Application of tanshinone I in preparation of medicine for treating AIDS (acquired immune deficiency syndrome)

Abstract

The invention relates to an application of tanshinone I in preparation of a medicine for treating AIDS (Acquired Immune Deficiency Syndrome), and a tanshinone I compound is used for preparing the medicine for treating the AIDS and is applied to treatment of the AIDS. The Tanshinone I compound can significantly reduce the efficiency of programmed ribosome frameshift when the mRNA of HIV-1 translates a Gag-Pol fusion protein, so that the translation efficiency of the Pol protein is greatly reduced, thereby causing the ratio imbalance of Gag and Pol in cells, causing the virus not to be effectively packaged, and finally causing the sharp reduction of the yield of the virus, and having a good anti-HIV effect.

Description

Technical Field

[0001] The present invention relates to the field of preparation of antiviral drugs, and in particular to the use of Tanshinone I in the preparation of drugs for treating AIDS. Tanshinone I inhibits the synthesis of viruses by interfering with programmed ribosomal frameshifting, and ultimately exerts an antiviral effect. Background Art

[0002] HIV-1 virus particles are spherical, with a diameter of about 100-120 nanometers. The core of the virus particle contains two single-stranded RNAs. The HIV-1 genome has a long terminal repeat sequence (LTR) at each end. Between the two LTRs, the genes encode key proteins that constitute the virus structure. The genome is 9.7kb in length and contains 9 open reading frames, of which 3 structural genes (Gag, Pol, Env) encode structural proteins and enzymes, and 6 regulatory protein genes (Tat, Rev, Nef, Vif, Vpu, Vpr) encode regulatory proteins and accessory proteins. Gag protein encodes matrix protein (MA, p17), capsid protein (CA, p24) and nucleocapsid protein (NC, p15), and plays a core role in the assembly and maturation of virus particles. Pol protein encodes reverse transcriptase (RT, including RNase H activity), protease (PR) and integrase (IN), which are essential in the process of viral replication. The Env protein encodes the envelope glycoprotein gp160, which can be cleaved into the envelope protein gp120 and the transmembrane protein gp41, and is involved in the fusion of the virus with the host cell. The Tat and Rev proteins are crucial for the regulation of HIV gene expression. The Tat protein enhances the transcriptional activity of viral genes, and the Rev protein promotes the transport of unspliced ​​or incompletely spliced ​​viral mRNA from the nucleus to the cytoplasm. The Nef, Vif, Vpr, and Vpu proteins are involved in viral replication, infectivity, cytopathic effect, and assembly and release of viral particles.

[0003] -1PRF is a mechanism that regulates protein translation. The characteristic mRNA sequences of some viruses contain programmed ribosomal frameshift (PRF) signals. When a ribosome encounters these signals during protein translation, it tends to pause translation. This causes most ribosomes to continue translating in the original reading frame, while a small fraction will slip on the mRNA, causing a frameshift and start translating in a new reading frame, allowing the virus to translate two proteins from a single RNA template. In some cases, -1PRF signals regulate the expression of two different polypeptides during embryonic development, while in other cases they result in truncated protein products that may function differently than the full-length protein.

[0004] The HIV-1 virus cleverly exploits the host cell's ribosome programmed frameshifting machinery to synthesize its own proteins. The -1PRF mechanism of HIV-1 allows HIV-1 to express Gag-Pol proteins from a single Gag-encoding mRNA, which is essential for viral replication and infectivity. The -1PRF signal prompts the ribosome to frameshift, precisely controlling the synthesis ratio of Gag to Gag-Pol, and even small changes in PRF efficiency, whether it increases or decreases, can have a significant inhibitory effect on viral yield. The -1PRF mechanism of HIV-1 is regulated by specific mRNA sequences and structures. This mechanism involves two basic RNA elements: the slippery sequence and the downstream stem-loop structure. For HIV-1, this sequence is U UUU UUA. This sequence is the specific location where the ribosome undergoes -1PRF. The other element is the stimulatory signal, which is an mRNA secondary structure located a specific distance downstream of the slippery sequence. In HIV-1, this stimulatory signal is contained in a 69-base segment that forms an extended stem-loop structure that helps regulate the probability of a -1PRF event. This structure is distinct from the −1PRF signals of most other viruses, which typically form pseudoknots.

[0005] When the ribosome encounters this signal while translating HIV-1 Gag-Pol mRNA, it pauses at this sequence. This special RNA structure enables the ribosome to recognize this region and induces the ribosome to shift back one nucleotide in about 5% of cases. This shift allows the ribosome to skip the stop codon of the Gag protein, so that the original ORF sequence is converted into the coding sequence of the Pol protein, allowing the translation of the Gag-Pol fusion protein. After the frameshift, the ribosome continues to translate in the new reading frame, producing the p160Gag-Pol polyprotein of HIV-1. If -1PRF does not occur, the ribosome will maintain the original reading frame and encounter the stop codon of the Gag gene, resulting in only the production of p55Gag polyprotein. However, most ribosomes stop translating when they encounter the stop codon at the end of the Gag open reading frame, producing only Gag protein. In this way, the HIV-1 virus is able to ensure that there is enough Gag protein to build the viral structure while limiting the amount of Pol protein to maintain a delicate balance of viral replication and assembly. In HIV-1, the efficiency of -1PRF is about 5%, which is crucial for the formation of virions and the infectivity of the virus. Any factors that affect this mechanism, whether it is the mRNA structure of the virus itself or host factors, may have a significant impact on the virus's replication ability and infectivity.

[0006] The -1PRF mechanism of HIV-1 is a key factor in viral replication and evolution. It not only affects the transmission efficiency and latent state of the virus, but also plays an important role in the genetic diversity and adaptability of the virus. In-depth research on the -1PRF mechanism will help develop new antiviral treatment strategies and provide new approaches for the treatment and prevention of HIV-1 infection. Exploring drugs that can affect the efficiency of -1PRF and their mechanisms of action is a current and future research focus, which may provide new insights into drug design and treatment options for some viruses. Understanding the process of programmed ribosomal frameshifting and related mechanism research is of great significance for the development of antiviral drugs and clinical treatment. Summary of the invention

[0007] The purpose of the present invention is to provide a new use of Tanshinone I, namely, use of Tanshinone I in preparing a medicine for treating AIDS.

[0008] To achieve the above object, the technical solution of the present invention is as follows:

[0009] In the first aspect of the present invention, a drug Tanshinone I that can significantly reduce programmed ribosomal frameshift is found, and its general structural formula is shown in Formula I:

[0010]

[0011] The present invention constructs a stable cell line based on model cells, which can characterize the efficiency of programmed ribosomal frameshifting by calculating the value of Firefly Luciferase / Renilla Luciferase, and conducts large-scale drug screening and concentration gradient drug addition tests, as well as compound mechanism retrieval combined with experimental verification, as follows:

[0012] The present invention treats the stable cell line R-(-1PRF)-F-293T with Tanshinone I according to the concentration gradient, and detects the values ​​of Firefly Luciferase and Renilla Luciferase. Figure 2 As shown, the enzyme content of Renilla Luciferase did not change significantly after treatment with Tanshinone I, while the enzyme content of Firefly Luciferase was significantly reduced, and the ratio of the two was significantly reduced, indicating that this type of compound significantly reduces the -1PRF efficiency of HIV-1, which can be close to half.

[0013] The present invention treats the stable cell lines R-(-1PRF)-F-Hela and R-(-1PRF)-F-THP1 with Tanshinone I according to the concentration gradient, and detects the values ​​of Firefly Luciferase and Renilla Luciferase. Figure 3 , Figure 4 As shown, the enzyme content of Firefly Luciferase was significantly reduced and the efficiency of -1PRF was significantly reduced after treatment with Tanshinone I, indicating that this type of compound has an inhibitory effect on the frameshift efficiency of different types of cells.

[0014] In the present invention, the pNL43-dE plasmid was transfected into 293T cells, and after being treated with Tanshinone I, the expression level of Gag-Pol protein in the cells was detected by Western blot technology. Figure 5 As shown, with the increase of the concentration of Tanshinone I, the expression of Gag protein did not change significantly, and the content of Gag-Pol fusion protein was significantly reduced, indicating that this type of compound can significantly reduce the efficiency of programmed ribosomal frameshifting by changing the ratio of Gag-Pol to Gag protein, and can affect the efficiency of programmed ribosomal frameshifting in real HIV-1 virus.

[0015] After the pNL-43-dE mutant plasmid is transferred into cells, the cells are treated with Tanshinone I to detect the production of viral particles. Figure 6 As shown, the p24 protein in the supernatant decreased significantly after Tanshinone I treatment, indicating that this type of compound has a significant antiviral effect.

[0016] After the pNL-43-dE mutant plasmid was transferred into cells, the cells were treated with Tanshinone I, and the changes in the supernatant virus content in the culture medium were detected by Real-time PCR. Figure 7 As shown, after treatment with Tanshinone I, the virus content in the supernatant gradually decreased, and could be reduced by 50%, indicating that this type of compound reduces viral packaging in cells, resulting in a decrease in virus production.

[0017] In summary, the present invention has found that this compound can significantly inhibit the efficiency of programmed ribosomal frameshifting, thereby reducing the content of Gag-Pol fusion protein, thereby disrupting the production ratio of Gag-Pol protein, resulting in the inability of the virus to assemble effectively, reducing the amount of virus production, and ultimately achieving an antiviral effect.

[0018] The second aspect of the present invention provides the use of a tanshinone I compound in the preparation of a medicament for treating AIDS.

[0019] The third aspect of the present invention provides the use of a tanshinone I compound in the preparation of an antiviral drug and / or a virus particle assembly inhibitor and / or a virus programmed-1 ribosomal frameshift inhibitor, wherein the virus is HIV-1.

[0020] The beneficial effects of the present invention are mainly reflected in:

[0021] (1) The present invention discovered that Tanshinone I can significantly reduce the efficiency of programmed ribosomal frameshifting, disrupt the production ratio of Gag-Pol protein, resulting in the inability of the virus to effectively assemble and causing the efficiency of virus production in the cell to decrease, and has a good anti-HIV-1 virus effect.

[0022] (2) The present invention utilizes Tanshinone I compound to prepare a drug for treating AIDS. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the general structural formula of Tanshinone I of the present invention;

[0024] Figure 2 The content of firefly luciferase and Renilla luciferase and their ratio, i.e., the change of -1PRF efficiency, after treating R-(-1PRF)-F-293T cells with Tanshinone I at concentration gradient (final concentration of 0, 2, 4, 8 μM) for 6 h; (A) is the comparison of the content of firefly luciferase and Renilla luciferase after concentration gradient treatment, and (B) is the ratio of the content of firefly luciferase / Renilla luciferase after concentration gradient treatment;

[0025] Figure 3 The changes in the contents of firefly luciferase and Renilla luciferase and their ratio, i.e., -1PRF efficiency, after Tanshinone I (Tanshinone I) was treated with a concentration gradient (final concentration of 0, 2, 4, 8 μM) in R-(-1PRF)-F-Hela cells for 6 h; (A) is the comparison of the contents of firefly luciferase and Renilla luciferase after the concentration gradient treatment, and (B) is the ratio of the contents of firefly luciferase / Renilla luciferase after the concentration gradient treatment; Figure 4The changes in the contents of firefly luciferase and Renilla luciferase and their ratio, i.e., -1PRF efficiency, after treating R-(-1PRF)-F-THP1 cells with Tanshinone I at concentration gradients (final concentrations of 0, 2, 4, and 8 μM) for 6 h; (A) is the comparison of the contents of firefly luciferase and Renilla luciferase after the concentration gradient treatment, and (B) is the ratio of the contents of firefly luciferase / Renilla luciferase after the concentration gradient treatment; Figure 5 After the pNL43Envelop mutant plasmid was transfected into 293T cells, the cells were treated with Tanshinone I at different concentration gradients, and the expression of Gag-Pol in the cells was detected by Western blot.

[0026] Figure 6 This is a statistical chart of the changes in the p24 protein content in the culture supernatant detected by ELISA kit after THP1 cells were transfected with pNL43 Envelop mutant plasmid for 24 hours and treated with Tanshinone I at a concentration gradient (final concentration of 0, 1, 2, 4, 8 μm) for 24 hours;

[0027] Figure 7 This is a statistical graph of the changes in the content of viral particles in the culture medium supernatant detected by Real-time PCR after 293T cells were transfected with the pNL43 Envelop mutant plasmid for 24 hours and treated with Tanshinone I at concentration gradients for 24 hours. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0029] In the following examples, pLVX-Renilla Luciferase-(-1PRF)-Firefly Luciferase was constructed by the laboratory itself, and the Renilla Luciferase-(-1PRF)-Firefly Luciferase fragment was constructed into the pLVX-Puro plasmid. The pLVX-Puro plasmid was purchased from TAKARA, and the pNL43-dE plasmid was purchased from Addgene.

[0030] In the following examples, R-(-1PRF)-F-293T cells were constructed by the laboratory itself, and the modified viral genome and pspAX2 and pMD2G plasmids were co-transfected into 293T cells, packaged into HIV pseudoviruses and then infected with 293T cells. After a series of monoclonal screening and verification, a stable cell line was finally obtained, which can characterize the efficiency of programmed ribosomal frameshifting by calculating the value of Firefly Luciferase / Renilla Luciferase, and was named R-(-1PRF)-F-293T.

[0031] Embodiment 1:

[0032] First, Tanshinone I was added to R-(-1PRF)-F-293T cells at a concentration gradient (final concentration of 0, 2, 4, 8 μM) for 6 hours, and the expression levels of Renilla Luciferase and Firefly Luciferase were detected by a multifunctional microplate reader. Figure 2 The results show that after treatment with Tanshinone I, the enzyme content of Renilla Luciferase did not change significantly, indicating that the protein translation of Renilla Luciferase was not greatly affected, while the enzyme content of Firefly Luciferase decreased significantly, indicating that its protein synthesis was significantly reduced. The ratio was calculated and it was found that the ratio decreased more significantly with the increase of Tanshinone I concentration, indicating that the compound can significantly inhibit the efficiency of programmed glycosome frameshifting.

[0033] Embodiment 2:

[0034] First, Tanshinone I was added to R-(-1PRF)-F-Hela cells at a concentration gradient (final concentration of 0, 2, 4, 8 μM) and treated for 6 hours. The expression levels of Renilla Luciferase and Firefly Luciferase were detected by a multifunctional microplate reader. Figure 3 It was shown that after treatment with Tanshinone I, the enzyme content of Firefly Luciferase decreased significantly with the increase of Tanshinone I concentration, and the ratio of Firefly Luciferase to Renilla Luciferase decreased significantly, indicating that the compound can significantly inhibit the efficiency of programmed glycosome frameshifting.

[0035] Embodiment 3:

[0036] First, Tanshinone I was added to R-(-1PRF)-F-THP1 cells at a concentration gradient (final concentration of 0, 2, 4, 8 μM) and treated for 6 hours. The expression levels of Renilla Luciferase and Firefly Luciferase were detected by a multifunctional microplate reader. Figure 4 It was shown that after treatment with Tanshinone I, the enzyme content of Firefly Luciferase decreased significantly with the increase of Tanshinone I concentration, and the ratio of Firefly Luciferase to Renilla Luciferase decreased significantly, indicating that the compound can significantly inhibit the efficiency of programmed glycosome frameshifting.

[0037] Embodiment 4:

[0038] First, the constructed pNL43-dE plasmid was transformed into 293T cells. After 24 hours, different concentrations of Tanshinone I were added to the cells for 24 hours, and the expression level of Gag-Pol was detected by Western blot. Figure 5 It was shown that after treatment with Tanshinone I, the expression level of Gag-Pol fusion protein decreased significantly with the increase of Tanshinone I concentration, indicating that the compound can significantly inhibit the efficiency of programmed glycosome frameshifting, thereby reducing the expression level of the fusion protein Gag-Pol.

[0039] Embodiment 5:

[0040] See also Figure 6 , by transfecting pNL43-dE plasmid into THP1 cells, and then adding Tanshinone I for 24 hours after 24 hours, p24 ELISA was used to detect the changes in the amount of viral particles in the supernatant. The results showed that the content of viral particles in the supernatant of cells treated with Tanshinone I (concentrations of 1, 2, 4, and 8 μM) was significantly lower than that in the control group DMSO (concentration of 0 μM), which showed that Tanshinone I can effectively inhibit the generation of HIV-1 viral particles.

[0041] Embodiment 6:

[0042] See also Figure 7, by transfecting pNL43-dE plasmid into 293T cells, and then adding different concentrations of Tanshinone I for 24 hours, extracting total RNA from the cell supernatant, and detecting the content of HIV virus particles by real-time PCR. The results showed that compared with the control group (without Tanshinone I), the virus content in the experimental group (with Tanshinone I) was significantly reduced with the concentration of Tanshinone I, that is, Vidofludimus can inhibit the production of HIV-1 virus.

[0043] The above results indicate that Tanshinone I of the present invention can significantly reduce the efficiency of programmed glycosome frameshifting, thereby reducing the expression of HIV viral protein Gag-Pol, causing disruption of the synthesis ratio of proteins required by the HIV-1 virus itself, thereby leading to the death of the HIV virus and ultimately achieving an antiviral effect.

Claims

1. Application of tanshinone I compound in the preparation of medicine for treating AIDS.

2. The use according to claim 1, characterized in that: The general structural formula of the tanshinone I compound is:

3. The use of a tanshinone I compound in the preparation of an antiviral drug, characterized in that: The virus is HIV-1.

4. The use of a tanshinone I compound in the preparation of a virus particle assembly inhibitor, characterized in that: The virus is HIV-1.

5. The use of tanshinone I compound in the preparation of viral programmed-1 ribosomal frameshift inhibitors, characterized in that: The virus is HIV-1.

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