Application of verofenib in killing pine wood nematodes

Abstract

The invention relates to an application of verofenib in killing pine wood nematodes, the verofenib can effectively kill the pine wood nematodes, and the median lethal concentration of the verofenib to the pine wood nematodes within 24 hours is 66.74 mu mol / L. As a selective inhibitor of serine-threonine protein kinase, the verofenib can effectively inhibit the activity of the serine-threonine protein kinase of the pine wood nematodes. The compound has a remarkable inhibition effect on movement and reproduction of pine wood nematodes, and has a good treatment effect on pine wilt. The method has the advantages of being simple in operation method, low in cost, high in nematicidal activity and the like, has a good application prospect in the aspect of controlling pine wood nematode diseases, and can be applied to pesticide preparations for killing pine wood nematodes.

Description

Technical Field:

[0001] The present invention relates to the application of vemurafenib in killing Bursaphelenchus xylophilus, belonging to the technical field of forest pest control. Background Art:

[0002] Pine wilt disease (PWD), also known as pine wilt blight, is a devastating forest disease caused by the infection of Bursaphelenchus xylophilus (PWN). This disease is characterized by a high lethality rate, a fast transmission speed, a complex pathogenic mechanism, and difficulty in prevention and control. It has caused huge economic and ecological losses worldwide. Many countries around the world, including China, have listed Bursaphelenchus xylophilus as an important quarantine object. Currently, there are still many defects in the control means for Bursaphelenchus xylophilus, and no effective and clear control plan has been formed. Therefore, in-depth research on nematicidal components with high efficiency and low toxicity has become the key content in the research work of pine wilt disease control. Summary of the Invention:

[0003] The purpose of the present invention is to overcome the deficiencies of the prior art and provide the application of vemurafenib in killing Bursaphelenchus xylophilus. Vemurafenib can be used for the treatment of human melanoma. Its biological safety has been clinically tested, the cost is relatively low, it has high nematicidal activity, and the action site in nematodes is clear.

[0004] In order to achieve the above-mentioned invention purpose, the vemurafenib solution with Bursaphelenchus xylophilus killing activity provided by the present invention is prepared by dissolving vemurafenib in an aqueous solution containing 5 wt% DMSO, and can effectively kill Bursaphelenchus xylophilus.

[0005] The structural formula of vemurafenib is:

[0006]

[0007] The present invention also provides the application of vemurafenib in killing Bursaphelenchus xylophilus. Vemurafenib can effectively inhibit the activity of serine / threonine protein kinase of Bursaphelenchus xylophilus and kill Bursaphelenchus xylophilus.

[0008] The application described in the present invention is to dissolve vemurafenib in an aqueous solution containing 5 wt% DMSO for use, and the concentration of vemurafenib is 20 - 100 μmol / L.

[0009] In the experiment of killing Bursaphelenchus xylophilus with vemurafenib, about 200 adult Bursaphelenchus xylophilus were added to a 96-well plate, and 10-50 μmol / L vemurafenib solution was added for treatment respectively. The Bursaphelenchus xylophilus treated with 5% DMSO was used as the negative control. After culturing in the dark at 25 °C for 24 h, the number of dead nematodes was counted under a stereomicroscope and the corrected mortality was calculated. The above experiment was repeated. The nematodes were considered dead when they were stiff and motionless under the field of view, otherwise they were considered alive. The results were statistically analyzed using SPSS 17.0. The calculation formula is as follows:

[0010]

[0011] The results showed that the median lethal concentration of vemurafenib against Bursaphelenchus xylophilus at 24 h was 66.74 μmol / L, and the 95% confidence interval was 47.71-140.94 μmol / L.

[0012] The median lethal concentrations (LC50) of vemurafenib against Bursaphelenchus xylophilus at 48 h and 72 h were 35.38 μmol / L and 20.20 μmol / L respectively. After treating Bursaphelenchus xylophilus with a low-concentration vemurafenib solution (50 μmol / L) for 48 h, the motility, reproductive ability and development level of the nematodes were significantly reduced. In addition, the experimental results showed that vemurafenib had a good therapeutic effect on pine seedlings infected with Bursaphelenchus xylophilus. After 45 days of treatment, compared with the control group of pine seedlings without vemurafenib treatment, the control group of pine seedlings treated with low-concentration vemurafenib (50 μmol / L) did not show any wilting symptoms.

[0013] In previous studies, the applicant of the present invention found that knocking down the expression level of the serine / threonine protein kinase gene stk in Bursaphelenchus xylophilus by RNAi technology would lead to a significant decrease in the motility, reproduction and development of the nematodes, indicating that the serine / threonine protein kinase encoded by stk in Bursaphelenchus xylophilus plays an important role in regulating the life activities of Bursaphelenchus xylophilus. Therefore, the present invention takes serine / threonine protein kinase as the action target and finds a selective inhibitor vemurafenib, which can effectively inhibit the activity of serine / threonine protein kinase of Bursaphelenchus xylophilus and kill Bursaphelenchus xylophilus.

[0014] Compared with the prior art, the present invention innovatively uses vemurafenib to kill Bursaphelenchus xylophilus. As a selective inhibitor of serine / threonine protein kinase, vemurafenib can effectively inhibit the activity of serine / threonine protein kinase of Bursaphelenchus xylophilus, has a significant inhibitory effect on the motility and reproduction of Bursaphelenchus xylophilus, and has a good therapeutic effect on pine wilt disease, laying a solid theoretical foundation for the development of new and efficient nematicidal drugs and providing a potential drug action target; the operation method is simple, the biological safety is high, the cost is low, the nematicidal activity is high, and it has a good application prospect in controlling Bursaphelenchus xylophilus disease, and can be applied to nematicidal pesticide preparations. Specific implementation manner:

[0015] The following further elaborates on the present invention in conjunction with specific embodiments.

[0016] Vemurafenib of the present invention is a commercially available product, and the vemurafenib solution of the present invention is obtained by dissolving vemurafenib in an aqueous solution of 5% wt DMSO.

[0017] Example 1:

[0018] This example relates to an experiment on the inhibitory effect of vemurafenib on serine / threonine protein kinase of Bursaphelenchus xylophilus (PWN-STK), specifically:

[0019] In the experiment on the inhibitory effect of vemurafenib on the serine / threonine protein kinase of Bursaphelenchus xylophilus, the gene stk encoding the serine / threonine protein kinase of Bursaphelenchus xylophilus (WormBase ID: BXYJ5.030106300) was ligated with the expression vector pET-15b to construct a recombinant expression vector, and the recombinant expression vector was transformed into Escherichia coli Rosetta(DE3) to construct an engineered bacterium. The IPTG induction method was used to induce the overexpression of PWN-STK in the engineered bacterium. After collecting the cells of the engineered bacterium, the cells were ultrasonically disrupted, and the cell lysate was centrifuged at 12,000 rpm and 4 °C for 20 minutes, and the supernatant was retained. The supernatant was purified by nickel ion affinity chromatography to obtain recombinant PWN-STK. In the experimental group, vemurafenib solutions with different final concentrations (50 nmol / L–125 nmol / L) were added to the recombinant PWN-STK in advance. After mixing evenly, the mixture was pre-treated in a 25 °C water bath for 10 minutes, and then centrifuged at 12,000 rpm for 2 minutes to retain the supernatant as the test enzyme solution for preparing the reaction system; under the same conditions, in the control group, the recombinant PWN-STK enzyme solution untreated with the vemurafenib solution was used as the test enzyme solution to prepare the reaction system. The reaction system (2 mL) for the determination of serine / threonine protein kinase activity: 20 mmol / L Tris-Cl (pH 7.5), 5 mmol / L MgCl2, 300 μg / mL bovine serum albumin, 2.67 mmol / L phosphoenolpyruvate, 70.94 μmol / L reduced coenzyme I (NADH), 1.45 mmol / L ATP, 31 U / mL lactate dehydrogenase, 31 U / mL pyruvate kinase, and 113 μg / mL test enzyme solution. After mixing the reaction system evenly, the reaction was carried out in a 25 °C water bath for 10 minutes, and then the oxidation level of NADH was measured at a wavelength of 340 nm. Within 10 minutes, a decrease in the absorbance of NADH at a wavelength of 340 nm by 0.1 was defined as one enzyme activity unit (U), and the specific activity of the enzyme (U / mg) was defined as: the enzyme activity unit possessed by every 1 mg of enzyme protein. In this enzyme activity determination experiment, each reaction was repeated three times. The results of SPSS analysis of the experimental data showed that under the action of the 125 nmol / L vemurafenib solution, the specific activity of the recombinant PWN-STK enzyme in the experimental group decreased by about 70% compared with that in the control group, and the half inhibitory concentration of vemurafenib on the activity of the recombinant PWN-STK enzyme was 92 nmol / L. This indicates that vemurafenib has a very significant inhibitory effect on the activity of PWN-STK enzyme.

[0020] Example 2:

[0021] This example relates to the experiment on the effects of vemurafenib on the movement and reproduction of Bursaphelenchus xylophilus and the treatment effect on pine wilt disease, specifically as follows:

[0022] 1. In the experiment on the effects of vemurafenib on the movement and reproduction of Bursaphelenchus xylophilus, the concentration of the vemurafenib solution in the experimental group was set at 50 μmol / L. 200 Bursaphelenchus xylophilus were added to the vemurafenib solution and incubated in the dark at 25°C for 48 hours. Under the same conditions, Bursaphelenchus xylophilus treated with an equal volume of sterile water or a 5 wt% DMSO (dimethyl sulfoxide) aqueous solution were regarded as the blank control group and the negative control group, with three biological replicates for each treatment. The experimental results are as follows:

[0023] (1) Effects of vemurafenib on the movement of Bursaphelenchus xylophilus: 15 nematodes were randomly selected from each group and the number of head swings per minute was counted under a microscope. The results showed that the number of head swings per minute of the nematodes in the vemurafenib treatment group was 17.07, while the number of swings of the nematodes in the blank control group and the negative control group was 37.81 and 34.93 times. This indicates that the motility of the nematodes in the vemurafenib treatment group was significantly weaker than that of the control group nematodes.

[0024] (2) Effects of vemurafenib on the reproduction of Bursaphelenchus xylophilus: 200 treated Bursaphelenchus xylophilus were inoculated onto a PDA plate covered with Botrytis cinerea mycelia. After dark incubation at 25°C for 8 days, the nematodes were separated by the Baermann funnel method, and the number of nematodes was observed and counted under a stereomicroscope. The results showed that the numbers of Bursaphelenchus xylophilus in the experimental group, the blank control group, and the negative control group were 2500, 5000, and 4800 respectively. The number of nematodes in the experimental group was only about 50% of that in the control group, indicating that vemurafenib can significantly inhibit the reproductive ability of Bursaphelenchus xylophilus.

[0025] 2. Experiment on the therapeutic effect of vemurafenib on pine wilt disease: A 10 μL suspension containing 200 J4-stage Bursaphelenchus xylophilus was inoculated into the wounds of 30-day-old Pinus thunbergii seedlings. 24 hours later, 10 μL of a vemurafenib solution (concentration of 0.050 mmol / L or 0.100 mmol / L) was inoculated again at the wound site of the Pinus thunbergii seedlings for treatment, serving as the experimental group; under the same conditions, Pinus thunbergii seedlings inoculated with an equal volume of sterile water or a 5 wt% DMSO aqueous solution again were used as the control group. The wilting symptoms of the Pinus thunbergii seedlings in different treatment groups were observed regularly. The results showed that 45 days after the second inoculation, the Pinus thunbergii seedlings in the control group died completely, while the Pinus thunbergii seedlings in the experimental group treated with vemurafenib did not show any wilting symptoms. This shows that a lower concentration of vemurafenib solution has a good therapeutic effect on pine wilt disease.

[0026] Example 3:

[0027] This example relates to an experiment for determining the nematocidal activity of vemurafenib against Bursaphelenchus xylophilus, specifically:

[0028] Approximately 100 pine wood nematodes were separately added to 96-well plates containing vemurafenib solutions with concentrations of 10, 20, 30, 40, and 50 μmol / L, respectively, and then they were placed in a dark environment at 25 °C for continued cultivation. At 24 hours, 48 hours, and 72 hours after treatment, the mortality rates of pine wood nematodes were calculated separately through a stereomicroscope. Under the same conditions, pine wood nematodes treated with a 5 wt% DMSO aqueous solution were used as the control group. The corrected mortality rate of pine wood nematodes was calculated using the following formula:

[0029]

[0030] The experimental results showed that at 24 hours, the corrected mortality rates of pine wood nematodes treated with vemurafenib solutions at concentrations of 10, 20, 30, 40, and 50 μmol / L were 18.98%, 25.82%, 34.42%, 39.80%, and 45.9%, respectively, while at this time, there was basically no nematode death in the control group. Through analysis with SPSS 22 software, the median lethal concentrations (LC50) of vemurafenib for pine wood nematodes at 24 hours, 48 hours, and 72 hours were 66.74 μmol / L, 35.38 μmol / L, and 20.20 μmol / L, respectively.

Claims

1. Use of vemurafenib in killing Bursaphelenchus xylophilus, characterized in that, The median lethal concentration of vemurafenib against Bursaphelenchus xylophilus within 24 hours was 66.74 μmol / L.

2. The application of vemurafenib in killing Bursaphelenchus xylophilus according to claim 1, wherein Vemurafenib can effectively inhibit the activity of serine / threonine protein kinase of Bursaphelenchus xylophilus and kill Bursaphelenchus xylophilus.

3. The use of vemurafenib according to claim 1 in killing Bursaphelenchus xylophilus, characterized in that, Vemurafenib can effectively inhibit the movement, reproduction and development of Bursaphelenchus xylophilus and kill Bursaphelenchus xylophilus.

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