CN116602947B - Small molecular compound and application thereof in preparation of medicines for treating FLT3 mutant leukemia - Google Patents

Abstract

The invention discloses a small molecular compound and application thereof in preparing a medicament for treating FLT3 mutant leukemia, and belongs to the field of biological medicines. The small molecular compound drug17 is a small molecular compound designed on the basis of a binding site screened by a wild type and mutant FLT3 activation state structure model constructed by a homologous molecular model. Experiments prove that drug17 can inhibit the growth of FLT3-ITD mutant leukemia cells in vitro, inhibit the phosphorylation level of FLT3, promote apoptosis and cause cycle arrest. And the drug17 has a certain degree of inhibition effect on the activity of FLT3 protein kinase at the level of the biochemical reaction, and the IC50 can reach the micromolar level. The small molecular compound drug17 provided by the invention is expected to reduce the drug resistance problem of secondary TKD region mutation, and provides a new hope for accurately targeting treatment of FLT3 mutation positive AML patients.

Description

Small molecular compound and application thereof in preparation of medicines for treating FLT3 mutant leukemia

Technical Field

The invention relates to the field of biological medicine, in particular to a small molecular compound and application thereof in preparing medicines for treating FLT3 mutant leukemia.

Background

Acute myeloid leukemia (Acute myeloid leukemia, AML) accounts for about 80% of acute leukemia in adults, and is an important type of leukemia. In the past 40 years, except that the cure rate of acute promyelocytic leukemia is remarkably improved due to the introduction of targeted therapy in AML, the treatment progress of other AML types is quite slow, and the scheme of high-dose cytarabine chemotherapy or allogeneic hematopoietic stem cell transplantation is mainly adopted to induce chemotherapy and subsequent risk layering consolidation treatment. Under this strategy, AML patients under 60 years old have a 5-year survival rate of only about 40%, whereas AML patients over 60 years old have a 5-year survival rate of less than 10%. Under the era of precise medicine, active exploration of targeted therapies is expected to improve the prognosis of such AML.

FLT3 (Fms-like tyrosine kinase, FMS-like tyrosine kinase 3) is a member of the subfamily of receptor tyrosine kinases III (receptor tyrosine kinase III, RTK III), with mutations in up to 30% of adult AML. FLT3 has mainly two mutation types in AML, namely an internal tandem repeat (ITD) mutation accounting for about 2/3 and a kinase domain (TKD) point mutation accounting for about 1/3. Both types of mutations are gain-of-function mutations, which can lead to the sustained autophosphorylation of FLT3 receptor without depending on FLT3 ligand, aberrant activation of signaling pathway, involvement in leukemia development and maintenance of cell aberrant proliferation capacity. Meanwhile, compared with a mutant negative patient, the FLT3-ITD positive AML patient has poorer response to induced chemotherapy, higher recurrence rate, and shorter total survival time without recurrence. The influence of FLT3-TKD mutation on the prognosis of AML patients is still controversial, but FLT3-ITD positive AML patients are subjected to early targeting treatment and then partial secondary FLT3-TKD mutation leads to acquired drug resistance and treatment failure, and brings great challenges for the subsequent treatment of AML. Since FLT3 mutation is the most common molecular mutation in AML, and mutated patients have poor prognosis under traditional treatment strategies, FLT3 naturally becomes one of the most attractive therapeutic targets in AML, hopefully improving the prognosis of this class of AML.

There are more than ten small molecule inhibitors currently available for FLT3 receptor kinase and entering clinical trials or approved batches. Although encouraging clinical efficacy is achieved to varying degrees, each inhibitor presents different problems. In addition to the application of pharmacokinetic changes in humans, important plagued problems include targeting non-specific problems, toxic and side effects after off-targeting problems, secondary mutation drug resistance and the like. The reason for this is that there is a deficiency in design, which is the core symptom. Because these targeted inhibitors were obtained by biochemical or cell level specific experimental screening or by designing, screening and optimizing other tyrosine kinase proteins as structural models, even though recently targeted drugs specific to FLT3 kinase proteins are designed, they are often based on the structural biology alone on the FLT3 protein crystal structure (1 RJB) which is wild-type and in a self-inhibiting state.

Therefore, based on the latest cognition of the functions and structures of the FLT3 kinase protein and the mutant thereof, the structure of the FLT3 self-inhibition and activation state is analyzed again, particularly the structure characteristic analysis of the mutant is performed, the unique structure characteristic of the FLT3 is used as the basis to search for new small molecule binding sites and screen and optimize specific small molecule inhibitors, a new scientific thinking is brought to the development of novel FLT3 small molecule targeted inhibitors, and the novel FLT3 small molecule targeted inhibitors are hopefully selected for the treatment of FLT3 mutant AML patients.

Disclosure of Invention

The invention aims to provide a small molecular compound and application thereof in preparing medicines for treating FLT3 mutant leukemia, so as to solve the problems in the prior art. The small molecular compound drug17 provided by the invention can be used as an inhibitor of FLT3 kinase, plays roles in resisting FLT3-ITD and drug-resistant mutant acute myelogenous leukemia, is expected to reduce the drug resistance problem of secondary TKD region mutation, and provides a new hope for accurately targeting treatment of FLT3 mutation positive AML patients.

In order to achieve the above object, the present invention provides the following solutions:

the invention provides an application of a small molecular compound in preparing a medicament for treating FLT3 mutant leukemia or inhibiting FLT3 protein phosphorylation, wherein the small molecular compound has a chemical structure as follows:

further, the FLT3 mutant leukemia is FLT3 mutant acute myeloid leukemia.

Further, the FLT3 mutation includes a FLT3-ITD mutation, a FLT3-TKD mutation, a FLT3-ITD-TKD mutation and a FLT3-ITD-F691L mutation.

Further, the FLT3 protein is a FLT3 mutated protein.

Further, the FLT3 mutation includes a FLT3-ITD mutation, a FLT3-TKD mutation, a FLT3-ITD-TKD mutation and a FLT3-ITD-F691L mutation.

Further, the medicament also comprises pharmaceutically acceptable auxiliary materials.

Further, the dosage forms of the medicament include injection, tablet, pill and granule.

The invention discloses the following technical effects:

the invention searches for a novel small molecule binding site specific to FLT3 based on a wild type and mutant (FLT 3-ITD, FLT3-ITD and D835Y double mutant and FLT3-ITD and F691L double mutation) FLT3 activation state structure model constructed by a homologous molecular model, and identifies and obtains a small molecule compound drug17 through computer virtual screening, optimization and subsequent biochemical, cellular and horizontal activity verification. Proved by verification, the small molecular compound drug17 can be used as an inhibitor of FLT3 kinase to play roles in resisting FLT3-ITD and drug-resistant mutant acute myelogenous leukemia. Experiments prove that drug17 can inhibit the growth of FLT3-ITD mutant leukemia cells in vitro, inhibit the phosphorylation level of FLT3, promote apoptosis and cause cycle arrest. And the drug17 has a certain degree of inhibition effect on the activity of FLT3 protein kinase at the level of the biochemical reaction, and the IC50 can reach the micromolar level.

The small molecular compound drug17 provided by the invention is expected to reduce the drug resistance problem of secondary TKD region mutation, and provides a new hope for accurately targeting treatment of FLT3 mutation positive AML patients.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a wild-type FLT3 self-inhibition model and an activation state molecular dynamics model of FLT3-ITD, FLT3-ITD partner D835Y, and FLT3-ITD partner F691L;

FIG. 2 shows OD values measured using the CCK8 assay after 100. Mu.M single concentrations of different small molecule drugs were used to treat normal 32D cells and 32D-ITD mutant stable transformants 72; wherein 0 represents DMSO control, 1-25 represent the number of small molecule compounds initially screened; drug17 corresponds to number 17, which has stronger inhibitory effect on 32D-ITD mutant stable transgenic strain than normal 32D cells at 100. Mu.M;

FIG. 3 shows the cell viability detected by CCK8 after 72 hours of treatment with drug17 at the same concentration gradient in normal 32D and 32D-ITD mutant stable transformants, resulting in drug17 IC50 for 32D and 32D-ITD of 50.23. Mu.M and 30.5. Mu.M, respectively, and the experiment was repeated three times and a near effect, showing statistics of three replicates from one of the experiments;

FIG. 4 shows the half inhibitory concentration (IC 50) of drug17 on normal 32D cells, 32D-ITD mutant stable transformants, FLT3 mutant leukemia cell lines (MV-4-11, molm 13) and non-FLT 3 mutant leukemia cell lines (THP-1, HL60, K562); three replicates, representing P < 0.01;

FIG. 5 shows the ability of drug17 to inhibit FLT3 kinase activity (A) based on Homogeneous Time Resolved Fluorescence (HTRF) technique, with a small molecule AC220 group as control (B), and a concentration gradient set to detect FLT3 kinase activity; the result shows that drug17 has a certain degree of inhibition effect on FLT3 protein kinase activity under in vitro conditions, and the IC50 can reach the micromolar level;

FIG. 6 shows the detection of FLT3 phosphorylation levels by cell harvest, protein lysis, protein purification and WB after 6 hours of action on 293T-FLT3-ITD stable transformants using DMSO, drug17 and AC220, respectively; DMSO, AC220 as control group, the results indicate that drug17 has inhibitory effect on FLT3 phosphorylation;

FIG. 7 shows the change in apoptosis detected by flow cytometry in the cells harvested after drug17 and sorafenib are applied to the 32D parental strain and 32D-ITD stable transgenic strain, as compared to the change in apoptosis between the drug-treated and non-drug-loaded groups; the result shows that drug17 and sorafenib are similar to each other and can induce apoptosis effects of 32D-ITD stable transgenic strain and parent strain cells to a certain extent, wherein the 32D-ITD stable transgenic cell strain is more sensitive to apoptosis induced by the lead micromolecules than the parent strain, and high-proportion late apoptosis is induced;

FIG. 8 shows cell cycle detection by flow cytometry of harvested cells after drug17 action on 32D parental and 32D-ITD stable transgenic plants, comparing cell cycle changes between drug-treated and non-drug-loaded groups; the results indicate that drug17 in comparison to the 32D parent strain causes a significant G0/G1 phase arrest in the 32D-ITD stable cells.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

The inventor has studied extensively and intensively, and has utilized the supercomputer of 'light of Taihu' to perform molecular dynamics simulation on FLT3 wild type, FLT3-ITD accompanied with D835Y double mutant and FLT3-ITD accompanied with F691L double mutant proteins for the first time, and explored a novel small molecule binding site specific to FLT 3. Unlike the currently mainstream design concept based on the structural biology of the FLT3 protein crystal structure in the wild type and in the self-inhibition state, the inventor utilizes the newly discovered structural characteristics of the mutant type and the intermediate non-activation state of the wild type FLT3 protein, and obtains the highly sensitive and highly specific FLT3 small molecule inhibitor (9E) -9-hydrozinylidenefluorene-2, 3-diene (nci number VCN-092758) through the high-throughput screening of the public database of the national cancer research center, the compound molecular library of the chinese country and other databases by computer virtual screening software according to the mutant type constructed by the homologous molecular model, the wild type FLT3 activation state structural model (fig. 1) and the existing self-inhibition model of the wild type FLT3, wherein the novel small molecule binding site of the discovered FLT3 is obtained by the novel mutant type and the wild type FLT3 activation state structural characteristics of the wild type FLT3 protein, and the novel FLT3 activation state structural characteristics are obtained by cell level and biochemical level screening and optimization, and the chemical structural formula is:

experimental materials used in the examples of the present invention:

experimental cells:

human Molm-13, MV4-11, THP-1, K562, HL60, 293T cells and mouse 32D cells were all purchased from North Nanophyte. The mouse 32D-FLT3-ITD stable transgenic cell and 293T-FLT3-WT/ITD stable transgenic cell are built internally.

Experimental reagent:

AC220, sorafenib, available from shanghai Tao Su biochemical technologies limited; drug17 was constructed by ABI Chem (germany). Recombinant Murine IL-3 available from PEPROTECH corporation (USA); CCK8 reagent was purchased from syn-chemical company (japan); annexin V-APC apoptosis detection kit, cell cycle detection kit purchased from biogammes (USA); HTRF kit was purchased from cisbio corporation (France); anti-pFLT3 antibodies were purchased from Cell Signaling Technology company (USA); the HAtag mouse mAb was purchased from gold company (china); horseradish peroxidase-labeled goat anti-rabbit IgG (h+l) was purchased from beyotidme corporation (china).

The experimental method comprises the following steps:

construction of FLT3-WT/ITD mutant plasmid

The plasmid vector pLenti-III-EF1 alpha (LV 043) is purchased from AppliedBiological Materials (abm) Inc. (Canada), and is constructed by designing enzyme cutting sites at two ends (5 '-end BamHI-3' -end XbaI), amplifying target sequences, connecting and transforming to construct target plasmids, and the constructed plasmids are verified by gene sequencing to be successful target FLT3-WT/ITD mutant plasmids.

2. Construction of mouse 32D-FLT3-ITD stable transgenic cell and 293T-FLT3-WT/ITD stable transgenic cell

(1) FLT3-WT/ITD mutant lentiviral package

1) Selecting 293T cells to spread on a 10cm cell culture dish for subculture to grow to logarithmic phase, adjusting cell strain state, and uniformly growing until the cells occupy 80% of dish bottom volume (cell number is 7×10) ⁶ Individual/dish).

2) 2 hours before the virus starts to package, the original medium is discarded and replaced with 5mL of fresh DMEM complete medium.

3) Lentiviral packaging system is shown in Table 1, two 1.5mL EP tubes were used, and after labelling tube A, B, 500. Mu.L of serum-free medium DMEM was added, and 40. Mu.L of liposome Lipo 2000 was added to tube B, followed by gentle stirring and mixing. And (3) respectively adding the tube A into the packaging system shown in the table 1, and lightly blowing and uniformly mixing.

TABLE 1 lentiviral packaging System

4) The liquid in the pipe B is sucked, the pipe A is slowly dripped dropwise until all the liquid is dripped, the liquid is gently flushed and mixed for three times, and the mixture is placed in an incubator at 37 ℃ for incubation for 30 minutes. And (3) dropwise adding the mixed solution of the two pipes A and B into a cell culture dish slowly and gently, uniformly mixing by a cross method, and placing the mixed solution in an incubator for culture.

5) After 6 hours of culture, the original culture medium was completely discarded in a waste liquid treatment device containing a disinfectant, and 5mL of fresh DMEM complete culture medium was added to complete cell replacement.

6) After 24 hours of incubation, the lentiviral-containing cultures were collected for the first time based on 15mL centrifuge tubes, stored at 4℃under sealing, and 5mL fresh DMEM complete medium was added.

7) After 48 hours and 72 hours of incubation, respectively, the second and third viruses were collected. Centrifuging all collected virus liquid at 1500 rpm at 4deg.C for 15 min, filtering supernatant with 0.22 μm filter, removing cell debris, and storing in refrigerator at-80deg.C.

(2) FLT3-WT/ITD lentivirus infection 293T and mouse 32D cells and stable strain screening

1) After completion of the packaging of the FLT3-WT/ITD mutant lentiviruses described above, appropriate amounts of lentiviruses were taken to infect the mouse 32D cells and 293T cells.

2) The culture medium containing puromycin was started to incubate 72 hours after infection until all negative control cells died.

3) The 293T/32D cells with good growth are taken for lysis, whether the cells stably express the FLT3 wild type/mutant type proteins or not is identified by detecting whether the HA-tag is expressed or not through Western Blot, and the cells capable of stably expressing the FLT3 wild type/mutant type proteins are identified as the mouse 32D-FLT3-ITD stable transgenic cells and the 293T-FLT3-WT/ITD stable transgenic cells.

Method for detecting cell viability by CCK8 method

The CCK8 method is a reliable and sensitive method for detecting cell viability. In CCK-8 reagent-8 will react in the presence of the electron carrier 1-Methoxy PMS to give the water-soluble formazan dye +.>-8, the cell viability can be reflected by detecting its absorbance at 450 nm.

Htrf technology to detect the inhibitory Capacity of drugs to FLt3 kinase Activity

Kinase activity detection is based on Homogeneous Time Resolved Fluorescence (HTRF) technology, in which acceptor fluorescent molecules are labeled on FLT3 substrates, donor fluorescent molecules are labeled on phosphorylation modified specific antibodies, and fluorescence energy is transferred when FLT3 phosphorylates itself, thereby obtaining recognition signals. And (3) measuring the enzymatic reaction efficiency by setting the concentration gradient of the acceptor fluorescent molecular marker and the donor fluorescent molecular marker, and drawing an enzymatic reaction kinetic curve.

Example 1 Targeted inhibition capability of drug17

Drug17 is more sensitive at the cellular level to cell lines with ITD mutations than to cell lines with less FLT3 mutations

Through the virtual screening result in the small molecule library in the early stage, tens of small molecule medicines are constructed. Based on the inhibition of these small molecule drugs at 100. Mu.M on the 32D parent strain and the 32D-ITD mutant strain, ten more drugs more sensitive to the 32D-ITD mutant strain were initially screened (FIG. 2). Drug17 was also confirmed to exhibit a more pronounced inhibitory effect on the 32D-ITD mutant strain in the multi-concentration gradient, and this difference was statistically significant, by drug sensitivity assay with the multi-concentration gradient (fig. 3).

Subsequently, the present invention examined the growth inhibitory capacity of drug17 against cell lines with or without FLT3 mutation. The results showed that drug17 had lower half inhibitory concentration (IC 50) for the myeloid leukemia cell lines (MV-4-11, molm 13) accompanied by FLT3 mutation than for the myeloid leukemia cell lines (THP-1, HL60, K562) without FLT3 mutation as a whole (FIG. 4).

Drug17 is effective in inhibiting FLT3 kinase activity at the level of biochemical activity

FLT3 is a member of the tyrosine kinase family, which promotes cell proliferation and survival by phosphorylating downstream pathway signaling proteins by exerting its own kinase activity. HTRF technology labels acceptor fluorescent molecules on FLT3 substrate, donor fluorescent molecules on phosphorylation modified specific antibodies, and fluorescence energy is transferred when FLT3 autophosphorylates, thereby obtaining recognition signals. The invention sets the concentration gradient of drug17 to detect FLT3 kinase activity, and sets the concentration gradient of AC220 as a control to calculate the IC50 concentration of drug inhibition. Through detection, drug17 has obvious inhibition effect on FLT3 protein kinase activity under in vitro conditions, and IC50 can reach a micromolar level (101.147 mu M) (FIG. 5).

Example 2Drug killing of FLT3 mutated cells by inhibiting FLT3 phosphorylation, promoting apoptosis and cycle arrest

Drug17 was effective in reducing FLT3 phosphorylation levels

The phosphorylation capacity of FLT3 reflects in part its ability to activate downstream signaling pathways. The invention utilizes the constructed 293T-FLT3-ITD cell strain to detect the FLT3 phosphorylation inhibition capability of drug 17. The invention uses DMSO, drug17 (100 mu M) and AC220 (10 nM) to act on 293T-FLT3-ITD cell lines in the same state respectively, then collects cells, and detects the expression level of the phosphorylated protein of FLT3 after lysis. The results indicate that after 6 hours of treatment of 293T cell line with ITD mutations with drug17, the phosphorylation level of FLT3 was significantly reduced compared to the DMSO group (FIG. 6).

Drug17 has obvious pro-apoptotic and cycle-blocking effects on FLT3 mutated cells

Induction of apoptosis of tumor cells is one of the main modes of action of antitumor drugs. To investigate the effect of drug17 on the apoptotic effect of AML cells harboring FLT3 mutations, the invention treated the 32D parental strain and the 32D-ITD mutant strain in the same state with 3 μm drug17 and 20nM sorafenib, respectively, for 48h, and apoptosis was detected by flow cytometry. Flow results show that drug17 is similar to the sorafenib effect, and can induce apoptosis effect of the 32D-ITD stable transgenic strain and parent strain cells to a certain extent, wherein the 32D-ITD stable transgenic cell strain is more sensitive to apoptosis effect induced by the lead small molecules than the parent strain, and high-proportion late apoptosis is induced (figure 7). The cycle-blocking effect is also one of the common mechanisms of antitumor drugs. The present invention examined the cell cycle of 3. Mu.M drug17 after 48h of treatment of cells of the 32D and 32D-ITD mutant in the same state. As a result, drug17 was found to cause a significant G0/G1 phase blocking effect on 32D-ITD cell lines (FIG. 8).

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims (5)

1. An application of a small molecular compound in preparing a medicament for treating FLT3 mutant leukemia is characterized in that the small molecular compound has the chemical structure as follows:

2. the use according to claim 1, wherein the FLT3 mutated leukemia is FLT3 mutated acute myeloid leukemia.

3. The use according to claim 1, wherein the FLT3 mutation comprises a FLT3-ITD mutation, a FLT3-TKD mutation, a FLT3-ITD-TKD mutation and a FLT3-ITD-F691L mutation.

4. The use according to claim 1, wherein the medicament further comprises pharmaceutically acceptable excipients.

5. The use according to claim 1, wherein the dosage form of the medicament comprises injections, tablets, pills and granules.