Small molecule drug targeting PABPC1 and application thereof in chronic myelogenous leukemia
阅读说明：本技术 一种靶向pabpc1的小分子药物及其在慢性髓系白血病中的应用 (Small molecule drug targeting PABPC1 and application thereof in chronic myelogenous leukemia ) 是由 余佳 马艳妮 周凡琦 孙晨光 于 2021-11-12 设计创作，主要内容包括：本发明公开了一种靶向PABPC1的小分子药物及其在慢性髓系白血病中的应用,所述小分子药物为小分子化合物ML324,本发明首次发现小分子化合物ML324对慢性髓系白血病具有显著的治疗效果,体内和体外的验证实验均表明了小分子化合物ML324对慢性髓系白血病细胞的生长具有显著的抑制作用,本发明为临床上开发治疗慢性髓系白血病的给药方案提供了一种全新的思路。(The invention discloses a small molecular drug targeting PABPC1 and application thereof in chronic myelogenous leukemia, wherein the small molecular drug is a small molecular compound ML324, the invention discovers that the small molecular compound ML324 has obvious treatment effect on the chronic myelogenous leukemia for the first time, and in-vivo and in-vitro verification experiments show that the small molecular compound ML324 has obvious inhibition effect on the growth of chronic myelogenous leukemia cells, and the invention provides a brand new thought for clinically developing a drug delivery scheme for treating the chronic myelogenous leukemia.)
1. The application of the PABPC 1-targeted small molecule compound in preparing the medicines for treating and/or preventing chronic myelogenous leukemia is characterized in that the small molecule compound is ML324, and the structural formula of the small molecule compound is shown as the formula (I):
2. the use according to claim 1, wherein the small molecule compound inhibits proliferation and growth of chronic myeloid leukemia cells;
preferably, the small molecule compound inhibits proliferation and growth of chronic myeloid leukemia cells by inhibiting expression of PABPC 1.
3. The use of claim 1, wherein the medicament consists of a therapeutically effective amount of a small molecule compound of formula (I) and a pharmaceutically acceptable carrier and/or adjuvant;
preferably, the small molecule compound is used at a concentration of 0.5-5 mg/kg.
4. A pharmaceutical composition for treating and/or preventing chronic myeloid leukemia, wherein said pharmaceutical composition comprises a therapeutically effective amount of small molecule compound as claimed in claim 1.
5. The pharmaceutical composition according to claim 4, further comprising a pharmaceutically acceptable carrier and/or adjuvant;
preferably, the pharmaceutically acceptable carrier and/or adjuvant comprises diluent, binder, surfactant, humectant, adsorption carrier, lubricant, filler, disintegrant.
6. A method for screening a candidate drug for treating and/or preventing chronic myelogenous leukemia, which comprises the steps of:
(1) using PABPC1 as a drug target, and adopting an ALPHA screen technology to screen a small molecular compound with an inhibiting effect on the combination of PABPC1 and polyA at a high throughput;
(2) carrying out experimental verification on the small molecular compound obtained by screening in the step (1), and screening the small molecular compound which can inhibit the proliferation of chronic myelogenous leukemia cells, and/or promote the apoptosis of the chronic myelogenous leukemia cells, and/or reduce the proportion of the chronic myelogenous leukemia cells in peripheral blood as a candidate drug;
preferably, the verification experiment comprises a cell proliferation experiment, an apoptosis experiment and a chronic myelogenous leukemia animal model experiment.
7. The method according to claim 6, wherein the candidate drug for treating and/or preventing chronic myelogenous leukemia screened in step (2) is a small molecule compound ML324, and the structural formula of the small molecule compound is shown as formula (I):
8. use of a small molecule compound targeting PABPC1 in the preparation of a reagent, wherein the small molecule compound is a small molecule compound according to claim 1; the agent is useful in any one or more of the following aspects:
(1) inhibiting proliferation of chronic myeloid leukemia cells in vitro;
(2) promoting the apoptosis of chronic myeloid leukemia cells in vitro;
preferably, the chronic myeloid leukemia cells comprise K562, MEG-01, K562/G01, JVM-3.
9. Use of a small molecule compound targeting PABPC1 for the manufacture of a medicament for the treatment and/or prevention of imatinib-resistant chronic myeloid leukemia patients, wherein said small molecule compound is as claimed in claim 1.
10. The use according to claim 9, wherein the medicament consists of a therapeutically effective amount of a small molecule compound as claimed in claim 1 and a pharmaceutically acceptable carrier and/or adjuvant.
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a PABPC 1-targeted small-molecule medicine and application thereof in chronic myelogenous leukemia, and more particularly relates to a PABPC 1-targeted small-molecule medicine ML324 and application thereof in chronic myelogenous leukemia.
Background
Chronic Myelogenous Leukemia (CML) is a myeloproliferative disease of stem cell origin driven by hematopoietic progenitor cells in the blood system, and is also a common myeloproliferative tumor, accounting for about 15% of the incidence of adult leukemia. The genetic characteristic is that more than 90 percent of patients have characteristic Philadelphia chromosome, namely t (9; 22) (q 34; q11) chromosome translocation to form BCR/ABL fusion genes (Taverna S, Giallombando M, Pucci M, et al. C. cut in inhibition in vitro and in vivo bacterial cells growth: a void role for exogenous delivery of miR-21[ J ]. Oncott, 2015,6(26): 21918-. The high tyrosine kinase activity plays an important role in the occurrence and development of chronic myelogenous leukemia, and is a specific target for treating the current chronic myelogenous leukemia. The fusion gene encodes P210 protein, so that the tyrosine kinase activity of the fusion gene is enhanced, a downstream signal transduction path is activated, apoptosis is inhibited, and the fusion gene plays an important role in promoting the occurrence and the development of chronic myelogenous leukemia.
Currently, imatinib, which is a first-line therapeutic for chronic myeloid leukemia, is an inhibitor of BCR/ABL Tyrosine Kinases (TKIs). Although a significant effect can be obtained after the treatment of imatinib in most patients with Philadelphia chromosome positive chronic myelogenous leukemia, drug resistance is the main cause of failure of imatinib in the treatment of chronic myelogenous leukemia (Giles FJ, cortex JE, Kantariian HM, et al. Accerarted and viral phases of chronic myelogenous leukemia [ J ]. Hematol Oncol Clin North Am,2004,18(3): 753-774.). Up to 25% of patients are statistically resistant to imatinib (de Lavallade H, applied JF, Khorashad JS, et al, Imatinib for newlydiagnosis patients with chronic myeloid leukemia: infection of refractory responses in an interaction to-patient analysis [ J ]. Clin Oncol,2008,26(20):3358 and 3363). With the accumulation of clinical application, the TKIs drug resistance phenomenon is increasing, so that the search for new candidate drugs with low side effects and anti-chronic myelogenous leukemia effect is urgently needed in the field.
Based on the current situation of the prior art, the inventor of the present application researches, through experiments, the regulation and control effects of PABPC1 in the occurrence and development of chronic myelogenous leukemia, and further obtains 7997 small molecule compounds (including FDA drugs, bioactive compounds and natural small molecules) by screening multiple compound libraries with PABPC1 as a target, wherein the screened small molecule compound ML324 can be directly combined with PABPC1 in vitro, and further cell experiments and animal experiments prove that ML324 can have an inhibitory effect on chronic myelogenous leukemia through a target RNA binding protein PABPC1 at a cell level and an animal level.
Disclosure of Invention
The invention provides a small molecule drug ML324 targeting PABPC1 in order to make up for the technical defects of the prior art in the current field, and provides a brand new thought for clinically developing a drug delivery scheme for treating chronic myelogenous leukemia.
The above object of the present invention is achieved by the following technical solutions:
the invention provides application of a small molecule compound targeting PABPC1 in preparing a medicament for treating and/or preventing chronic myelogenous leukemia.
Further, the small molecule compound is ML324, and the structural formula of the small molecule compound is shown as formula (I):
the small molecular compound is ML324, CAS number 1222800-79-4 and molecular formula C21H23N3O2The molecular weight is 349.43, the structural formula is shown in formula (I), and the compound is named as N- (3- (Dimethylamino) propyl) -4- (8-hydroxyquinolin-6-yl) benzamide; the small molecular compound is one of the compounds screened by the inventor of the application at high flux by adopting an ALPHA screen technology with PABPC1 as a target spot, has an inhibiting effect on the combination of PABPC1 and polyA, and further experimental verification shows that the small molecular compound has an inhibiting effect on the combination of PABPC1 and polyAThe small molecular compound can obviously inhibit the proliferation and growth of chronic myelogenous leukemia cells and can obviously reduce the proportion of the chronic myelogenous leukemia cells in peripheral blood.
Further, the small molecule compound inhibits the proliferation and growth of chronic myelogenous leukemia cells;
preferably, the small molecule compound inhibits proliferation and growth of chronic myeloid leukemia cells by inhibiting expression of PABPC 1.
Further, the medicine consists of a small molecular compound shown as a formula (I) with a therapeutically effective amount and a pharmaceutically acceptable carrier and/or auxiliary material;
preferably, the small molecule compound is used at a concentration of 0.5-5 mg/kg.
In the embodiment of the present invention, the concentration of the small molecule drug is preferably 2.5mg/kg, and the ability to significantly inhibit the proliferation and growth of chronic myelogenous leukemia cells is already exerted when the concentration of the small molecule drug is 2.5mg/kg, so it should be clear to those skilled in the art that the concentration of the small molecule drug of the present invention is not limited to 0.5-5 mg/kg.
In a second aspect of the present invention, there is provided a pharmaceutical composition for the treatment and/or prevention of chronic myeloid leukemia.
Further, the pharmaceutical composition comprises a therapeutically effective amount of a small molecule compound as described in the first aspect of the invention.
Further, the pharmaceutical composition also comprises a pharmaceutically acceptable carrier and/or an auxiliary material;
preferably, the pharmaceutically acceptable carrier and/or adjuvant comprises diluent, binder, surfactant, humectant, adsorption carrier, lubricant, filler, disintegrant.
Further, the diluents include (but are not limited to): lactose, sodium chloride, glucose, urea, starch, water, and the like.
Further, the adhesive includes (but is not limited to): starch, pregelatinized starch, dextrin, maltodextrin, sucrose, acacia, gelatin, methyl cellulose, carboxymethyl cellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, alginic acid, alginate, xanthan gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, and the like.
Further, the surfactants include (but are not limited to): polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, stearic acid monoglyceride, cetyl alcohol, etc.
Further, the humectants include (but are not limited to): glycerin, starch, and the like.
Further, the adsorbent carrier includes (but is not limited to): starch, lactose, bentonite, silica gel, kaolin, bentonite, etc.
Further, the lubricants include (but are not limited to): zinc stearate, glyceryl monostearate, polyethylene glycol, pulvis Talci, calcium stearate and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolaurocyanate, sodium lauryl sulfate, magnesium lauryl sulfate, etc.
Further, the fillers include (but are not limited to): mannitol (granular or powdery), xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polymeric sugar, coupling sugar, glucose, lactose, sucrose, dextrin, starch, sodium alginate, laminarin powder, agar powder, calcium carbonate, sodium bicarbonate, etc.
Further, the disintegrant includes (but is not limited to): crosslinked vinyl pyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, crosslinked sodium carboxymethyl cellulose, soybean polysaccharide, etc.
In a third aspect of the present invention, there is provided a method for screening a candidate drug for the treatment and/or prevention of chronic myelogenous leukemia.
Further, the method comprises the steps of:
(1) using PABPC1 as a drug target, and adopting ALPHAScreen technology to screen a small molecular compound with an inhibiting effect on the combination of PABPC1 and polyA in a high throughput manner;
(2) carrying out experimental verification on the small molecular compound obtained by screening in the step (1), and screening the small molecular compound which can inhibit the proliferation of chronic myelogenous leukemia cells, and/or promote the apoptosis of the chronic myelogenous leukemia cells, and/or reduce the proportion of the chronic myelogenous leukemia cells in peripheral blood as a candidate drug;
preferably, the verification experiment comprises a cell proliferation experiment, an apoptosis experiment and a chronic myelogenous leukemia animal model experiment.
Further, the candidate drug screened in the step (2) for treating and/or preventing chronic myelogenous leukemia is a small molecular compound ML324, and the structural formula of the small molecular compound is shown as the formula (I):
furthermore, no relevant research for screening drugs aiming at PABPC1 in chronic myelogenous leukemia exists at present, the invention firstly uses PABPC1 as a target spot to screen a research for treating and/or preventing chronic myelogenous leukemia micromolecule compound, and screens the micromolecule compound ML324 which has a significant inhibiting effect on the combination of PABPC1 and polyA, and cell experiments and animal experiments prove that the micromolecule compound ML324 can significantly inhibit the proliferation and growth of chronic myelogenous leukemia cells, can significantly reduce the proportion of chronic myelogenous leukemia cells in peripheral blood, and has a significant treatment effect on chronic myelogenous leukemia.
The ALPHASCEN TECHNIQUE, together with the ALPHASCEN METHOD, is a pharmaceutical activity testing technology developed based on the principle of biomolecular substance interaction, such as antigen-antibody reaction, protein DNA interaction, protein interaction, etc., and forms a complex of donor microbeads, acceptor microbeads and interacting molecules through the interaction between molecules. The 680nm laser is used for exciting the donor beads to release singlet oxygen molecules, the energy transfer cascade reaction is triggered, the acceptor beads emit light waves of 520-620 nm, and the quenching resistance is high. If there is no specific interaction between the biomolecules and the monomeric oxygen will not diffuse to the receptor beads, no signal will be generated.
The fourth aspect of the invention provides the use of a small molecule compound targeting PABPC1 in the preparation of a reagent.
Further, the small molecule compound is the small molecule compound as described in the first aspect of the present invention; the agent is useful in any one or more of the following aspects:
(1) inhibiting proliferation of chronic myeloid leukemia cells in vitro;
(2) promoting the apoptosis of chronic myeloid leukemia cells in vitro;
preferably, the chronic myeloid leukemia cells comprise K562, MEG-01, K562/G01, JVM-3.
In the specific embodiment of the present invention, the chronic myelogenous leukemia cells are K562, MEG-01, K562/G01, and it should be clear to those skilled in the art that the chronic myelogenous leukemia cells of the present invention are not limited to K562, MEG-01, K562/G01.
The fifth aspect of the invention provides an application of a small molecule compound targeting PABPC1 in preparing a medicament for treating and/or preventing imatinib-resistant chronic myeloid leukemia patients.
Further, the small molecule compound is the small molecule compound as described in the first aspect of the present invention.
Further, the medicament consists of a therapeutically effective amount of the small molecule compound as described in the first aspect of the invention and a pharmaceutically acceptable carrier and/or adjuvant.
Further, said pharmaceutically acceptable carriers and/or adjuvants are well described in Remington's Pharmaceutical Sciences (19th ed.,1995) as needed to aid in the stability of the formulation or to aid in the activity or its bioavailability or to produce an acceptable mouthfeel or odor upon oral administration, and the formulations which may be used in such Pharmaceutical compositions may be in the form of their original compounds per se, or optionally in the form of their pharmaceutically acceptable salts, and the Pharmaceutical compositions so formulated may be selected as necessary for administration of the drug in any suitable manner known to those skilled in the art.
Further, the appropriate dose of the pharmaceutical composition may be prescribed in various ways depending on factors such as the formulation method, the administration mode, the age, body weight, sex, disease state, diet, administration time, administration route, excretion rate and response sensitivity of the patient, and a skilled physician can easily determine the prescription and the dose prescribed to be effective for the desired treatment or prevention in general.
Further, the pharmaceutical composition may further comprise conventional cosolvents, buffers, pH regulators, and the like, and if necessary, other materials may also be added to the pharmaceutical composition, and the pharmaceutical composition may be made into various dosage forms, including (but not limited to): the preparation comprises tablets, subcutaneous implants, vaginal or uterine cavity administration preparations, capsules, dropping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injections and the like, wherein the pharmaceutical compositions of the various dosage forms can be prepared according to the conventional method in the pharmaceutical field, and can be used for injection administration, and the injection administration comprises subcutaneous injection, intravenous injection, intramuscular injection, intracavity injection and the like; intraluminal, such as through the uterine cavity and vagina; respiratory administration, such as nasal administration; administration to the mucosa.
Compared with the prior art, the invention has the advantages and beneficial effects that:
(1) the invention discovers that the small molecular compound ML324 has obvious treatment effect on chronic myelogenous leukemia for the first time, and in-vivo and in-vitro verification experiments show that the small molecular compound ML324 has obvious inhibition effect on the proliferation and growth of chronic myelogenous leukemia cells;
(2) the invention firstly uses PABPC1 as a target spot to screen the small molecular compound for treating and/or preventing chronic myelogenous leukemia, and research results show that the small molecular compound ML324 is combined with a target RNA sequence polyA by inhibiting PABPC1, so that the proliferation and the growth of chronic myelogenous leukemia cells are obviously inhibited, and the effect of obviously inhibiting the disease process of the chronic myelogenous leukemia is further achieved;
(3) the application of the small molecular compound ML324 targeting the PABPC1 in preparing the medicine for treating and/or preventing chronic myelogenous leukemia is disclosed for the first time, provides a new method and a new idea for the clinical treatment of the chronic myelogenous leukemia, and has a very good clinical application prospect.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 shows a schematic diagram of ALPHAScreen;
FIG. 2 is a graph showing the results of a high throughput screening of 7997 small molecule compounds of the present invention;
FIG. 3 shows a schematic structural diagram of a small molecule compound ML 324;
FIG. 4 is a graph showing the results of in vitro binding of the small molecule compound ML324 to PABPC 1;
fig. 5 shows a graph of the results of detecting IC50 values of small molecule compound ML324 in different chronic myelogenous leukemia cell lines, wherein, a graph: k562 cells, panel B: MEG-01 cells;
FIG. 6 shows the results of the effect of small molecule compound ML324 on the proliferation and growth of hematopoietic stem progenitor cells in patients with primary CML, wherein, Panel A: patent 1#, B diagram: patent 2#, graph C: patent 3#, graph D: patent 4 #;
FIG. 7 is a graph showing the effect of a small molecule compound ML324 on the proliferative capacity of a TKI (BCR-ABL inhibitor) -resistant CML cell line;
FIG. 8 is a graph showing the results of the therapeutic effect of small molecule compound ML324 on a mouse model of CML, wherein, Panel A: experimental flow chart, panel B: results plot of ML324 effect on leukemia cell number in peripheral blood, panel C: results plot of the effect of ML324 on survival of CML mice.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. As will be understood by those of ordinary skill in the art: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The following examples are examples of experimental methods not indicating specific conditions, and the detection is usually carried out according to conventional conditions or according to the conditions recommended by the manufacturers.
Example 1 screening of Small molecule Compounds having inhibitory Effect on the binding of PABPC1 to PolyA
The RNA binding protein PABPC1 plays an important role in maintaining normal life activities of cells, PABPC1 plays a role in regulating RNA stability and translation by combining with a specific RNA sequence polyA, and the inventor finds that PABPC1 plays an important regulation function in the occurrence and development of chronic myelogenous leukemia in early experiments. Based on the fact, the invention provides a new candidate drug for the clinical treatment of chronic myelogenous leukemia by screening the small molecular compound which can inhibit the combination of the RNA binding protein PABPC1 and the target RNA polyA thereof.
1. In vitro detecting the binding of PABPC1 and ployA by ALPHA screen method, and screening small molecule compounds with inhibitory effect on the binding of PABPC1 and polyA
The ALPHA screen is shown in figure 1, and the specific experimental steps are as follows:
(1) using an assoy buffer: 50mM Tris-HCl (pH 7.4), 150mM NaCl, 0.1% BSA (pre-use formulation) diluted proteins PABPC1-his (Fitzgerald, 80R-3949), RNA polyA-bio (synthesized by Yihuiyuan, Beijing) to the desired concentration;
(2) using a sonic sample applicator in 384 microwell plates (PerkinElmer, OptiPlate)TM) A small molecule library (an activity screening platform of the pharmaceutical technology center of Qinghua university) is added for high-throughput screening; the assay buffer dilutes ML324 small molecules to the use concentration;
(3) add 5. mu.L of RNA (final concentration of 2nM) to 384 plates, add 5. mu.L of PABPC1 (final concentration of 15 nM); during large-scale screening, the final volume of the step is 10 mu L, and the volume of the small molecule library is 20nL and is ignored; in ML324 in vitro experiment, the volume of the small molecule drug is 5 muL, and the final volume of the step is 15 muL;
(4) after the sample adding is finished, covering by using a dark black film, and centrifuging at the normal temperature of 1000rpm for 1 min;
(5) incubation for 1.5 hours at room temperature;
(6) prepare beads mix (ALPHA screen kit, PerkinElmer, 6760619M) in the dark: using the assay buffer to configure the beads mix, wherein the final concentration of the Donor and Acceptor beads is 20 mu g/mL, and the total volume is 10 mu L;
(7) adding 10 mu L of beads mix into the 384-well plate incubated in the step (5), covering by using a dark black film, and centrifuging at the normal temperature of 1000rpm for 1 min;
(8) incubating for 1h at room temperature;
(9) reading by a microplate reader: the emission wavelength was 680 nm.
2. Results of the experiment
The results show that 7997 small molecule compounds (including FDA drugs, bioactive compounds and natural small molecules) are obtained by high-throughput screening, wherein the binding inhibition effect of the small molecule compound ML324 on PABPC1 and polyA is more obvious (see figure 2). The CAS number of the small molecular compound ML324 is 1222800-79-4, and the molecular formula is C21H23N3O2The molecular weight is 349.43, the structural formula is shown in figure 3, and the N- (3- (Dimethylamino) propyl) -4- (8-hydroxyquinolin-6-yl) benzamide is obtained.
Example 2 verification of the in vitro binding Capacity of Small molecules ML324 to PABPC1
This example is based on the small molecule compound ML324 screened in example 1, and it is tested whether PABPC1 and the small molecule ML324 can be directly combined in vitro by micro thermal surge test (MST).
1. Experimental methods
(1) The binding affinity between PBABC1 and the small molecule ML324 was determined using a NanoTemper monoliths nt.115 instrument (NanoTemper Technologies, Germany);
(2) PABPC1 protein was first adjusted to a concentration of 10. mu.M and labeled with Monolith NT.115protein Labeling Kit RED-NHS (NanoTemper Technologies, Germany);
(3) after completion of the protein labeling assay, PABPC1 was diluted with binding buffer (20mM HEPES, pH 7.4, 100mM NaCl, 0.005% Tween 20) to ensure a fluorescence intensity of PABPC1 of approximately 500RU for MST detection; in this experiment, the final concentration of PABPC1 mixed with ML324 was 20 nM;
(4) 1: 2, diluting ML324 to 16 concentration gradients, and then incubating with equivalent diluted PABPC1 for 3h, 8h and 16h at room temperature;
(5) after incubation, the sample was loaded into a capillary tube that was quality treated and measured in a NanoTemper Monolith nt.115 instrument; binding force KD values were determined by NanoTemper Monolith affinity software (NanoTemper Technologies, Germany) using a 1: 1 binding mode for fitting.
3. Results of the experiment
The results show that the small molecule compound ML324 obtained by screening of the invention can be directly combined with PABPC1 in vitro, and the results of affinity assay show that the combining force of ML324(CAS 1222800-79-4) with PABPC1 in vitro is 23.2nM (see figure 4).
Example 3 CCK8 experiment to determine the IC50 value of the small molecule compound ML324 in two cancer cell lines
1. Experimental Material
The two cancer cell lines are chronic myelogenous leukemia cell lines, namely a K562 cell line and an MEG-01 cell line, wherein the K562 cell line is derived from laboratory stock of an applicant; MEG-01 cell line was purchased from the national biomedical laboratory cell resource library (BMCR) of the institute of basic medicine, national academy of medical sciences;
2. experimental methods
(1) The concentration gradient for the ML324 small molecule effect was set as: 100. mu.M, 10. mu.M, 1. mu.M, 100nM, 10 nM; background DMSO 0.1%;
(2) configuring the ML324 small molecule concentration in the step (1) by 10X by using a culture medium;
(3) collecting cells K562 and MEG-01 cultured for 2 days, centrifuging at 800rpm for 5min, resuspending the cells in 1mL of the medium and calculating the cell concentration by using CountStar, and diluting the cells to 9000 cells/90. mu.L;
(4) spreading the cells diluted in the step (3) in a 96-well plate, wherein each well is 90 mu L, each concentration is provided with 3 multiple wells, and 100 mu L of PBS buffer solution is added into the peripheral wells of the cells;
(5) adding 10 μ L of the drug diluted in the step (2) to the cells; patting and mixing, and culturing in incubator (37 deg.C, 5% CO)2) Cultivation methodCulturing for 48 hours;
(6) after 48 hours, adding 10 μ L of CCK8 Assay reagent (DOJINDO, NV546) into each well of cells under the condition of keeping out of the sun, placing the cells into an incubator at 37 ℃ for acting for 2 hours, removing bubbles in the cell suspension before detection, patting the cells gently and mixing the cells uniformly;
(7) reading and detecting by a Bio-Tek microplate reader: the emission wavelength is 450nm-630 nm; IC50 values were calculated for Graphpad prism 6.0.
3. Results of the experiment
The results show that the IC50 value of the small molecule compound ML324 is 3.755 μ M in K562 cells; in MEG-01, IC50 value of small molecule compound ML324 is 4.767 μ M (see FIGS. 5A and 5B), indicating that small molecule compound ML324 has inhibitory effect on chronic myelogenous leukemia cell line.
Example 4 detection of the proliferation inhibitory Capacity of Small molecule Compound ML324 on cells of CML patients 1 magnetic bead sorting of bone marrow CD34 Positive hematopoietic Stem and progenitor cells of CML patients
(1) CML patient information: 4 CML patients are all BCR-ABL (p210) positive treatment CML patients, which are respectively expressed as Patient 1#, Patient 2#, Patient 3#, and Patient4 #;
(2) preparing an aseptic sorting buffer: PBS, 2% FBS, 0.4% 0.5M EDTA, 1% double antibody;
(3) isolation of bone marrow mononuclear cells from CML patients: bone marrow blood samples were diluted well with 4 volumes of EDTA-PBS, 2: 1 volume ratio was applied to HISTOPAQUE (Sigma, 10771) along the wall, slowly increased and decreased at room temperature, and centrifuged at 680g for 20 min; sucking out the white membrane layer and the upper and lower parts of the white membrane layer, sorting buffer to dilute and wash off HISTOPAQUE, centrifuging at room temperature of 1600rpm for 10min, sucking and discarding the supernatant;
(4) the magnetic bead antibody (America, whirly, 130, 046, 702) marks CD34+ cells (operation without light): each 1 × 108Adding 300 μ L sorting buffer, 100 μ L blocker, 100 μ L CD34+ magnetic bead heavy suspension cell precipitate into each cell, mixing well, incubating at 4 deg.C for 30min, and shaking gently to mix well cell suspension every 10 min; 50mL sorting buffer washes 1 cell, centrifugates 10min at 1500rpm room temperature, sucks and discards the supernatant;
(5) sorting CD34+ cells (dark operation): 3mL sorting Buffer is used for resuspending the cells, a cell filter is used for filtering cell suspension, and then 1mL sorting Buffer is used for washing the filter for 3 times to obtain 6mL single cell suspension; an adsorption column, namely LS Columns (401), is arranged on a magnetic frame, namely MidiMACS Separator (130. the day and the whirly, 042. the. 130. the. 042. the. 302), and 3mL of a sorting buffer rinses the column (avoiding generating bubbles and preventing the liquid in the column from cutting off); passing 6mL of the single cell suspension through an adsorption column, washing the adsorption column by 3mL of a sorting buffer, and discarding an effluent; the adsorption column is detached, 3mL of sorting buffer is used for eluting the adsorption column for 2 times, and the residual liquid is rapidly pushed out with great force by a push rod to collect cells into a new 15mL centrifuge tube;
(6) CD34+ cell culture
Cell concentration was calculated using CountStar; take 1X 105Carrying out CD34-APC (Mercebioscience, SAB4700161) flow antibody staining on the cells, and carrying out flow detection on the positive rate of CD 34;
the media formulation used to culture bone marrow CD34+ cells was as follows: IMDM-Gluta MAX (Gibco, 31980097), 10% FBS, 1% Penicillin-Streptomyces (Gibco, 10378016), 55 μ M2-Mercaptoethanol (Sigma, M6250), 10ng/mL IL-6(Peprotech, AF-200-06), 20ng/mL IL-3(Peprotech, AF-213-13), 100ng/mL FLT3L (Peprotech, AF-300-19), 50ng/mL TPO (Peprotech, AF-300-18), 100ng/mL SCF (Peprotech, AF-300-07); the experimental treatment was carried out within 2-4 days of cell culture.
2. CCK8 Assay for initial diagnosis of proliferation of hematopoietic stem and progenitor cells in CML patients
CCK8 Assay measures the proliferative growth capacity of hematopoietic stem and progenitor cells of CML patients at the initial visit after treatment with the small molecule compound ML 324. The specific experimental steps are as follows:
(1) dividing 5 96-well plates into 0h, 24h, 48h, 72h and 96h groups, setting an experimental group (5 mu M ML324 group) and a control group (0.1% DMSO group) on each plate, setting 5 multiple wells on each group, wherein each well has about 5000 cells, planting 100 mu L of the cell suspension of the experimental group and the cell suspension of the control group on each plate according to the same set sequence, adding 100 mu L of PBS buffer solution into peripheral wells, tapping, uniformly mixing, placing in an incubator (37 ℃, 5% CO and 5 ℃), and placing in a container2) Medium culture;
(2) adding 10 μ L of LCCK8 solution (DONGRENCHE, CK04) into 100 μ L of cell suspension 0 hr, 24 hr, 48 hr, 72 hr, and 96 hr after plating, beating, mixing, and culturing in incubator (37 deg.C, 5% CO)2) And (3) incubating for 2h, removing bubbles in the cell suspension, patting and uniformly mixing, and detecting the absorbance of each sample hole at the wavelength of 450nm and 630nm by using a Bio-Tek microplate reader. Selecting 3 multi-pore values as three biological replicates, calculating Mean + -SD by Graphpad prism6.0, and comparing the difference of dehydrogenase activity of each group of cells at different time points<0.05，**p<0.01，***p<0.001。
3. Results of the experiment
The results show that the cell proliferation capacity is obviously reduced after 5 mu M ML324 is added into the hematopoietic stem progenitor cells of the CML patient at the initial diagnosis compared with the control group, which indicates that the small molecular compound ML324 obtained by screening of the invention can obviously inhibit the proliferation and growth of the cells of the CML patient (see figures 6A-6D).
Example 5 detection of proliferation inhibitory Activity of Small molecule Compound ML324 in CML-resistant Strain
1. Experimental Material
The TKI (BCR-ABL inhibitor) drug-resistant CML cell strain K562/G01 (human chronic myelogenous leukemia cell imatinib-resistant cell, KG cell for short) described in this example was provided by the institute of hematology of Chinese academy of medicine sciences.
2. Experimental methods
CCK8 Assay detects the cell proliferation condition of TKI drug-resistant CML cell strain K562/G01 after being treated by small molecule compound ML324, and the specific experimental steps are as follows:
(1) the medium formulation used for culturing KG cells was as follows: RPMI 1640+ 10% FBS + 1% double antibody, and the culture conditions are sterile, 37 ℃ and 5% CO2And a saturated humidity;
(2) cell plating and absorbance detection calculation; selecting 3 multi-well values as three biological replicates, calculating Mean ± SD by Graphpad prism6.0, comparing the difference of dehydrogenase activity of each group of cells at different time points, [ p ] 0.05, [ p ] 0.01, [ p ] 0.001.
3. Results of the experiment
The result shows that the cell proliferation capacity of KG cells is obviously reduced after 5 mu M ML324 is added compared with that of a control group, and the result shows that the small molecular compound ML324 can obviously inhibit the proliferation and growth of the TKI-resistant CML cell strain K562/G01 (see figure 7).
Example 6 therapeutic Effect of the Small molecule Compound ML324 on a mouse model of CML
1. Experimental Material
The C57 mouse described in this example was purchased from vintongli, inc, and was a 6-8 week old female mouse; cell suspensions described in this example: bone marrow cells were removed from C57 Mouse femurs, treated with 5-FU and stably transfected with MSCV-BCR-ABL-GFP plasmid (which was constructed by the method described in Methods Mol biol. 2016; 1438:225-43.doi:10.1007/978-1-4939-7Cell suspension per mL.
2. Experimental grouping and experimental methods
The experimental flow chart of this example is shown in fig. 8A, and the detailed experimental steps are as follows:
(1) taking 10C 57 mice, injecting the cell suspension into the mice through tail vein within 24h after gamma-ray irradiation with 10Gy lethal dose, and randomly dividing the cell suspension into a control group and an ML324 treatment group after disease attack, wherein 5 cells are used per group;
(2) after about 10 days of tail vein injection, peripheral blood is taken from the tail vein of the mouse, mononuclear cells are obtained by red cracking, PBS (phosphate buffer solution) is used for cleaning cells and is prepared into single cell suspension, and the flow detection shows that the proportion of GFP + cells is about 10 percent, namely the mosaic rate of leukemia cells, and the sign of the chronic myelogenous leukemia of the mouse is prompted. Then, the mice were randomly divided into two groups, and the mice in the control group and the ML 324-treated group were injected with PBS and ML324(2.5mg/kg) via tail vein, respectively, once at the same time point every day, and after eight consecutive days of injection, peripheral blood was taken from the tail vein of the mouse, and the ratio of GFP + cells in the single nuclear cells of the peripheral blood of the mouse, i.e., the chimerism rate of leukemia cells was measured by flow. Values within groups were Mean ± SD, Graphpad prism6.0 compared differences in GFP + cell ratios for each group, # p <0.05, # p <0.01, # p < 0.001;
(3) the survival number of each group of mice was counted during two months of CML cell transplantation, and a survival curve was plotted.
3. Results of the experiment
The results show that compared with a control group, the proportion of leukemia cells in the peripheral blood of a mouse with chronic myelogenous leukemia treated by ML324 is remarkably reduced, the survival period of the mouse is remarkably prolonged, and the mouse is less prone to disease attack (see a figure 8B and a figure 8C), which shows that the micromolecule compound ML324 obtained by screening can inhibit the generation and the development of the chronic myelogenous leukemia, and further shows that ML324 is expected to become a novel compound for treating the chronic myelogenous leukemia and has better clinical application prospect.
The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.