Class of triterpene structures, analogs thereof, and use thereof in preparation of antiviral drugs

Abstract

Use of a class of triterpene structures and analogs thereof in the preparation of antiviral drugs. The antiviral effect of the triterpene compounds is produced on the basis of the inhibition of programmed ribosomal frameshifting. The compounds are used in the preparation of antiviral drugs and in the treatment of certain viruses. The compounds inhibit the proliferation of HIV-1 in cells and have a good antiviral effect.

Description

A type of triterpenoid structure and its analogs and their application in preparing antiviral drugs Technical Field

[0001] The present invention relates to a class of triterpenoid structure compounds and analogs thereof, which inhibit the synthesis of viral proteins by interfering with ribosome stability and ultimately exert antiviral effects. Background Art

[0002] Viruses are tiny organisms composed of nucleic acid (DNA or RNA) encased in a protein coat. They cannot survive independently and require host cells to replicate and reproduce. Therefore, viruses must utilize various machinery within the host's body to synthesize the substances they need. Among these, viruses utilize the host's ribosomes to synthesize the proteins they need for packaging. In eukaryotes, ribosomes are divided into large and small subunits, 60S and 40S. These two subunits must organically combine to form a complete ribosome and maintain stability during translation to effectively interpret the codons on mRNA and synthesize proteins.

[0003] HIV (human immunodeficiency virus) is a virus that attacks the human immune system, ultimately leading to AIDS (acquired immunodeficiency syndrome). HIV weakens the immune system by destroying and attacking key immune cells called CD4+ T lymphocytes, making the body more susceptible to other infections. HIV treatment primarily involves antiretroviral drugs (ARVs) to control viral replication, boost the patient's immune system, and slow the damage the virus causes to the body. These drugs are divided into different categories, including: Nucleotide reverse transcriptase inhibitors (NRTIs): These drugs inhibit viral growth by preventing the virus from replicating its genetic material, RNA, when infecting new cells. Non-nucleotide reverse transcriptase inhibitors (NNRTIs): These drugs also block viral replication, but they do so through a different mechanism. Protease inhibitors (PIs): These drugs prevent the virus from producing new viral particles after infection, thereby slowing the progression of infection. Integrase inhibitors (INSTIs): These drugs prevent the virus from integrating its genetic material into the host cell's DNA, thereby halting viral replication. CCR5 antagonists: These drugs slow infection by blocking the virus from entering the CCR5 receptor on CD4 cells. However, no small molecule compound has yet been found that can target the ribosome to treat HIV.

[0004] HIV utilizes the host ribosome's programmed frameshifting mechanism to synthesize its own proteins. Programmed frameshifting doesn't occur at any random location within the viral mRNA, but rather requires a specific RNA sequence that contains two characteristic features: a slippery sequence and a stem-loop sequence. These sequence features are found in all seven currently identified viruses. The slippery sequence typically consists of a seven-nucleotide sequence, XXXYYYZ, where X and Y represent identical sequences. The stem-loop structure, located downstream of the slippery sequence, forms an RNA secondary structure that effectively promotes programmed frameshifting.

[0005] Taking HIV-1 as an example, the mechanisms and biological roles of programmed ribosome frameshifting are further illustrated. In HIV-1, the -1PRF signal is located between the gag and pol open reading frames (ORFs). The HIV-1 gag-pol mRNA contains a circular sequence (stem loop) consisting of a seven-nucleotide slippery sequence (U UUU UUA) and a downstream RNA stem loop. When the ribosome encounters this signal during translation, it pauses at this sequence. In most cases, the ribosome continues translation in the original reading frame until the stop codon is reached, producing the Gag protein. However, 5% of ribosomes can shift backward by one nucleotide in this region, generating a new reading frame downstream. This new reading frame encodes the Pol protein, which is then translated into the Gag-Pol fusion protein. Gag is a viral structural protein, and Pol is a key enzymatic protein. Therefore, the production of the Pol protein is crucial for the maturation and packaging of HIV-1. Under the further action of the viral protease, the Gag protein is cleaved into structural proteins such as p17, p24, and p7, and the Pol protein is cleaved into enzyme proteins such as reverse transcriptase and integrase. HIV-1 utilizes this mechanism to strictly control the production ratio of Gag to Pol proteins to 20:1, which is required for the packaging of a complete virus.

[0006] Therefore, from the perspective of viruses, ribosomes play an important role in their physiological processes. If small molecule compounds that affect ribosome programmed frameshifting can be designed, it will have a very positive effect in antiviral treatment. Summary of the Invention

[0007] Based on the special structure of the large and small ribosome subunits, the present invention discovered a new compound through virtual design combined with experimental verification. Its general structural formula is shown in Formula I. This compound can significantly reduce the efficiency of nuclear programmed glycosome frameshifting during the ribosome translation of the HIV viral protein Gag-Pol, resulting in a decrease in the efficiency of nuclear programmed glycosome frameshifting, making it impossible for the HIV-1 virus to use the ribosome to synthesize the proteins it needs, thereby leading to the death of the HIV virus and ultimately achieving an antiviral effect.

[0008] The first aspect of the present invention is to design a small molecule compound that can significantly reduce programmed ribosomal frameshifting, the general structural formula of which is shown in Figure 1 and Formula I:

[0009] Among them, A1, A2, A3, A4 are independently selected from glucose, rhamnose, fucose, digitose, oleandose, etc., or are missing; R1, R2, R3, R4 are independently selected from H, CH3, OH or CHO.

[0010] In a second aspect of the present invention, antiviral drugs are prepared using this small molecule compound; the triterpene structure and its analogs inhibit the synthesis of viral proteins in cells, thereby achieving an antiviral effect.

[0011] The triterpene structure and its analogs are one or more of Strophanthidin, Glucodigifucoside, Deslanoside and Convallatoxin.

[0012] After the pNL-43-dE mutant plasmid was transfected into cells, the cells were treated with four small molecule compounds, strophanthidin, glucodigifucoside, deslanoside, and convallatoxin, to detect the production of viral particles. As shown in Examples 5 and 6, the p24 protein and viral particles in the supernatant were significantly reduced after treatment with strophanthidin, glucodigifucoside, deslanoside, and convallatoxin, indicating that these compounds have significant antiviral effects.

[0013] The benefits of the present invention are mainly reflected in:

[0014] (1) The present invention designs a small molecule compound that can significantly affect programmed ribosomal frameshifting, and utilizes these small molecule compounds to play a role in anti-HIV-1 virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG1 is a general structural formula of a triterpene structure and its analogs of the present invention;

[0016] FIG2 is the structural formula of four small molecule compounds used in the embodiments of the present invention according to the general structural formula;

[0017] FIG3 shows the changes in the Flu / Rlu ratio after pLVX-Rennila Luciferase-(-1PRF)-Firefly Luciferase was transfected into Hela cells and treated with four different compounds.

[0018] FIG4 shows the changes in the Flu / Rlu ratio after pLVX-Rennila Luciferase-(-1PRF)-Firefly Luciferase was transfected into THP1 cells and treated with four different compounds.

[0019] FIG5 shows the changes in the Flu / Rlu ratio after pLVX-Rennila Luciferase-(-1PRF)-Firefly Luciferase was transfected into PBMC cells and treated with four different compounds.

[0020] FIG6 shows the expression of Gag-Pol in 293T cells after pNL43Envelop mutant plasmid was transformed into the cells and then treated with four different compounds, and Western blot was used to detect the expression of Gag-Pol in the cells.

[0021] FIG7 is a statistical graph showing the changes in the p24 protein content in the culture supernatant detected by ELISA kit after cells were transfected with pNL43Envelop mutant plasmid for 24 hours and then treated with corresponding compounds for 24 hours.

[0022] Figure 8 is a statistical graph showing changes in viral particle content in the culture supernatant detected by Real-time PCR 24 hours after cells were transfected with the pNL43Envelop mutant plasmid and then treated with the corresponding compound for 24 hours. Specific implementation method:

[0023] The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and embodiments.

[0024] In the following embodiments, four small molecule compounds, namely, Strophanthidin, Glucodigifucoside, Deslanoside, and Convallatoxin, are used as examples to further illustrate the technical effects of the present invention. The structures of the four small molecule compounds are shown in FIG2 .

[0025] In the following examples, pLVX-Rennila Luciferase-(-1PRF)-Firefly Luciferase was constructed by the laboratory itself, and the Rennila 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.

[0026] Example 1:

[0027] The constructed pLVX-Rennila Luciferase-(-1PRF)-Firefly Luciferase was first transfected into Hela cells. Twenty-four hours later, the four compounds were added (at a final concentration of 50 nM) for 6 hours. The expression levels of Rennila Luciferase and Firefly Luciferase were then measured using a multi-functional microplate reader. The results, shown in Figure 3, show that the ratio of Firefly Luciferase to Rennila Luciferase decreased significantly after treatment with the four compounds, indicating that the compounds significantly reduce the efficiency of programmed glycosome frameshifting.

[0028] Example 2:

[0029] The constructed pLVX-Rennila Luciferase-(-1PRF)-Firefly Luciferase was first transfected into THP1 cells. Twenty-four hours later, the four compounds were added (at a final concentration of 50 nM) for 6 hours. The expression levels of Rennila Luciferase and Firefly Luciferase were then measured using a multi-functional microplate reader. The results, shown in Figure 4, show that the ratio of Firefly Luciferase to Rennila Luciferase decreased significantly after treatment with the four compounds, indicating that the compounds significantly reduce the efficiency of programmed glycosome frameshifting.

[0030] Example 3:

[0031] The constructed pLVX-Rennila Luciferase-(-1PRF)-Firefly Luciferase was first transfected into PBMC cells. Twenty-four hours later, the cells were treated with the four compounds (final concentration of 50 nM) for 6 hours. The expression levels of Rennila Luciferase and Firefly Luciferase were then measured using a multi-functional microplate reader. The results showed that the ratio of Firefly Luciferase to Rennila Luciferase decreased significantly after treatment with the four compounds, indicating that the compounds significantly reduce the efficiency of programmed glycosome frameshifting.

[0032] Example 4:

[0033] The constructed pNL43-dE plasmid was first transfected into 293T cells. Twenty-four hours later, the four compounds were added (final concentration 50 nM) and treated for 6 hours. Gag-Pol expression was then assessed by Western blot. The results, shown in Figure 6, show that treatment with each of the four compounds significantly reduced the expression of the Gag-Pol fusion protein, indicating that the compounds significantly reduce the efficiency of programmed glycosome frameshifting, leading to decreased expression of the fusion protein Gag-Pol.

[0034] Example 5:

[0035] Please refer to Figure 7. 293T cells were transfected with the pNL43-dE plasmid and, 20 hours later, strophanthidin, glucodigifucoside, deslanoside, and convallatoxin (final concentration of 50 nM) were added for 12 hours. The amount of viral particles in the supernatant was then measured by p24 ELISA. The results showed that the amount of viral particles in the supernatant of cells treated with strophanthidin, glucodigifucoside, deslanoside, and convallatoxin was significantly lower than that in the DMSO control group. This result indicates that strophanthidin, glucodigifucoside, deslanoside, and convallatoxin can effectively inhibit the production of HIV-1 virions.

[0036] Example 6:

[0037] Refer to Figure 8. TPH1 cells were transfected with the pNL43-dE plasmid. 20 hours later, strophanthidin, glucodigifucoside, deslanoside, and convallatoxin (final concentration 50 nM) were added for 12 hours. Total RNA was extracted from the cell supernatant, and the HIV virion content was measured by real-time PCR. The results showed that compared to the DMSO control group, the viral content in the experimental group treated with strophanthidin, glucodigifucoside, deslanoside, and convallatoxin was significantly reduced, indicating that strophanthidin, glucodigifucoside, deslanoside, and convallatoxin can inhibit HIV-1 production.

[0038] The above results show that the triterpenoid structure and its analogs of the present invention can significantly reduce the efficiency of programmed glycosome frameshifting, thereby reducing the expression level of HIV viral protein Gag-Pol, rendering HIV-1 virus unable to synthesize the proteins it needs, thereby leading to the death of HIV virus and ultimately achieving an antiviral effect.

Claims

1. Use of a triterpenoid structure and its analogs in the preparation of antiviral drugs, characterized in that: The triterpene structure and its analogues are shown in the general formula of formula I: Wherein, A1, A2, A3, A4 are independently selected from glucose, rhamnose, fucose, digitose, oleandose or missing; R1, R2, R3, R4 are independently selected from H, CH3, OH or CHO.

2. The use according to claim 1, characterized in that The triterpene structure and its analogs are one or more of Strophanthidin, Glucodigifucoside, Deslanoside, Convallatoxin or similar compounds with structural formula characteristics.

3. The use according to claim 1, characterized in that The virus is HIV-1 virus.

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