Inhibitory compounds targeting homogentisate solubilase and uses thereof

Abstract

The present application relates to the field of agricultural chemistry and environmental protection, in particular to a kind of with homogentisic acid solanonyl transferase (HST) as molecular target inhibitory compound and its application.A kind of with homogentisic acid solanonyl transferase as molecular target inhibitory compound, it is characterized in that: compound is the compound shown in the following structure, in the formula, R1, R2, R3 And R4 Respectively Br.The compound has significant inhibitory effect on phytoplankton growth at low concentration, and shows strong herbicidal effect on weeds such as eustreptosin and portulaca oleracea.The AlphaFold prediction analysis further confirms that these compounds inhibit the synthesis of plastoquinone by binding with HST, thereby effectively interfering with photosynthesis.The present application can be applied to algae and weed control in agriculture and aquaculture, and has wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agrochemistry and environmental protection, in particular to an inhibitory compound with homogentisate solanesyltransferase (HST) as a molecular target and its application. BACKGROUND

[0002] Homogentisate solanesyltransferase (HST) is a new potential molecular target of herbicides, which has important application value in improving killing efficiency and dealing with weeds with strong tolerance to existing chemicals. HST plays a key role in plant biosynthesis, catalyzing the isopentenylation and decarboxylation of homogentisic acid to generate 2-methyl-6-solanyl-1,4-benzoquinol, which is the first intermediate in the biosynthesis of plastoquinone. As an important compound in the photosynthetic electron transport chain, plastoquinone directly affects photosynthesis in plants. By inhibiting the activity of HST, the synthesis of plastoquinone can be effectively blocked, thereby interfering with the normal progress of photosynthesis. This inhibition not only causes the growth of plants to stop, but also causes the albino phenomenon, ultimately leading to their death. Therefore, developing a compound that inhibits HST provides a new strategy for the control of algae and weeds in the environment such as agriculture and aquaculture. SUMMARY

[0003] The present application aims to provide an inhibitory compound with homogentisate solanesyltransferase (HST) as a molecular target and its application.

[0004] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0005] An inhibitory compound with homogentisate solanesyltransferase as a molecular target, the compound being a compound as shown in the following structure,

[0006]

[0007] In the formula, R1, R2, R3 and R4 are Br, respectively.

[0008] An application of the inhibitory compound with homogentisate solanesyltransferase as a molecular target, the compound being applied in inhibiting homogentisate solanesyltransferase.

[0009] An application of the inhibitory compound with homogentisate solanesyltransferase as a molecular target, the compound being applied in preparing a plant and phytoplankton inhibitor.

[0010] The compound is applied in preparing a growth inhibitor for Echinochloa crus-galli, Portulaca oleracea and diatoms.

[0011] An inhibitor of homogentisate solanesyltransferase, said inhibitor comprising the compound.

[0012] The concentration of the solution (aqueous solution) of the inhibiting compound is 3-5 mg / L.

[0013] Use of the inhibitor of homogentisate solanesyltransferase, said inhibitor for inhibiting the growth of plants and phytoplankton.

[0014] Use of the inhibitor, said inhibitor being sprayed to the plants to be treated, the minimum application amount being 50 mg / L.

[0015] The present application has the following advantages:

[0016] The biphenyl compound of the present application inhibits the synthesis of plastoquinone by binding with HST, thereby hindering the process of photosynthetic electron transport, effectively interfering with photosynthesis. The compound has a significant growth inhibition effect on phytoplankton at a low concentration, and exhibits a strong herbicidal effect on weeds such as Echinochloa crus-galli and Portulaca oleracea. Furthermore, the present application can be applied to the control of algae and weeds in agriculture and aquaculture, and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The compound provided by the embodiment of the present application and the structure prediction of homogentisate solanesyltransferase (HST) and its substrate docking effect diagram are shown in the following figure; wherein, A is the three-dimensional structure of HST constructed using ColabFold; B is the docking study by AutoDock, which shows that homogentisic acid binds with HST. The amino acids of the active site of HST are as follows: LEU is leucine, GLY is glycine, VAL is valine, and TYR is tyrosine. Homogentisic acid docks inside the active site and interacts with these amino acids in different ways; C. shows the binding of three structurally similar molecules (i.e. TBB-BP, 2,2'-dihydroxybiphenyl and 3,3',5,5'-tetrabromobiphenyl) with HST.

[0018] Figure 2 The inhibition rate of compound TBB-BP of different concentrations provided by the embodiment of the present application over time after treating phytoplankton

[0019] Figure 3 The inhibition rate of compound 2,2'-dihydroxybiphenyl of different concentrations provided by the embodiment of the present application over time after treating phytoplankton

[0020] Figure 4 The inhibition rate of compound 3,3',5,5'-tetrabromobiphenyl of different concentrations provided by the embodiment of the present application over time after treating phytoplankton

[0021] Figure 5 The maximum photosynthetic efficiency Fv / Fm response curves of different concentrations of compound TBB-BP provided by the embodiments of the present application over time after treating phytoplankton.

[0022] Figure 6 The plant growth conditions of different concentrations of compound TBB-BP provided by the embodiments of the present application after treating Portulaca oleracea and Echinochloa crus-galli. DETAILED DESCRIPTION

[0023] The specific embodiments of the present application are further described in conjunction with examples, and it should be noted that the specific embodiments described herein are only for the purpose of illustrating and explaining the present application, and are not limited to the present application.

[0024] In the following examples, compound TBB-BP is used, which can be obtained by preparation as described in CN108684665B. It can also be prepared according to conventional chemical synthesis.

[0025] Example 1

[0026] Interaction of compound with homogentisate solubilizing enzyme

[0027] The full-length of the homogentisate solubilizing enzyme HST coding gene of plants was obtained. The AlphaFold prediction technology was used to analyze the structure of the interaction between the compound (TBB-BP) shown in the structural formula of the present application and the HST protein.

[0028] The homogentisate solubilizing enzyme HST gene sequence (NCBI reference sequence: XM_002178060.1) was obtained from plant genome data, and its complete sequence was determined using Takara SMARTer RACE 5' / 3' kit (TaKaRa). The complete open reading frame of HST is 1164 bp long, including a 22-amino-acid hypothetical transit peptide sequence. The three-dimensional structure of the HST sequence was constructed using ColabFold, and the normal substrate of HST, homogentisic acid (the substrate of HST), and the present compound were docked to HST using AutoDock. In order to ensure the accuracy of the docking prediction, two molecules similar in structure to the present compound (i.e., 2,2'-dihydroxybiphenyl and 3,3',5,5'-tetrabromobiphenyl) were used as controls, and their binding sites with HST were determined according to the docking method of the present compound. All structures were visualized using PyMOL (http: / / www.pymol.org) (see Figure 1 A-C).

[0029] From Figure 1As can be seen from Figures A and B, the present compound TBB-BP has the same active site as homogentisic acid. The LEU_203, TYR_207, LEU_243 and GLY_246 regions of HST are the potential active sites of homogentisic acid, and these amino acids interact with homogentisic acid through hydrogen bonds and hydrophobic bonds. The present compound TBB-BP can interact with three of these amino acids (i.e. LEU_203, LEU_243 and GLY_246) through two halogen bonds, two hydrophobic bonds and one hydrogen bond, and has similar site competition ability as homogentisic acid. The results show that 4-BP can compete with the HST active site of homogentisic acid, thereby inhibiting the subsequent biosynthesis of plastoquinone-9.

[0030] To further verify the target site of TBB-BP, two molecules similar in structure to the present compound TBB-BP, i.e. 2,2'-dihydroxybiphenyl and 3,3',5,5'-tetrabromobiphenyl, were used to perform the same analysis. Although they can also interact with the above-mentioned site, they only bind to two amino acids. 2,2'-dihydroxybiphenyl interacts with LEU_203 and TYR_207 through hydrophobic bonds and hydrogen bonds, while 3,3',5,5'-tetrabromobiphenyl interacts with LEU_203 and LEU_243 through hydrophobic bonds and halogen bonds. It is the weakened interaction ability of 2,2'-dihydroxybiphenyl and 3,3',5,5'-tetrabromobiphenyl with the homogentisic acid binding site that makes them unable to sufficiently compete with homogentisic acid, and thus unable to inhibit the growth of phytoplankton. These data demonstrate that TBB-BP can compete with the HST active site of homogentisic acid.

[0031] Example 2

[0032] The inhibitory effect of these three structurally similar molecules on phytoplankton was further studied by adding different concentrations of TBB-BP, 2,2'-dihydroxybiphenyl and 3,3',5,5'-tetrabromobiphenyl prepared in water to the culture of phytoplankton Phaeodactylum tricornutum CCMP 2561 in the exponential growth phase, and observing the growth dynamics of the phytoplankton, with the phytoplankton culture without the addition of these compounds as a control. The change in fluorescence intensity (excitation and emission wavelengths of 440 nm and 680 nm, respectively) in the culture system was measured every day, and the inhibition rate was calculated, i.e. inhibition rate = (FC-FT) / FC x 100%, where FT and FC represent the fluorescence intensity of the phytoplankton culture with and without the addition of the compound, respectively.

[0033] From the results shown in Figure C, it can be seen that the present compound TBB-BP has a stronger inhibitory effect on the growth of phytoplankton than 2,2'-dihydroxybiphenyl and 3,3',5,5'-tetrabromobiphenyl. This is because TBB-BP can interact with three amino acids in the HST active site of homogentisic acid through two halogen bonds, two hydrophobic bonds and one hydrogen bond, while 2,2'-dihydroxybiphenyl and 3,3',5,5'-tetrabromobiphenyl can only interact with two amino acids. Therefore, TBB-BP has a stronger inhibitory effect on the growth of phytoplankton than 2,2'-dihydroxybiphenyl and 3,3',5,5'-tetrabromobiphenyl. Figure 2It is evident that at concentrations of 0-0.125 mg / L, the compound TBB-BP showed no significant inhibitory effect on phytoplankton. However, at concentrations above 0.5-5 mg / L, the inhibition rate exceeded 80% after 72 hours, indicating that high concentrations of the compound significantly inhibited phytoplankton growth. At a concentration of 0.25 mg / L, although the compound also inhibited phytoplankton growth, the inhibition rate after 72 hours was only 40%. In conclusion, certain concentrations of TBB-BP can inhibit phytoplankton growth.

[0034] Depend on Figure 3 It is evident that within the tested concentration range (0-13.5 mg / L), compound 2,2'-dihydroxybiphenyl did not significantly inhibit phytoplankton growth, indicating that compound 2,2'-dihydroxybiphenyl does not have an inhibitory effect on phytoplankton growth.

[0035] Depend on Figure 4 It is evident that within the tested concentration range (0-10 mg / L), compound 3,3',5,5'-tetrabromobiphenyl did not significantly inhibit phytoplankton growth, indicating that compound 3,3',5,5'-tetrabromobiphenyl does not have an inhibitory effect on phytoplankton growth.

[0036] Depend on Figure 1 As can be seen from Example 1 above, the interaction between TBB-BP and similar compounds with homosuccinate solanesyltransferase (HST) is relatively weak. This weak interaction of similar compounds results in a weaker effect of 2,2'-dihydroxybiphenyl and 3,3',5,5'-tetrabromobiphenyl on the homosuccinate binding site. Consequently, when they interact with phytoplankton HST and homosuccinate, they cannot occupy the homosuccinate binding site, thus failing to inhibit HST function. Consequently, phytoplankton growth is not inhibited. However, the binding ability of the compound of this invention to the homosuccinate binding site is comparable to that of homosuccinate to its binding site. It can compete with the HST active site of homosuccinate, thereby inhibiting HST function and thus inhibiting phytoplankton growth.

[0037] Simultaneously, the inhibitory effect of the compound of this invention on phytoplankton was further tested, with the common phytoplankton *Phaeodactylum tricornutum* CCMP 2561 selected as the test plant. The cells were cultured in f / 2 medium at 20°C, with a light intensity of 60 μmol m⁻² s⁻¹ and a light-to-dark cycle of 12 h:12 h. Phytoplankton in the logarithmic growth phase were used as the treatment. Different concentrations of the compound TBB-BP were added to the above-mentioned treatments, with final concentrations in the culture medium of 0 mg / L, 0.25 mg / L, 0.5 mg / L, 1 mg / L, 1.8 mg / L, 2.5 mg / L, 5 mg / L, and 10 mg / L, respectively. A control group was prepared using DMSO as the solvent. Cells were then cultured under the above conditions, and the cell count and photosynthetic parameter Fv / Fm were measured at 0 h, 24 h, 48 h, and 72 h. Cells were fixed with glutaraldehyde and counted using flow cytometry. Phytoplankton photosynthesis was analyzed and evaluated using chlorophyll fluorescence parameters. Chlorophyll fluorescence Fv / Fm was measured using a WATER-PAM chlorophyll fluorometer. The maximum photosynthetic efficiency of photosynthetic system II, Fv / Fm = (Fm - F0) / Fm, was measured after 20 minutes of dark adaptation (see [reference]). Figure 5 ).

[0038] Depend on Figure 5 It can be seen that at concentrations below 1 mg / L, Fv / Fm showed no significant difference compared to the control group. However, at concentrations of 3.0 mg / L and 5.0 mg / L, Fv / Fm decreased to 0, indicating that high concentrations of the compound significantly inhibited photosynthesis. At concentrations of 1.0 mg / L and 1.8 mg / L, although the Fv / Fm values ​​decreased, they rebounded after 8 hours, and showed no significant difference compared to the control group at 48 hours and 72 hours.

[0039] In summary, a certain concentration of TBB-BP can compete with the HST active site of homogentisic acid, thereby inhibiting HST function. Since HST is a key protein in the plastoquinone pathway, an important compound in the electron transport chain of the photosynthetic system, inhibition of HST hinders plastoquinone synthesis, directly affecting photosynthetic electron transport in plants. This leads to a decrease in the photosynthetic parameter Fv / Fm to 0, impairing plant photosynthesis and consequently inhibiting phytoplankton growth.

[0040] Example 3

[0041] The inhibitory effect of compounds on weeds

[0042] The common weeds such as Echinochloa crus-galli and Portulaca oleracea were taken as the research objects to test the inhibition effect of the compound on weeds. The seeds of Echinochloa crus-galli and Portulaca oleracea were planted at room temperature, and were cultured for 4 days according to the conventional cultivation method. The experimental design included foliar spraying treatment, compound prepared by water, and the concentration was set as 1 mg / L, 5 mg / L and 50 mg / L, and the foliar spraying was carried out when the weeds grew from seeds to seedlings for 4 days. After spraying, the whitening degree and the growth condition of the plants were observed after 2 days, and the herbicidal effect of the compound was evaluated (see Figure 6 ).

[0043] It can be seen from Figure 6 that the compound can cause the yellowing of the leaf tip of the plant, the problem of the plant not being upright, and the growth of the plant being stagnant within 72 hours after spraying. The effect of foliar spraying is significant, indicating that the compound can more effectively enter the plant body through the leaves to play a role.

[0044] In summary, the compound of the present application can inhibit the activity of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme of the enzyme

Claims

1. An application of a compound with the structure shown below, characterized in that, The application of this compound in inhibiting urinary solanesyltransferase. In the formula, R1, R2, R3 and R4 are Br respectively.

2. The application of an inhibitor of urinary solanesyltransferase, characterized in that: The inhibitor contains a compound with the structure shown below. In the formula, R1, R2, R3 and R4 are Br; The inhibitor is used to inhibit phytoplankton growth; the concentration of the compound in the inhibitor is 3-5 mg / L.

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