Application of pramipexole as sapovirus helicase ATPase activity inhibitor

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a novel medical application of a dopamine receptor stimulant pramipexole (PPX) as an inhibitor of the activity of sapovirus 2C RNA helicase ATPase, and the inhibitor is pramipexole. In-vitro enzymology experiments prove that the pramipexole can remarkably inhibit the ATP enzyme activity of the SaV 2C RNA helicase, and the inhibition effect of the pramipexole is quantitatively verified through a malachite green-ammonium molybdate colorimetric method. The invention further discloses that pramipexole and SaV-2C RNA helicase form stable interaction (the binding energy is-126.53 kJ / mol) through molecular docking, and the key interaction comprises four hydrogen bonds mediated by Asp47 and Glu48 residues, hydrophobic interaction between the four hydrogen bonds and Leu30, Ala31 and Trp46, and electrostatic force generated by the four hydrogen bonds and Glu48. The invention has the potential of preventing and treating sapovirus infection diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically providing a new application of the known compound pramipexole (CAS No. 191217-81-9) in the treatment of Sapporo virus, particularly its mechanism of action in inhibiting the activity of the viral 2C helicase ATPase and its drug development strategy.

[0002] The present invention also relates to the application of the above-described method. Background Technology

[0003] Sapporo virus (SaV) is a non-enveloped, positive-sense, single-stranded RNA virus belonging to the Caliciviridae family. It is a major pathogen causing acute gastroenteritis (AGE) in humans and animals. Globally, SaV infection accounts for approximately 10% of diarrhea cases and causes about 23,000 deaths annually in children under the age of five. Besides humans, pigs are also a major host for this virus. The potential for cross-species transmission has raised concerns about zoonotic spillovers, food safety, and public health. Furthermore, surveillance data from the past decade shows a significant upward trend in SaV infection in several countries, including Japan, South Korea, and Thailand, increasing global attention. SaV infection is primarily transmitted through the fecal-oral route or via contaminated food, with main clinical manifestations including vomiting and diarrhea. Currently, there are no specific vaccines or antiviral drugs for the prevention or treatment of SaV infection.

[0004] Like other positive-sign single-stranded RNA viruses, the Sapporo virus genome contains multiple cis-acting RNA elements responsible for regulating key processes including RNA replication, translation, and capsid assembly. The non-structural protein 2C RNA helicase of Sapporo virus possesses nucleoside triphosphatase (NTPase) activity and is a classic superfamily 3 (SF3) helicase. Extensive research on viruses with conserved SF3 helicase sequences has demonstrated that the ATPase activity of helicases is essential for coronavirus replication and proliferation; therefore, targeting or screening drugs against these helicases is one of the ideal strategies for developing anti-Sapporo virus drugs.

[0005] Pramipexole is a non-ergotamine D3 dopamine receptor agonist. Currently, clinically available pramipexole-containing drugs include pramipexole hydrochloride. It has shown good efficacy in the clinical treatment of Parkinson's disease, but its role as an antiviral drug, especially as a viral RNA helicase inhibitor, has not been previously reported.

[0006] Based on the aforementioned research background, this invention, through in vitro enzymatic experiments, confirms that pramipexole can significantly inhibit the ATPase activity of Sapporo virus (SaV) 2C RNA helicase, and its inhibitory effect is quantitatively verified by the malachite green-ammonium molybdate colorimetric method. Molecular docking prediction reveals that pramipexole forms a stable interaction with SaV-2C RNA helicase (binding energy -126.53 kJ / mol). Key interactions include four hydrogen bonds mediated by Asp47 and Glu48 residues, hydrophobic interactions with Leu30, Ala31, and Trp46, and electrostatic forces generated with Glu48. These results indicate that pramipexole has the potential to prevent and treat Sapporo virus infection. Summary of the Invention

[0007] In view of this, the present invention provides a novel application of the known compound pramipexole in the treatment of Sapporo virus. The results of this invention demonstrate that SaV-2C possesses helicase activity in vitro, and further show that pramipexole can inhibit the ATPase activity of SaV-2C in vitro, thereby inhibiting viral genome replication.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention prepares SaV-2C by codon optimization and the addition of the solubilizing tag MBP. After induction with 0.2 mM IPTG, the expression level of SaV-2C in Escherichia coli is significantly enhanced.

[0009] This invention enriches SaV-2C protein using Ni-NTA affinity chromatography, and further purifies it using gel filtration chromatography to obtain the target protein in high purity.

[0010] Through high-throughput screening and enzyme kinetics verification, this invention is the first to discover that pramipexole can target and inhibit the ATPase activity of SaV-2C helicase, and its effect is dose-dependent.

[0011] This invention utilizes Discovery Studio for molecular docking, revealing the binding mode of pramipexole to the SaV-2C helicase: the stability of pramipexole with Sav-2C is predicted to be primarily driven by four hydrogen bonds mediated by Asp47 and Glu48 residues. Furthermore, it also depends on hydrophobic interactions with Leu30, Ala31, and Trp46 residues, as well as the electrostatic force generated by Glu48; these key interactions synergistically stabilize the formed complex. Attached Figure Description

[0012] Figure 1 Express and purify the SaV-2C protein with the fusion tag MBP.

[0013] Figure 2 (A) SaV-2C has ATPase activity, while the tag protein MBP does not; (B) Pramipexole (PPX) can inhibit the ATPase activity of SaV-2C helicase in a concentration-dependent manner.

[0014] Figure 3 Spatial structural model of the interaction between pramipexole and SaV-2C, and the amino acids involved in the key role.

[0015] Figure 4 Oral toxicity prediction results and toxicity model reports for pramipexole. Detailed Implementation

[0016] Example 1: Expression and purification of SaV-2C protein 1. Experimental Materials The optimization of the Escherichia coli BL21(DE3), LB medium, IPTG, and SaV-2C gene expression system for Escherichia coli was completed in our laboratory.

[0017] 2. Buffer Preparation (1) Lysis buffer: 50 mM HEPES, 150 mM NaCl, pH 7.5 (2) Ni-NTA column eluent: 50 mM HEPES, 150 mM NaCl, 20-500 mM imidazole, pH 7.5 (3) Gel filtration chromatography buffer: 50 mM HEPES, 150 mM NaCl, pH 7.5 3. Experimental Procedure (1) Take 100 μl of bacterial culture expressing SaV-2C protein and inoculate it into 100 ml of LB medium. Shake for 12-16 h to activate the bacteria. Inoculate 1 L of LB medium at a ratio of 1:100 to induce large-scale growth. Incubate at 220 rpm and 37 °C until OD. 600 When the concentration reaches between 0.6 and 0.8, cool to room temperature, add a final concentration of 0.2 mM IPTG, and induce expression overnight (18-20 h) at 20°C and 180 rpm. Collect the bacterial cells by centrifugation at 4°C and 4000 rpm for 30 min.

[0018] (2) The bacterial cells were resuspended in lysis buffer. After sonicating on ice for 30 minutes, the cell lysis buffer was centrifuged at 1,2000 rpm for 20 minutes and the supernatant was collected.

[0019] (3) Slowly add the filtered supernatant to the Ni-NTA column that has been pre-equilibrated with lysis buffer to allow the protein to bind to the resin, and then rinse the Ni-NTA column with lysis buffer.

[0020] (4) The bound proteins were eluted using a gradient elution buffer with a concentration of 20-500 mM imidazole.

[0021] (5) After eluting and concentrating the protein, high-purity target protein was obtained by gel filtration chromatography.

[0022] (6) The purified protein samples and target components were analyzed by electrophoresis using 12% SDS-PAGE.

[0023] The results are as follows Figure 1 As shown, the SaV-2C protein with the MBP tag was successfully purified.

[0024] Example 2: Lead compound screening 1. Experimental software / website Discovery Studio 2019, ZINC 20, AlphaFold2, SAVES 2. Operation process (1) Download the FDA compound library from the ZINC20 database.

[0025] (2) 3D structural prediction of SaV-2C was performed using AlphaFold2. The structural quality of the model was assessed using the SAVES structural analysis and validation server.

[0026] (3) The model with the highest confidence score was processed by adding hydrogen atoms, etc. using Discovery Studio 2019.

[0027] (4) Perform ADMET property prediction on the compound library and evaluate it according to the five principles of drug-likeness (Lipinski) and Veber's rule. Small molecules that do not conform to the above rules are excluded.

[0028] (5) The LibDock rigid docking method was used to perform molecular docking of the SaV-2C structure, and the top 20% of the compound structures in terms of LibDock score were retained.

[0029] (6) Calculate the receptor-ligand binding energy and identify the 10 compounds with the lowest binding energy as candidate inhibitors.

[0030] Example 3: ATPase activity inhibition experiment 1. Experimental Materials Purified SaV-2C helicase (purity >95%), malachite green, ammonium molybdate, and pramipexole (Shanghai Haohong Biomedical Technology Co., Ltd., CAS 104632-26-0) 2. Reaction system 50mM HEPES (pH 7.5), 150mM NaCl, 2mMMgCl2, 4mM ATP, 10μM SaV-2C protein 3. Experimental Procedure (1) Prepare the reaction solution according to the reaction system.

[0031] (2) Add gradient concentrations of pramipexole (0-30 mM), incubate at room temperature for 60 minutes, and then terminate the reaction with 40 mM EDTA.

[0032] (3) Take 40 μl and transfer it to a 96-well plate, mix it with malachite green-ammonium molybdate reagent, and incubate for 15 min.

[0033] (4) The absorbance was measured at 620 nm to quantify the release of inorganic phosphorus based on the formation of the phosphate molybdate complex.

[0034] (5) All measurements were repeated at least three times.

[0035] The results are as follows Figure 2 As shown, (A) compared with the control group MBP, SaV-2C wild-type exhibits higher ATPase activity, unaffected by the MBP tag. (B) When the pramipexole concentration is ≥20 mM, the inhibition rate is >50%.

[0036] Example 4: Molecular docking and toxicity prediction 1. Experimental software / website Discovery Studio 2019, ProTox-3.0 2. Operation process (1) The binding model of pramipexole and SaV-2C helicase was displayed by docking with Discovery Studio.

[0037] (2) Predict the key amino acid residues and major interaction forces involved in the binding of pramipexole to Sav-2C.

[0038] (3) The toxicological properties of pramipexole were predicted using ProTox-3.0. The website calculates the median lethal dose (LD50) of the compound and predicts its toxicity level (1-6, with higher levels indicating lower toxicity) using toxicological models such as organ toxicity, carcinogenicity, mutagenicity, cytotoxicity, and immunotoxicity.

[0039] The results are as follows Figure 3 , 4As shown, the stability of pramipexole with Sav-2C is primarily driven by four hydrogen bonds mediated by Asp47 and Glu48 residues. Furthermore, it depends on the hydrophobic interactions between Leu30, Ala31, and Trp46, as well as the electrostatic forces generated by Glu48. Toxicity analysis showed that pramipexole has an LD50 of 800 mg / kg and a predicted toxicity level of 4, classifying it as a low to moderately toxic compound. Toxicity predictions indicate that pramipexole may exhibit some neurotoxicity and inhalation toxicity, but it is not carcinogenic, mutagenic, or immunotoxic.

Claims

1. Use of pramipexole for the preparation of an inhibitor of the RNA helicase ATPase activity of the Zalop virus, characterized in that, The inhibitor is pramipexole.

2. Use according to claim 1, characterized in that, It inhibits ATPase activity by allosteric binding to the helicase, with an inhibition rate of >50% at a concentration of 20 mM.

3. Use according to claim 2, characterized in that, The helicase is the Sapporo virus 2C RNA helicase.

4. Use according to claim 3, characterized in that, The helicase is prepared by codon optimization and addition of a solubility tag MBP.

5. Use according to claim 2, 3, characterized in that, The binding site and interaction of pramipexole with the Sapporo virus 2C RNA helicase include: Asp47 and Glu48 form four hydrogen bonds; Leu30, Ala31 and Trp46 form hydrophobic interactions; and Glu48 generates an electrostatic force.

6. Use according to claim 2, characterized in that, The inhibitory activity is quantitatively detected by the ammonium molybdate-malachite green colorimetric method, with the following specific conditions: reaction system: 50 mM HEPES (pH 7.5), 150 mM NaCl, 2 mM MgCl2, 4 mM ATP, 10 μM SaV-2C protein; inhibitor concentration gradient: 0-30 mM; detection wavelength: 620 nm (inorganic phosphorus release amount).

7. A method for inhibiting the RNA helicase ATPase activity of a sapovirus, comprising incubating the inhibitor of claim 2 with the system of claim 5, characterized in that, Incubation at room temperature for 60 min can improve the inhibition rate.

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