Application of a homoharringtonine compound as a drug for preparing AIDS treatment

Abstract

The invention relates to the use of a homoharringtonine compound as a drug for preparing a drug for treating AIDS. The beneficial effect is that the screened homoharringtonine can effectively reduce the occurrence of HIV-1PRF and can significantly inhibit the replication of HIV-1 virus in model cells, THP1 cells, and peripheral blood mononuclear cells, and inhibit the assembly of HIV-1 virus particles.

Description

Technical Field

[0001] The present invention relates to the use of homoharringtonine compounds, in particular to the use in the pharmaceutical field. Background Art

[0002] Many retroviruses, including human immunodeficiency virus type 1 (HIV-1) and some coronaviruses, such as severe acute respiratory syndrome virus and infectious bronchitis virus, use programmed -1 ribosomal frameshifting (-1PRF) to control the production ratio of viral structural and enzymatic proteins, favoring self-viral assembly.

[0003] In HIV virus, the -1PRF signal is located between the gag and pol open reading frames (ORFs). Ribosomes can recognize this region and induce it to shift one nucleotide backward in this region, thereby translating to generate Gag-Pol fusion protein, in which Gag protein is a viral structural protein and Pol is a key enzyme protein of the virus. The specific mechanism is as follows: During the translation of Gag-pol mRNA, most ribosomes terminate at the stop codon at the end of the gag open reading frame, producing only Gag protein.

[0004] However, since HIV-1 gag-pol mRNA contains a circular sequence (Stem loop) consisting of a seven-nucleotide sliding sequence (U UUU UUA) and a downstream RNA stem loop, when the ribosome encounters this signal during the translation process, the ribosome will pause in this sequence, which can induce about 5% of the ribosome to move backward by one nucleotide, thereby skipping the termination codon of the Gag protein and converting the subsequent ORF sequence into the coding sequence of the Pol protein, thereby producing the Gag-Pol fusion protein. Under the further action of the viral protease protease, the Gag protein is cut into structural proteins such as p17, p24, and p7, and the Pol protein is cut into enzyme proteins such as reverse transcriptase and integrase. HIV uses this mechanism to strictly control the production ratio of Gag-Pol protein to 20:1, which is conducive to the assembly of the virus itself.

[0005] Despite the great progress made in current antiretroviral therapy, AIDS caused by HIV-1 is still a global health problem, which makes it urgent to develop new drugs against HIV-1 and study new therapeutic targets for this virus. The frameshift efficiency of HIV-1 controls the ratio of Gag:Pol. The deviation of this ratio, especially the reduction of frameshift efficiency, will lead to a decrease in the content of Pol protein and thus make it impossible to produce the enzyme protein required by the virus in the cell. Therefore, changing the efficiency of -1PRF can become a new target for anti-HIV-1.

[0006] Homoharringtonine is an alkaloid extracted from the leaves and bark of the Cephalotaxus truncatum tree. The structural formula of homoharringtonine is:

[0007]

[0008] Homoharringtonine has been used in the preparation of drugs for acute myeloid leukemia and chronic myeloid leukemia since the 1970s, and was approved by the FDA for marketing in the United States in 2012. However, there are currently no authorized patents or literature indicating that homoharringtonine can be used to treat HIV-1. Summary of the invention

[0009] The object of the present invention is to provide a new use of homoharringtonine compounds, that is, a new application in the pharmaceutical field.

[0010] In fact, the present invention relates to the use of a homoharringtonine compound as a drug for preparing AIDS treatment.

[0011] To achieve the above object, the technical solution of the present invention is as follows:

[0012] First, construct a dual luciferase reporter gene, such as Figure 1 As shown, the -1PRF sequence from HIV-1 is inserted between the coding sequences of Renilla luciferase (Rluc) and Firefly luciferase (Fluc). It can be seen that Rluc can be transcribed and translated normally and can be used as an internal reference, while FLuc can only be translated when the ribosome produces a -1 frameshift in the HIV-1 frameshift signal region. Therefore, the present invention can use the change in the ratio of Fluc / Rluc to judge the change in -1PRF efficiency.

[0013] The present invention constructs the sequence into pLVX-neomycin plasmid, packages it into virus with pspAX2 and pMD2G, infects Hela and 293 cells, screens them with G418, constructs stable cell lines, and conducts large-scale drug screening.

[0014] The present invention uses nearly 2,000 FDA-approved drugs to act on the dual-luciferase cell line. When the Fluc / Rluc ratio decreases significantly, it indicates that the drug has an inhibitory effect on -1PRF efficiency, so new targets and drugs can be screened. Through this method, the compound homoharringtonine obtained by the present invention can significantly reduce the Fluc / Rluc ratio in model cells, and further demonstrates that it has a significant inhibitory effect on the production of HIV-1 virus at the cellular level.

[0015] The technical points of the present invention are as follows:

[0016] (1) The length and sequence of -1PRF; the present invention uses a 212 bp HIV-1PRF sequence, which includes a slippery sequence and a stem-loop structure, and the sequence is as follows: The bold black sequence is the slippery sequence and the stem-loop sequence, and the underlined sequence is the location of the stop codon that appears when -1PRF does not occur, which means that only Rennila protein can be produced in the cell. Only when -1PRF occurs will the TAA stop codon shift frame and translate the downstream Firefly luciferase protein.

[0017] (2) The chemical structure of homoharringtonine is as follows Figure 2 As shown. Homoharringtonine is an alkaloid extracted from the leaves and bark of Cephalotaxus truncatula. Homoharringtonine has been used in the preparation of drugs for acute myeloid leukemia and chronic myeloid leukemia since the 1970s, and was approved by the FDA for marketing in the United States in 2012. However, there are currently no authorized patents or literature indicating that homoharringtonine can be used to treat HIV-1.

[0018] (3) Homoharringtonine significantly reduced the efficiency of HIV-1-1PRF in Hela and 293 cells stably transfected with Rlu-(-1PRF)-Flu. The Rennila luciferase and Firefly luciferase values ​​were detected by a multifunctional microplate reader. Figure 3 It was shown that the Fluc / Rluc value of cells treated with homoharringtonine was significantly decreased compared with the control group treated with dimethyl sulfoxide (DMSO).

[0019] (4) When pLVX-Rlu-(-1PRF)-Flu was transfected into THP1 cells and normal human peripheral blood mononuclear macrophages 48 hours later, they were treated with homoharringtonine and the Rennila luciferase and Firefly luciferase values ​​were detected by a multifunctional microplate reader. Figure 4 As shown, the Fluc / Rluc values ​​of cells treated with homoharringtonine were also significantly decreased.

[0020] (5) When the 293 cells stably transfected with Rlu-(-1PRF)-Flu were treated with different concentrations of homoharringtonine in the present invention, Figure 5 As shown, the first column is the DMSO control group, and the second to seventh columns are the treatment results of homoharringtonine at concentrations of 1, 2, 5, 10, 20, and 50 nM, respectively. It was found that homoharringtonine can significantly reduce the -1PRF efficiency at 5 nM.

[0021] (6) Homoharringtonine has been found to be useful for treating leukemia. Therefore, the present invention selected small molecule compounds fludarabine and mitoxantrone from the drug library that can also treat leukemia to treat cells to observe whether they can affect the -1PRF mechanism of HIV cells and thus affect the production of HIV virus. Figure 6 The results of the present invention show that compared with the significant decrease in -1PRF efficiency after homoharringtonine treatment, the -1PRF of cells after fludarabine and mitoxantrone treatment did not significantly decrease, indicating that they cannot act on the -1PRF mechanism of HIV and cannot affect the packaging of the virus. Therefore, from this result, the present invention can see that drugs that can treat leukemia may not be able to treat HIV, and there is no direct correlation between the two.

[0022] (7) After the pNL4-3-Envolope mutant plasmid is transfected into 293 cells, the plasmid can package HIV-1 particles with mutant outer membrane proteins. It is not infectious, but it can characterize the production of viral particles in cells and can be used to determine the EC of the drug. 50 By this method, the present invention measured the EC of homoharringtonine to inhibit HIV-1 production 50 The concentration is 857nM (such as Figure 7 shown).

[0023] (8) 24 hours after 293T cells were transfected with the pNL4-3-Envolope mutant plasmid, the present invention treated the cells with different concentrations of homoharringtonine for 12 hours and then collected the cells to detect the expression of Gag-Pol fusion protein in the cells by Western blot. Figure 8As shown, when the drug concentration is above 20 nM, the Gag-Pol fusion protein is basically undetectable in the present invention.

[0024] (9) In addition to the -1PRF sequence of HIV, the present invention also constructed the -1PRF sequences of three viruses, SIV, RSV, and MMTV, between Rennila luciferase and Firefly luciferase. After these plasmids were transfected into 293 cells for 48 hours, they were treated with homoharringtonine and it was found that this compound could also reduce the -1PRF efficiency of these different viruses ( Fig. 9 ), indicating that this drug has a broad spectrum of efficiency in inhibiting the virus's -1PRF. As long as the virus has a -1PRF mechanism, homoharringtonine is a potential therapeutic drug.

[0025] (10) The present invention obtains immunodeficient mice NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ (NSG), separates human hematopoietic stem cells and transplants them into mice, and detects the differentiation of hematopoietic stem cells in mice after two weeks of continuous culture, and finally obtains mice with humanized hematopoietic cells. Therefore, the present invention can use this model to evaluate the effect of drugs in vivo. The present invention purifies HIV virus and injects it into the blood system of mice through the tail vein. After 48 hours, the present invention also injects homoharringtonine into mice through the tail vein. After another 48 hours, the present invention detects the content of HIV virus particles in the blood of mice. Fig.10 The results showed that the number of HIV virus particles in the blood of mice injected with homoharringtonine was significantly lower than that of the control group. This result shows that in animal experiments, homoharringtonine can also significantly inhibit the replication of HIV.

[0026] The beneficial effects of the present invention are:

[0027] (1) The homoharringtonine screened by the present invention can effectively reduce the occurrence of HIV-1PRF and can significantly inhibit the replication of HIV-1 virus in model cells, THP1 cells, and peripheral blood mononuclear cells, and inhibit the assembly of HIV-1 virus particles.

[0028] (2) The present invention has discovered a new indication for a leukemia treatment drug, homoharringtonine, which has an anti-HIV-1 effect. Because the drug is already on the market, the invention can significantly reduce the capital investment in safety evaluation in the early stages of drug development and reduce the risk of drug development.

[0029] (3) The present invention found that homoharringtonine has a broad spectrum of -1PRF inhibition. As long as the virus has a -1PRF mechanism, homoharringtonine is a potential therapeutic drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of dual luciferase reporter gene;

[0031] Figure 2 is a schematic diagram of the chemical structure of homoharringtonine;

[0032] Figure 3 This is a schematic diagram of the change in the Fluc / Rluc ratio of Hela and 293 cells stably transfected with pLVX-Rennila luciferase-(-1PRF)-Firefly luciferase after being treated with homoharringtonine for 6 hours;

[0033] Figure 4 This is a schematic diagram of the change in the Fluc / Rluc ratio after THP1 and human peripheral blood mononuclear cells were transfected with the pLVX-Rennila luciferase-(-1PRF)-Firefly luciferase plasmid and treated with homoharringtonine for 6 hours;

[0034] Figure 5 Schematic diagram of the change in Fluc / Rluc ratio after pLVX-Rennila luciferase-(-1PRF)-Fireflyluciferase stably transfected cells were treated with different concentrations of homoharringtonine;

[0035] Figure 6 This is a schematic diagram of the changes in the Fluc / Rluc ratio after pLVX-Rennila luciferase-(-1PRF)-Firefly luciferase stably transfected cells were treated with fludarabine, mitoxantrone, and homoharringtonine;

[0036] Figure 7 EC5 was calculated for peripheral blood mononuclear cells transfected with pNL43 envelop mutant plasmid for 24 h and then treated with homoharringtonine for 6 h. The changes of viral particles in the culture supernatant were detected by p24 ELISA. 0 Schematic diagram of

[0037] Figure 8 This is a schematic diagram of the expression of Gag-Pol fusion protein in peripheral blood mononuclear cells detected by Western blot after they were transfected with pNL43 envelop mutant plasmid for 24 hours and then treated with homoharringtonine for 12 hours;

[0038] Fig. 9This is a schematic diagram of the change in the Fluc / Rluc ratio after the pLVX-Rennilaluciferase-(-1PRF)-Fireflyluciferase plasmid of SIV, RSV, and MMTV was transfected into cells and treated with homoharringtonine for 6 hours;

[0039] Fig.10 Schematic diagram of the changes in HIV production in mouse blood viruses after treatment with homoharringtonine in animal experiments. DETAILED DESCRIPTION

[0040] The present invention is further described below in conjunction with embodiments. The description of the following embodiments is only used to help understand the present invention. It should be noted that for ordinary persons in the art, without departing from the principle of the present invention, the present invention can also be modified in some ways, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

[0041] Embodiment 1:

[0042] like Figure 3 As shown, a homoharringtonine compound is used as a drug for preparing a drug for treating AIDS. The homoharringtonine compound is used to reduce the efficiency of HIV-1PRF, reduce the content of Pol protein, and make the virus unable to be packaged into a complete particle in the cell, thereby causing a sharp decrease in the production of HIV. The present invention utilizes this mechanism of HIV to screen out compounds that can significantly promote HIV-1PRF in the FDA-approved drug library, thereby obtaining potential drugs that can treat HIV.

[0043] The present invention firstly establishes a pLVX-Rennila luciferase-(-1PRF)-Firefly luciferase hela and pLVX-Rennila luciferase-(-1PRF)-Fireflyluciferase 293 stable cell lines that can indicate -1PRF efficiency, and then screens a compound, homoharringtonine, that can significantly reduce the efficiency of HIV-1PRF from more than 2,000 FDA-approved drugs for marketing, and proves at the stable cell line level that the compound can inhibit the -1PRF efficiency of HIV.

[0044] Embodiment 2:

[0045] like Figure 4As shown, when homoharringtonine is used, the constructed pLVX-Rennila luciferase-(-1PRF)-Firefly luciferase is firstly transfected into THP1 and PBMC cells, and after homoharringtonine is added to the cells, the expression changes of Renilla luciferase and Firefly luciferase are detected by a multifunctional microplate reader. The results show that after homoharringtonine treatment, the ratio of Fluc / Rluc is significantly reduced.

[0046] Embodiment 3:

[0047] like Figure 5 As shown, based on Example 1, the stably transfected cells pLVX-Rennila luciferase-(-1PRF)-Firefly luciferase 293 were treated with different concentrations of homoharringtonine compounds and the changes in fluorescence values ​​were measured. The results showed that when the inhibitory concentration of homoharringtonine reached 5nM, the ratio of Fluc / Rluc was significantly reduced. The expression results showed that homoharringtonine was able to significantly inhibit the efficiency of HIV-1PRF under this concentration condition.

[0048] Embodiment 4:

[0049] like Figure 6 As shown, based on Example 1, three small molecule compounds that can treat leukemia, fludarabine, mitoxantrone, and homoharringtonine were used to treat stably transfected cells pLVX-Rennila luciferase-(-1PRF)-Fireflyluciferase 293 and the changes in fluorescence values ​​were measured. The results showed that except for homoharringtonine, the -1PRF of cells treated with fludarabine and mitoxantrone did not decrease significantly.

[0050] Embodiment 5:

[0051] like Figure 7 As shown, human embryonic kidney 293 cells were transfected with pNL4-3 envelope mutant plasmid for 24 hours and then treated with different concentrations of homoharringtonine compounds for 12 hours. The changes in viral particles in the culture medium were detected by p24 ELISA, the absorbance was detected at OD450 by a spectrophotometer, and the EC of the homoharringtonine compound was calculated. 50 The results showed that the EC of homoharringtonine compounds 50 =8.57nM.

[0052] Embodiment 6:

[0053] like Figure 8As shown, embryonic kidney 293 cells were transfected with pNL4-3 envelope mutant plasmid for 24 hours, and then treated with different concentrations of homoharringtonine compounds for 12 hours, and then the cells were collected and lysed, and Western blot was used to detect the expression of Gag-Pol in the cells. The results showed that when the homoharringtonine compound was 5nM, the content of Gag-Pol in the cells was significantly reduced, and when the homoharringtonine compound reached 20nM, the present invention basically could not detect the presence of Gag-Pol fusion protein in the cells.

[0054] Embodiment 7:

[0055] like Fig. 9 As shown, in addition to the -1PRF sequence of HIV, the present invention also constructs the -1PRF sequences of four viruses, SIV, RSV, and MMTV, between Rennila lucifease and Firefly lucifease. After these plasmids were transferred into 293 cells for 48 hours, the cells were acted on by adding homoharringtonine, and the changes in the expression levels of Rennila lucifease and Firefly lucifease were detected by a multifunctional microplate reader. The results showed that after the treatment of homoharringtonine, the ratio of Fluc / Rluc in the -1PRF sequences constructed by these different viruses was significantly reduced. This result shows that the compound has a broad spectrum in inhibiting the -1PRF efficiency of the virus. As long as the virus has a -1PRF mechanism, homoharringtonine is a potential therapeutic drug.

[0056] Embodiment 8:

[0057] like Fig.10 As shown, the immunodeficient mouse NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ (NSG) was transplanted into the mouse after human hematopoietic stem cells were separated for two weeks to establish a mouse animal model of humanized blood cells. The HIV virus was purified and injected into the mouse blood system through the tail vein. After 48 hours, the present invention injected homoharringtonine into the mouse through the tail vein. After another 48 hours, the present invention detected the content of HIV virus particles in the mouse blood by ELISA. As can be seen from Figure 11, homoharringtonine can also significantly inhibit the replication of HIV in the blood of humanized mice.

[0058] In summary, the homoharringtonine screened by the present invention can effectively reduce the occurrence of HIV-1PRF and can significantly inhibit the replication of HIV-1 virus in model cells, THP1 cells, and peripheral blood mononuclear cells, and inhibit the assembly of HIV-1 virus particles; the present invention discovers a new indication for homoharringtonine, a leukemia treatment drug, that is, it has an anti-HIV-1 virus effect. Because the drug is already on the market, the invention can significantly reduce the capital investment in safety evaluation in the early stage of drug research and development, and reduce the risk of drug research and development; the present invention finds that homoharringtonine has a broad spectrum of inhibiting -1PRF, and as long as the virus has a -1PRF mechanism, homoharringtonine is a potential therapeutic drug.

Claims

1. Use of a homoharringtonine compound in the preparation of a drug for treating AIDS.

2. Use of a homoharringtonine compound in the preparation of an antiviral drug, wherein the virus is HIV-1.

3. Use of a homoharringtonine compound in the preparation of a virus particle assembly inhibitor, wherein the virus is HIV-1.

4. Use of a homoharringtonine compound in the preparation of a virus programmed-1 ribosomal frameshift inhibitor, wherein the virus is HIV-1.

5. The use according to any one of claims 1 to 4, characterized in that: The drug is administered by injection.

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