LU504411B1 - Small molecule compound and its application in preparing medicine for treating FLT3 mutant leukemia - Google Patents

Abstract

The invention discloses a small molecular compound and its application in preparing a medicine for treating FLT3 mutant leukemia, belonging to the field of biomedicine. The micromolecule compound drug 17 is a micromolecule compound designed by screening the binding sites of wild-type and mutant FLT3 activated state structural models based on homologous molecular models. Experiments show that drug 17 can inhibit the growth of FLT3-ITD mutant leukemia cells, inhibit the phosphorylation level of FLT3, promote cell apoptosis and lead to cycle arrest in vitro. Moreover, at the biochemical level, drug 17 can inhibit the activity of FLT3 protein kinase to a certain extent, and the IC50 can reach the micromolar level. The micromolecule compound drug 17 provided by the invention is expected to reduce the drug resistance problem of secondary TKD region mutation, and provides new hope for accurate targeted treatment of AML patients with FLT3 mutation.

Description

DESCRIPTION LU504411

Small molecule compound and its application in preparing medicine for treating FLT3 mutant leukemia

TECHNICAL FIELD

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

BACKGROUND

Acute myeloid leukemia (AML) accounts for about 80% of adult acute leukemia and is an important type of leukemia. In the past 40 years, except for acute promyelocytic leukemia in AML, the cure rate has been significantly improved due to the introduction of targeted therapy, and the treatment progress of other types of AML has been slow.

High-dose cytarabine chemotherapy or allogeneic hematopoietic stem cell transplantation is still mainly used to induce chemotherapy and subsequent risk stratification consolidation treatment. Under this strategy, the 5-year survival rate of AML patients under 60 years old is only about 40%, while that of AML patients over 60 years old is less than 10%. In the era of precision medicine, actively exploring targeted therapy is expected to improve the prognosis of this kind of AML.

FLT 3 (FMS-like tyrosine kinase 3) is a member of receptor tyrosine kinase Ill (RTK lll) subfamily, and there are mutations in up to 30% of adult AML. There are two main types of FLT3 mutations in AML, namely, about 2/3 internal tandem repeats (ITD) mutations and about 1/3 kinase domain (TKD) point mutations. Both types of mutations are function-acquired mutations, which can lead to continuous autophosphorylation of

FLT3 receptor, abnormal activation of signal pathway, participation in the occurrence and development of leukemia and maintenance of abnormal cell proliferation ability without relying on FLT3 ligand. At the same time, patients with FLT3-ITD positive AML hav&J504411 worse response to induced chemotherapy, higher recurrence rate, shorter recurrence-free survival and shorter total survival time than patients with mutation negative. The influence of FLT3-TKD mutation on the prognosis of AML patients is still controversial, but some patients with FLT3-ITD positive AML have FLT3-TKD mutation after early targeted therapy, which leads to acquired drug resistance and treatment failure, which brings great challenges to the follow-up treatment of AML. Because FLT3 mutation is the most common molecular mutation in AML, and the prognosis of patients with mutation is bad under the traditional treatment strategy, FLT3 naturally becomes one of the most attractive therapeutic targets in AML in order to improve the prognosis of this kind of AML.

At present, there are more than ten small molecule inhibitors that are suitable for

FLT3 receptor kinase and have entered clinical trials or been approved. Although encouraging clinical effects have been achieved in different degrees, each inhibitor has different problems. In addition to the pharmacokinetic changes after application in human body, important problems include the non-specificity of target, the side effects caused by off-target, and the secondary mutation resistance. Investigate its reason, the deficiency in design is its core crux. Because most of these targeted inhibitors were obtained by biochemical or cell-level specific experiments in the early stage, or by designing, screening and optimizing other tyrosine kinase proteins as structural models, even the recent targeted drug design specifically targeting FLT3 kinase proteins is often based on the structural biology of the wild-type FLT3 protein crystal structure (1RJB).

Therefore, based on the latest understanding of the function and structure of FLT3 kinase protein and its mutant, the self-inhibition and activation state structure of FLT3 are re-analyzed, especially the structural characteristics of the mutant itself. Based on the unique structural characteristics of FLT3, finding new small molecule binding sites and screening and optimizing specific small molecule inhibitors will bring new scientific ideas for the development of new FLT3 small molecule targeted inhibitors, and hopefully bring better choices for the treatment of AML patients with FLT3 mutation.

SUMMARY LU504411

The purpose of the present invention is to provide a small molecular compound and its application in preparing a medicine for treating FLT3 mutant leukemia, so as to solve the problems existing in the prior art.. The micromolecule compound drug 17 provided by the invention can be used as an inhibitor of FLT3 kinase, and plays an anti-FLT3-ITD and drug-resistant mutant acute myeloid leukemia role, which is expected to reduce the drug resistance problem of secondary TKD regional mutation and provide new hope for accurate targeted treatment of FLT3 mutation positive AML patients.

In order to achieve the above objectives, the present invention provides the following scheme:

The invention provides an application of a small molecular compound in preparing a medicine for treating FLT3 mutant leukemia or inhibiting FLT3 protein phosphorylation, and the chemical structure of the small molecular compound is as follows: >», ; ‘

Further, the FLT3 mutant leukemia is an FLT3 mutant acute myeloid leukemia.

Further, the FLT3 mutation includes FLT3-ITD mutation, FLT3-TKD mutation,

FLT3-ITD-TKD mutation and FLT3-ITD-F691L mutation.

Further, the FLT3 protein is a FLT3 mutant protein.

Further, the FLT3 mutation includes FLT3-ITD mutation, FLT3-TKD mutation,

FLT3-ITD-TKD mutation and FLT3-ITD-F691L mutation.

Further, the medicine also comprises pharmaceutically acceptable excipients.

Further, the dosage forms of the medicine include injections, tablets, pills and granules.

The invention discloses the following technical effects: LU504411

According to the invention, based on the structural models of wild-type and mutant

FLT3 activation states (FLT3-ITD, FLT3-ITD with D835Y double mutation and FLT3-ITD with F691L double mutation) constructed by homologous molecular models, a new small molecule binding site unique to FLT3 is searched, and a small molecule compound drug 17 is identified and obtained through computer virtual screening, optimization and subsequent biochemical, cellular and horizontal activity verification. It has been proved that drug 17, a small molecular compound, can be used as an inhibitor of FLT3 kinase and play an anti-FLT3-ITD and drug-resistant mutant acute myeloid leukemia role.

Experiments show that drug 17 can inhibit the growth of FLT3-ITD mutant leukemia cells, inhibit the phosphorylation level of FLT3, promote cell apoptosis and lead to cycle arrest in vitro. Moreover, at the biochemical level, drug 17 can inhibit the activity of FLT3 protein kinase to a certain extent, and the IC50 can reach the micromolar level.

The micromolecule compound drug 17 provided by the invention is expected to reduce the drug resistance problem of secondary TKD region mutation, and provides new hope for accurate targeted treatment of AML patients with FLT3 mutation.

BRIEF DESCRIPTION OF THE FIGURES

In order to explain the embodiments of the present invention or the technical scheme in the prior art more clearly, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention, and other drawings can be obtained according to these drawings without creative work for ordinary people in the field.

Fig. 1 shows the self-inhibition model of wild-type FLT3 and the molecular dynamics model of the activation state of FLT3-ITD, FLT3-ITD with D835Y and FLT3-ITD with

F691L;

Fig. 2 shows the OD values measured by CCK8 detection method after normal 32D cells and 32D-ITD mutant 72 were treated with different small molecule drugs at a single concentration of 100uM; Wherein, 0 represents DMSO control, and 1-25 represents the number of the small molecular compound preliminarily screened; Drug 17 correspondg/504411 to No.17, and its inhibitory effect on 32D-ITD mutant stably transformed plants is stronger than that of normal 32D cells at 100uM;

Fig. 3 shows the cell viability detected by CCK8 after 72 hours of treatment with drug 17 with the same concentration gradient in normal cells 32D and 32D-ITD mutant stable plants, and the IC50 of drug 17 to 32D and 32D-ITD are 50.23uM and 30.5uM respectively. The experiment is repeated three times and can get close results. The statistical results of three repeated samples in one experiment are shown in the figure.

Fig. 4 shows the semi-inhibitory concentration (IC50) of drug 17 on normal 32D cells, 32D-ITD mutant, FLT3 mutant leukemia cell lines (MV-4-11, Molm13) and FLT3-free mutant leukemia cell lines (THP-1, HL60, K562); Three repetitions, * * means P < 0.01;

Fig. 5 shows the inhibition ability of drug 17 on FLT3 kinase activity based on homogeneous time-resolved fluorescence (HTRF) technology (a), setting small molecule

AC220 group as control (b) and setting concentration gradient to detect FLT3 kinase activity, The results showed that drug 17 had a certain inhibitory effect on FLT3 protein kinase activity in vitro, and IC50 could reach micromolar level.

Fig. 6 shows that after DMSO, drug 17 and AC220 were used to treat 293T-FLT3-ITD stably for 6 hours, the phosphorylation level of FLT3 was detected by cell collection, protein lysis, protein purification and WB. DMSO and AC220 were used as control groups, and the results showed that drug 17 inhibited the phosphorylation of

FLT3.

Fig. 7 shows that after the 32D parent strain and the 32D-ITD stably transformed strain were treated with drug 17 and sorafenib, the received cells were detected by flow cytometry, and the changes of apoptosis between the drug-treated group and the drug-free group were compared. The results showed that both drug 17 and sorafenib could induce the apoptosis of 32D-ITD stably transformed cells and their parents to a certain extent, among which 32D-ITD stably transformed cells were more sensitive to the apoptosis induced by small lead molecules than their parents, and induced a high proportion of late apoptosis.

Fig. 8 shows the cell cycle detection by flow cytometry after the 32D parent stralrt)504411 and the 32D-ITD stably transformed strain were treated with drug 17, and the changes of cell cycle between the drug-treated group and the drug-free group were compared. The results showed that drug 17 could cause obvious G0/G1 phase arrest of 32D-ITD stable cells compared with 32D parent strain.

DESCRIPTION OF THE INVENTION

A number of exemplary embodiments of the present invention will now be described in detail, and this detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention.

It should be understood that the terminology described in the present invention is only for describing specific embodiments and is not used to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Intermediate values within any stated value or stated range, as well as each smaller range between any other stated value or intermediate values within the stated range are also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

Unless otherwise specified, 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 relates. Although the present invention only describes the preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe methods and/or materials related to the documents. In case of conflict with any incorporated document, the contents of this specification shall prevail.

It is obvious to those skilled in the art that many improvements and changes can bé/504411 made to the specific embodiments of the present invention without departing from the scope or spirit of the present invention. Other embodiments will be apparent to the skilled person from the description of the invention. The description and example of that present invention are exemplary only.

The terms "including", "comprising", "having" and "containing" used in this article are all open terms, which means including but not limited to.

After extensive and in-depth research, the inventors used the "Light of Taihu Lake" supercomputer for the first time to simulate the molecular dynamics of FLT3 wild type,

FLT3-ITD, FLT3-ITD with D835Y double mutant and FLT3-ITD with F691L double mutant proteins, and explored new small molecule binding sites unique to FLT3. Different from the current mainstream design idea that the crystal structure of wild-type and self-inhibiting FLT3 protein is based on structural biology, the inventors used the newly discovered structural characteristics of the intermediate inactive state of mutant and wild-type FLT3 protein to construct the structural model of mutant and wild-type FLT3 activated state according to the homologous molecular model (Figure 1) and the existing self-inhibiting model of wild-type FLT3 (Figure 1). The new small molecule binding sites unique to FLT3 were explored, and dozens of leading small molecule compounds were obtained through high-throughput screening in the public database of National Cancer

Research Center of the United States and the national compound molecular database of

China by computer virtual screening software. After screening and optimization at the cellular level and biochemical level, the FLT3 small molecule inhibitor (9E)-9-hydrazinylidenefluoride-2,3-diamine (drug 17, and the NCI number is

VCN-092758.) with high sensitivity and high specificity was obtained. The chemical structural formula is:

© >

Experimental materials used in the embodiment of the invention:

Experimental cells:

Human Molm-13, MV4-11, THP-1, K562, HL60, 293T cells and mouse 32D cells were all purchased from Beina Biological Company. Mouse 32D-FLT3-ITD stably transformed cells and 293T-FLT3-WT/ITD stably transformed cells were constructed internally.

Experimental reagent:

AC220 and sorafenib were purchased from Shanghai Taosu Biochemical

Technology Co., Ltd.; Drug 17 was built by ABI Chem (Germany). Recombinant Murine

IL-3 was purchased from PEPROTECH Company (USA); CCK8 reagent was purchased from Tongren Chemical Company (Japan); Annexin V- APC apoptosis detection kit, cell cycle detection kit purchased from biogems (USA); HTRF kit was purchased from cisbio (France); Anti-pFLT3 antibody was purchased from Cell Signaling Technology Company (USA). HA tag mouse mAb was purchased from Kingsley Company (China);

Horseradish peroxidase labeled goat anti-rabbit IgG(H+L) was purchased from Beyotime

Company (China).

Experimental method: 1. Construction of FLT3-WT/ITD mutant plasmid.

PLenti-Ill-EF1a(LV043) plasmid vector was purchased from Applied Biological

Materials (ABM) Inc. (Canada), and the target plasmid was constructed through site design (5'-end BamHI-3'-end Xbal), amplification of the target sequence, ligation and transformation. The constructed plasmid was verified to be a successful target FLT 3-WT by gene sequencing.

2. Construction of mouse 32D-FLT3-ITD stably transformed cells and)504411 293T-FLT3-WT/ITD stably transformed cells. (1) lentivirus package of FLT3-WT/ITD mutant 1) Select 293T cells to spread and grow in a 10cm cell culture dish to logarithmic growth stage, and after adjusting the state of the cell strain to be optimal, wait until the cells grow evenly to occupy 80% of the bottom volume of the dish (the number of cells is 7x106/dish). 2) 2 hours before the virus began to be packaged, the original medium was discarded and 5mL of fresh DMEM complete medium was used for liquid exchange. 3) The lentivirus packaging system is shown in Table 1. Take two 1.5mL EP tubes, label tubes A and B, and then add 500 u | of serum-free medium DMEM respectively.

First, add 40uL of liposome Lipo 2000 into tube B, and gently blow and mix. Take tube A, add them into the packaging system shown in Table 1, and gently blow and mix.

Table 1 lentivirus packaging system pMD2.G" 2.5uL

Base psPAX2 2 7.5uL d on DNA

A tube Target plasmid 10pL

Serum-free 500uL medium DMEM

Liposome Lipo 40uL 2000

B tube

Serum-free 500uL medium DMEM 1 Addgene plasmid # 12259; 2 Addgene plasmid # 12260 4) Suck the liquid from tube B, slowly add it into tube A drop by drop until all the liquid is dripped, gently purge and mix it evenly for three times, and place it in an incubator at 37°C for 30 minutes. Add the mixed solution of tube A and tube B gently and)504411 slowly into the cell culture dish, mix it evenly by cross method, and put it in an incubator for culture. 5) After 6 hours of culture, all the original culture medium was discarded in the waste liquid processor containing disinfectant, and 5mL of fresh DMEM complete culture medium was added to complete the cell exchange. 6) After 24 hours of culture, the culture containing lentivirus was collected for the first time based on a 15mL centrifuge tube, sealed and stored at 4°C, and 5 mL of fresh

DMEM complete medium was added. 7) After 48 hours and 72 hours of culture, the second and third viruses were collected. All the collected virus liquid was centrifuged at 1500 rpm at 4°C for 15 minutes, and the supernatant was filtered with a 0.22um filter to remove impurities such as cell debris, and then packaged and stored in a refrigerator at -80°C. (2)FLT3-WT/ITD lentivirus infects 293T and 32D cells of mice, and screening the stably transformed plants. 1) aft packaging that lentivirus of the FLT3-WT/ITD mutant, take a proper amount of lentivirus to infect 32D cells and 293T cells of mice. 2) After 72 hours of infection, culture medium containing puromycin was added for culture until all negative control cells died. 2) The well-growing 293T/32D cells were lysed, and the expression of HA-tag was detected by Western Blot to identify whether the cells stably expressed FLT3 wild-type/mutant protein. The cells that could stably express FLT3 wild-type/mutant protein were identified as mouse 32D-FLT3-ITD stably transformed cells and 293T-FLT3-WT/ITD stably transformed cells. 3. Detection of cell viability by 3.CCK8 method.

CCK8 method is a reliable and sensitive method to detect cell viability. WST-8 in

CCK-8 reagent will react in the presence of electron carrier 1-Methoxy PMS to generate water-soluble methine dye WST-8, and the cell survival can be reflected by detecting its absorbance at 450nm.

4 HTRF technology was used to detect the inhibitory ability of drugs on FLT3 kinadgJ504411 activity.

The detection of kinase activity is based on homogeneous time-resolved fluorescence (HTRF) technology. In the reaction, the receptor fluorescent molecule is labeled on FLT3 substrate, and the donor fluorescent molecule is labeled on the phosphorylated specific antibody. When FLT3 itself is phosphorylated, the fluorescence energy shifts, thus obtaining the recognition signal. By setting the concentration gradient of receptor fluorescent molecular marker and donor fluorescent molecular marker, the efficiency of enzymatic reaction was measured and the kinetic curve of enzymatic reaction was drawn.

Example 1 Targeting Inhibitory Ability of Drug17 1. Drug 17 is more sensitive to the cell line with ITD mutation than the cell line without FLT3 mutation at the cellular level.

Through that virtual screen results in the small molecule library in the early stage, the invention construct dozens of small molecule drugs. According to the inhibition of these small molecular drugs on the 32D parent strain and the 32D-ITD mutant strain at 100uM, more than ten drugs which are more sensitive to the 32D-ITD mutant strain were preliminarily screened out (Figure 2). Through the drug sensitivity test of multi-concentration gradient, it was confirmed that drug 17 showed more obvious inhibitory effect on 32D-ITD mutant in multi-concentration gradient, and the difference was statistically significant (Figure 3).

Subsequently, the present invention tested the growth inhibition ability of drug 17 on cell lines with or without FLT3 mutation. The results showed that the semi-inhibitory concentration (IC50) of drug 17 on myeloid leukemia cell lines (MV-4-11, Molm13) with

FLT3 mutation was lower than that of myeloid leukemia cell lines (THP-1, HL60, K562) without FLT3 mutation (Figure 4). 2. Drug 17 can effectively inhibit FLT3 kinase activity at biochemical level.

FLT3, a member of tyrosine kinase family, promotes cell proliferation and survival by exerting its own kinase activity to phosphorylate downstream pathway signal proteins.

HTRF technology labels the receptor fluorescent molecule on FLT3 substrate, and the donor fluorescent molecule is labeled on the phosphorylated specific antibody. Wheru504411

FLT3 itself is phosphorylated, the fluorescence energy shifts, thus obtaining the recognition signal. According to the invention, the concentration gradient of drug 17 is set to detect FLT3 kinase activity, and the concentration gradient of AC220 is set as a control to calculate the IC50 concentration of drug inhibition. After testing, drug 17 has obvious inhibitory effect on FLT3 protein kinase activity in vitro, and IC50 can reach micromolar level (101.147jM) (Figure 5).

Example 2 Drug kills FLT3 mutant cells by inhibiting FLT3 phosphorylation, promoting apoptosis and cycle arrest. 1. Drug 17 can effectively reduce the phosphorylation level of FLT3.

The phosphorylation ability of FLT3 partly reflects its ability to activate downstream signal pathways. In the invention, the constructed 293T-FLT3-ITD cell strain is used to detect the FLT3 phosphorylation inhibition ability of drug 17. In the invention, DMSO, drug 17(100uM) and AC220(10nM) are respectively used to act on 293T-FLT3-ITD cell lines in the same state, and then the cells are collected, and the phosphorylated protein expression of FLT3 is detected after lysis. The results showed that the phosphorylation level of FLT3 was significantly lower than that of DMSO group after 6 hours of treatment with drug 17 on 293T cell line with ITD mutation (Figure 6). 2. Drug 17 can obviously promote apoptosis and arrest cell cycle in FLT3 mutant cells.

Inducing apoptosis of tumor cells is one of the main modes of action of anti-tumor drugs. In order to explore the influence of drug 17 on apoptosis of AML cells carrying

FLT3 mutation, the 32D parent strain and 32D-ITD mutant strain in the same state were treated with 3uM drug 17 and 20nM sorafenib for 48 hours respectively, and the apoptosis was detected by flow cytometry. The results of flow cytometry showed that drug 17 was similar to sorafenib, and it could induce a certain degree of apoptosis in 32D-ITD stably transformed cells and their parents. Among them, 32D-ITD stably transformed cells were more sensitive to the apoptosis induced by small lead molecules than their parents, and induced a high proportion of late apoptosis (Figure 7). Cycle arrest effect is also one of the common mechanisms of anti-tumor drugs. The cell cycle of 32D and 32D-ITD mutant cells in the same state treated with 3uM drug 17 for 48 hout$)504411 was detected. It was found that drug 17 could induce obvious G0/G1 phase arrest in 32D-ITD cell line (Figure 8).

The above-mentioned embodiments only describe the preferred mode of the invention, and do not limit the scope of the invention. Under the premise of not departing from the design spirit of the invention, various modifications and improvements made by ordinary technicians in the field to the technical scheme of the invention shall fall within the protection scope determined by the claims of the invention.

Claims (10)

CLAIMS LU504411

1. An application of a small molecule compound in preparing a medicine for treating FLT3 mutant leukemia, characterized in that the chemical structure of the small molecule compound is: Te N. Ne N

2. The application according to claim 1, wherein the FLT3 mutant leukemia is FLT3 mutant acute myeloid leukemia.

3. The application according to claim 1, wherein the FLT3 mutation includes FLT3-ITD mutation, FLT3-TKD mutation, FLT3-ITD-TKD mutation and FLT3-ITD-F691L mutation.

4. The application according to claim 1, wherein the medicine further comprises pharmaceutically acceptable excipients.

5. The application according to claim 1, characterized in that the dosage forms of the medicine include injections, tablets, pills and granules.

6. An application of a small molecule compound in preparing a drug for inhibiting the phosphorylation of FLT3 protein, characterized in that the chemical structure of the small molecule compound is:

TS No X Da ï &,

7. The application according to claim 6, wherein the FLT3 protein is a FLT3 mutant protein.

8. The application according to claim 6, wherein the FLT3 mutation includes FLT3-ITD mutation, FLT3-TKD mutation, FLT3-ITD-TKD mutation and FLT3-ITD-F691L mutation.

9. The application according to claim 6, further comprising pharmaceutically acceptable adjuvants.

10. The application according to claim 6, characterized in that the dosage forms of the medicine include injections, tablets, pills and granules.