown indicate that amino acids at some sites in the MI9-14 peptide inhibitor can be replaced, and the peptide still maintains a similar affinity for M1AR after replacement. Figure 5 In A, replacing the leucine (L) at position 3 of MI9-14 with other aliphatic hydrophobic amino acids such as methionine (M) or valine (V) did not significantly decrease its affinity. d The values ​​were 2.50 μM and 2.88 μM, respectively. Figure 5 In B, replacing the threonine (T) at position 6 of MI9-14 with other hydrophobic amino acids such as phenylalanine (F) or isoleucine (I) results in an affinity similar to that of MI-914. d The values ​​were 2.68 μM and 2.84 μM, respectively. Figure 5 C shows that replacing the 7th alanine (A) in MI9-14 with any other amino acid, including nonpolar amino acids such as tryptophan (W), polar non-ionizing neutral amino acids such as glutamine (Q), acidic amino acids such as aspartic acid (D), or basic amino acids such as histidine (H), still maintains an affinity comparable to that of MI-914.

[0154] This result indicates that in MI9-14, the L at position 3 can be replaced by other aliphatic hydrophobic amino acids, including M (verified), V (verified), or isoleucine (I); the T at position 6 can be replaced by other hydrophobic amino acids, including F (verified), W, tyrosine (Y), I (verified), L, M, or V; and the A at position 7 can be replaced by any amino acid, including nonpolar amino acids such as W (verified), F, A, L, M, I, proline (P), or V, polar non-ionizing neutral amino acids such as Q (verified), cysteine ​​(C), glycine (G), asparagine (N), serine (S), Y, or T, acidic amino acids such as D (verified) and glutamic acid (E), or basic amino acids such as H (verified), arginine (R), or lysine (K).

[0155] Comparative Example 1: MI9-14 showed significantly better activity than the inhibitors MI9-17 and MI9-11.

[0156] MI9-14 has two important characteristics: (1) it enhances the hydrophobic interaction between key residue sites and the catalytic active pocket of MLL1 by introducing the non-natural amino acid ortholeucine (NLE); (2) it can target the key cysteine ​​residue on the "door loop" region of MLL1 and covalently bind to it. During the design and screening of MI9-14, two other inhibitory peptide molecules were also obtained: MI9-11 and MI9-17. MI9-11 has a CRTMQTARY sequence, and compared to MI9-14 (CRL{NLE}QTARY), the third and fourth amino acids of MI9-11 are less hydrophobic; MI9-17 has a ARL{NLE}QTARY sequence, and only the first amino acid differs from MI9-14. Due to the lack of cysteine, MI9-17 cannot covalently bind to the "door loop" region of MLL1.

[0157] The covalent binding of MI9-11 and MI9-17 to MLL1 was detected by MALDI-TOF MS / MS. The experimental method was consistent with the MALDI-TOF MS / MS detection procedure in Example 1.1, except that MI9-14 was replaced with MI9-11 and MI9-17, respectively. In this experiment, the theoretical molecular weight of the MLL1 protein fragment used was 18211.15 Da, the theoretical molecular weight of MI9-11 was 1094.3 Da, and the theoretical molecular weight of MI9-17 was 1091.3 Da. Therefore, the covalent binding of MI9-11 to MLL1 could be determined by detecting the changes in signal intensity at molecular weights of 18211.15 and 19305.45 Da; the covalent binding of MI9-17 to MLL1 could be determined by detecting the changes in signal intensity at molecular weights of 18211.15 and 19302.45 Da.

[0158] Subsequently, we used fluorescence polarization experiments to detect the binding affinity of MI9-11 and MI9-17 molecules to MLL1. The experimental method was the same as the operation steps of the fluorescence polarization experiment in 2.1 of Example 2, except that the 5-FAM fluorescently labeled MI9-14 was replaced with 5-FAM fluorescently labeled MI9-11 and 5-FAM fluorescently labeled MI9-17, respectively.

[0159] Also utilizes MTase-Glo TMThe Methyltansferase Assay Kit was used to detect the IC50 of MI9-11 and MI9-17 on the enzyme activity inhibition of 80 nM M1WRAD complex based on bioluminescence. The experimental method was the same as the experimental procedure for detecting the inhibitory efficiency of MI9-14 on MLL1 activity in Example 2, except that the MI9-14 molecule was replaced with MI9-11 or MI9-17 respectively.

[0160] like Figure 4 As shown in Figure A (right), MALDI-TOF MS / MS results indicate that the inhibitor peptide MI9-11 can also covalently bind to MLL1 (a signal peak at 19305 Da appears after 6 hours of incubation). However, fluorescence polarization experiments show that the reduced hydrophobicity of the third and fourth residues of MI9-11 significantly decreases its affinity for M1AR. The K+ binding capacity of MI9-11 to M1AR is... d Only 12.8±3.2μM ( Figure 4 B). Decreased affinity led to a reduced inhibitory efficiency of MI9-11 molecules on MLL1 enzyme activity, with an IC50 of 27.6 ± 4.3 μM. Figure 4 C) The half-inhibitory concentration (IC50) of the MI9-14 molecule is about 58 times higher than that of the MI9-14 molecule (IC50 = 0.48 ± 0.07 μM), which indicates that the high hydrophobicity of the key residues of the inhibitor peptide is crucial for the binding of the inhibitor molecule to inhibit the enzyme activity of MLL1.

[0161] Unlike MI9-11, fluorescence polarization experiments showed that MI9-17 has a high affinity for M1AR, with a Kd of 0.4 ± 0.03 μM. Figure 4 B). However, MALDI-TOF MS / MS experiments confirmed, as expected, that the MI9-17 molecule could not covalently bind to MLL1 (no signal peak at 19302.45 Da). Figure 4 (Left figure A). Because it cannot covalently bind to MLL1, although its affinity for M1AR is higher than that for MI9-14 (K... d The inhibitory efficiency of MI9-17 was approximately 6 times higher than that of MI9-14 (IC50 = 0.48 ± 0.07 μM), but the IC50 was still lower than that of MI9-14 (IC50 = 0.48 ± 0.07 μM), with an IC50 of 1.6 ± 0.3 μM. Figure 4 C), which indicates that the covalent binding of the inhibitor molecule to MLL1 significantly enhances the activity of the inhibitor.

[0162] In vitro enzyme activity inhibition assays showed that the enzyme activity inhibition efficiency of MI9-17 was only about 4 times that of MI9-14. Therefore, the effect of MI9-17 on the proliferation capacity of leukemia cells was also tested. We fused the C-terminus of the MI9-17 molecule with the cell-penetrating peptide TAT (GRKKRRQRRRPQ) to promote the effective cell-penetrating action of MI9-17. Subsequently, various leukemia cell lines, including MLL-AF9 fusion cell lines (MOLM-13 and NOMO-1) and the non-MLL fusion cell line K562 (BCR-ABL, CML), were treated with MI9-17-TAT (amino acid sequence ARL{NLE}QTARYGRKKRRQR RRPQ). After 72 h of treatment, the cell viability of each group was measured and calculated using CCK-8 assays to characterize the effect of MI9-17-TAT on leukemia cell proliferation. Specific experimental procedures are as follows:

[0163] (1) Three cell lines, MOLM-13, NOMO-1, and K562, were seeded in 96-well plates, with 100 μL of 2000 cells per well and 5 replicates per cell line. The culture medium for MOLM-13 and NOMO-1 was RPMI 1640 + 10% FBS, and the culture medium for K562 was RPMI 1640 + 20% FBS.

[0164] (2) After pre-culturing at 37°C in a 5% CO2 incubator for 24 hours, 100 μL of MI9-17-TAT, MI9-14-TAT and 0.4% DMSO diluted with the corresponding culture medium were added to each cell well (five replicates for each group) to make the final concentration of the inhibitor peptide 40 μM, and the cells were continued to be cultured at 37°C in a 5% CO2 incubator.

[0165] (3) After culturing for 72 hours, discard the old culture medium, add 100 μL of fresh culture medium and 10 μL of CCK-8 solution to each well, and be careful not to generate bubbles. At the same time, set up 100 μL of fresh culture medium with 10 μL of CCK-8 added as a blank control. Place the culture plate in an incubator and continue to incubate at 37°C for 2 to 4 hours. Then, read the absorbance at 450 nm using an ELISA reader.

[0166] (4) Calculate cell viability: Cell viability = [(absorbance of experimental wells - absorbance of blank wells) / (absorbance of control wells - absorbance of blank wells)].

[0167] like Figure 4As shown in Figure D, 40 μM MI9-14-TAT significantly inhibited the proliferation of MOLM-13 and NOMO-1 cells, and also had some inhibitory effect on K562 cells; while the same concentration of MI9-17-TAT did not show a significant inhibitory effect on the proliferation of MOLM-13, NOMO-1, and K562 cells. This further indicates that the covalent binding of the inhibitor molecule to MLL1 is crucial for enhancing the biological activity of the inhibitor.

[0168] In summary, the MI9-14 molecule exhibits significantly better inhibitory efficiency and biological activity against MLL1 than MI9-11, which has reduced hydrophobicity of key residues, and MI9-17, which cannot covalently bind to MLL1.

[0169] The sequences involved in this application are shown in Table 2 below. Because the sequences in this application involve non-natural amino acids, they may not be accurately represented in a computer-readable sequence listing. If the computer-readable XML sequence listing attached to this application differs from the information in Table 2, the sequence information in Table 2 shall prevail.

[0170] Table 2: Partial Sequence Information Related to This Application

[0171]

[0172]

[0173]

[0174]

Claims

1. A polypeptide whose amino acid sequence comprises CRX1{NLE}QX2X3RY or CRX1{NLE}QX2X3RY; wherein X1 is L, M, V, or I; X1 is preferably L; X2 is T, F, I, W, Y, L, M, or V; X2 is preferably T; X3 is any amino acid selected from the group consisting of: A, W, F, L, M, I, P, V, Q, C, G, N, S, Y, T, D, E, H, R, and K; X3 is preferably A.

2. A polypeptide, which is an MI9-14-TAT fusion polypeptide, that is, the C-terminus of MI9-14 fused with TAT; The amino acid sequence of MI9-14 includes CRX1{NLE}QX2X3RY or CRX1{NLE}QX2X3RY; wherein X1 is L, M, V or I, preferably L; X2 is T, F, I, W, Y, L, M or V, preferably T; X3 is any amino acid selected from the group consisting of A, W, F, L, M, I, P, V, Q, C, G, N, S, Y, T, D, E, H, R and K, preferably A; The amino acid sequence of the TAT is GRKKRRQRRRPQ (SEQ ID NO:2); The preferred amino acid sequence of the MI9-14-TAT fusion polypeptide is CRL{NLE}QTARYGRKKRRQRRRPQ.

3. The polypeptide of claim 1 or 2, which targets the catalytic active site of MLL1 to inhibit the activity of MLL1.

4. A pharmaceutical composition comprising a polypeptide as described in claim 1 or 2 and one or more pharmaceutically acceptable carriers.

5. A combination comprising the polypeptide as described in claim 1 or 2, and one or more therapeutic agents.

6. The combination of claim 5, wherein one or more therapeutic agents are anticancer agents, analgesics, or anti-inflammatory agents.

7. A method for treating a disease or condition in which one benefits from the inhibition of mixed lineage leukemia 1 (MLL1), comprising administering to a subject in need a therapeutically effective amount of the compound as claimed in claim 1 or 2.

8. The method of claim 7, wherein the disease or condition in which MLL1 is benefited by inhibition is chronic myeloid leukemia (CML), acute T-lymphoblastic leukemia (T-ALL), acute myeloid leukemia with MLL1 translocation rearrangement (AML), or acute lymphoblastic leukemia with MLL1 translocation rearrangement (ALL); wherein the disease or condition is preferably acute myeloid leukemia with MLL1 translocation rearrangement (AML) or acute lymphoblastic leukemia with MLL1 translocation rearrangement (ALL).

9. Use of the compound of claim 1 or 2 in the preparation of a medicament for treating a disease or condition in which the patient benefits by inhibiting mixed lineage leukemia 1 (MLL1).

10. The use as claimed in claim 9, wherein the disease or condition in which MLL1 is benefited by inhibition is chronic myeloid leukemia (CML), acute T-lymphoblastic leukemia (T-ALL), acute myeloid leukemia with MLL1 translocation rearrangement (AML), or acute lymphoblastic leukemia with MLL1 translocation rearrangement (ALL); wherein the disease or condition is preferably acute myeloid leukemia with MLL1 translocation rearrangement (AML) or acute lymphoblastic leukemia with MLL1 translocation rearrangement (ALL).

11. Use of the compound of claim 1 or 2 in combination with a second therapeutic agent in the preparation of a medicament for treating a disease or condition that can be treated by inhibiting mixed lineage leukemia 1 (MLL1).

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