Use of indolocarbazole compounds as chitin synthase inhibitors

Abstract

The application of indole carbazole compounds as chitin synthase inhibitors belongs to the technical field of pesticides. In order to solve the technical problems of the current agricultural fungicide field, such as the failure of traditional agents, the single chemical type of existing chitin synthase inhibitors, etc., the present application takes chitin synthase PsChs1 as a target, and finds that indole carbazole compounds and all have the effect of inhibiting chitin synthase, can be used as potential lead compounds of antifungal or oomycete drugs, realize agricultural application, and have the value of further modification.

Description

Technical Field

[0001] This invention belongs to the field of pesticide technology, specifically relating to the application of indolecarbazole compounds as chitin synthase inhibitors. Background Technology

[0002] The problem of fungal and oomycete resistance is becoming increasingly serious, with resistant strains spreading rapidly worldwide. As pathogens develop greater resistance to fungicides with traditional mechanisms of action, such as azoles and polyenes, the development of antifungal drugs with novel mechanisms of action has become an urgent need for industry and academia. This need is particularly prominent in agricultural production; for example, in the control of soybean root rot, *Phytophthora sojae* resistant to commonly used agents such as metalaxyl and fluoxetine has been detected in the field. Phytophthora sojae The presence of certain strains of *Phytophthora soyba* leads to a significant decrease in the effectiveness of traditional pesticides. Therefore, developing green and highly efficient fungicides based on novel molecular targets is of great significance for controlling important pathogens such as *Phytophthora soyba*.

[0003] In the development of antifungal drugs and pesticides, chitin synthase has long been considered a highly promising target. Chitin is a key structural component of the cell walls of most fungi and oomycetes, synthesized by chitin synthase. This enzyme is responsible for polymerizing the substrate uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) and exporting it extracellularly to form chitin microfibrils, thereby constructing and maintaining the integrity of the cell wall. Since chitin is not present in plants and mammals, inhibitors designed targeting this site are theoretically highly selective, effectively killing pathogens while remaining safe for the host and the environment; therefore, their research and development have attracted much attention. It is worth noting that chitin synthases are also widely found in arthropods (such as insects and mites) and mollusks (such as snails and slugs), and are important synthases for their exoskeleton or shell structures (Merzendorfer H. The cellular basis of chitin synthesis in fungi and insects: common principles and differences[J]. European journal of cell biology, 2011, 90(9):759-769.). Therefore, research on inhibitors targeting this site also has potential application value in the development of novel, selective insecticides and molluscicides.

[0004] Among numerous chitin synthases, PsChs1, derived from *Phytophthora sojae*, stands out as a representative target due to its clear, crucial, and unique biological functions. This enzyme catalyzes substrate polymerization to form chitin filaments, providing structural support for the oomycete cell wall and maintaining its integrity. Studies have confirmed that inhibiting PsChs1 activity directly blocks chitin biosynthesis, leading to cell wall damage and significantly reducing the pathogen's infectivity and viability. Therefore, PsChs1 is a key target with a well-defined mechanism and directly related to pathogenicity.

[0005] However, the development of agricultural fungicides currently faces severe challenges due to a lack of targets and structural homogeneity, which is one of the important reasons for the frequent occurrence of drug resistance in pathogens. PsChs1, as an important member of the chitin synthase family, has an active site that is somewhat conserved in pathogen populations. Furthermore, higher plants and mammals lack a chitin synthesis system, providing a structural basis for achieving highly selective inhibition. Although natural products such as nicotinic acid and polyoxin have been reported to inhibit PsChs1 through competitive substrate binding, these known inhibitors are mostly nucleoside peptide structures, with limited room for chemical scaffold modification. They are also susceptible to environmental influences in the field, exhibiting insufficient stability and persistence. Currently, commercially available agents targeting PsChs1 / oomycete chitin synthases and possessing novel small-molecule scaffolds are still relatively limited.

[0006] In recent years, the structural analysis and functional studies of PsChs1 have provided an important theoretical foundation for the development of chitin synthase inhibitors. Screening and rationally optimizing lead compounds targeting this type of target not only helps overcome existing resistance bottlenecks but may also provide new molecular mechanisms and application pathways for disease control. Exploring small-molecule inhibitors with novel chemical skeletons, greater optimization potential, and suitability for field application is of significant research and application value for expanding the chemical diversity and application potential of chitin synthase inhibitors. Summary of the Invention

[0007] To address the current technical challenges in the field of agricultural fungicides, such as the ineffectiveness of traditional agents and the limited chemical types of existing chitin synthase inhibitors, this invention targets chitin synthase PsChs1. Through screening, it was discovered that indolecarbazole compounds can inhibit chitin synthase PsChs1 and can serve as lead compounds for potential antifungal or oomycete drugs, enabling agricultural applications and possessing further potential for modification.

[0008] To solve the above-mentioned technical problems and achieve the corresponding technical effects, the present invention provides the following technical solution: The first object of this invention is to provide the use of indolecarbazole compounds as chitin synthase inhibitors, the structural formula of which is as follows: or As shown.

[0009] In one embodiment of the present invention, the chitin synthase is PsChs1.

[0010] In one embodiment of the present invention, the effective concentration of the indolecarbazole compound is 20 μM-50 μM.

[0011] A second objective of this invention is to provide the application of indolecarbazole compounds as active ingredients in the preparation of pesticide fungicides, wherein the structural formula of the indolecarbazole compounds is as follows: or As shown, the bactericide is used to kill fungi or oomycetes containing chitin synthase.

[0012] In one embodiment of the present invention, the chitin synthase is PsChs1.

[0013] A third object of the present invention is to provide the application of indolecarbazole compounds as active ingredients in the preparation of insecticides, wherein the structural formula of the indolecarbazole compounds is as follows: or As shown, the insecticide exerts its killing effect by inhibiting the chitin synthase of pests, which are arthropods or mollusks.

[0014] In one embodiment of the present invention, the chitin synthase is PsChs1.

[0015] The beneficial effects of this invention are: This invention targets chitin synthase PsChs1. Through molecular docking simulation screening, it was discovered that compounds Liu-DB-1-3 and Liu-DB-1-9 can occupy the chitin oligosaccharide transport channel of PsChs1, hindering the normal efflux of nascent chitin chains and thus interfering with the continuous catalytic process of PsChs1. This elucidates the inhibitory mechanism of compounds Liu-DB-1-3 and Liu-DB-1-9 on chitin synthase PsChs1. Furthermore, in vitro assays of PsChs1 inhibitory activity revealed that both compounds exhibited significant PsChs1 inhibitory activity, with inhibition rates exceeding 95% at a concentration of 20 μmol / L and 100% at a concentration of 50 μmol / L. Furthermore, enzyme kinetic analysis showed that the Ki values ​​of Liu-DB-1-3 and Liu-DB-1-9 were 0.26±0.06 μmol / L and 0.97±0.04 μmol / L, respectively, indicating that these two natural products have a strong affinity for PsChs1. The experiments of this invention confirm that compounds Liu-DB-1-3 and Liu-DB-1-9 can serve as lead compounds for potential antifungal or oomycete drugs, enabling agricultural applications. This invention enriches the chemical types of chitin synthase inhibitors, and subsequent research can use Liu-DB-1-3 and Liu-DB-1-9 as a starting point to develop novel antibacterial agents with stronger antibacterial efficacy. Attached Figure Description

[0016] Figure 1 The structural formula of compound Liu-DB-1-3; Figure 2 The structural formula of compound Liu-DB-1-9; Figure 3 This is a schematic diagram of the molecular docking between compound Liu-DB-1-3 and chitin synthase PsChs1. Figure 4 This is a schematic diagram of the molecular docking between compound Liu-DB-1-9 and chitin synthase PsChs1. Figure 5 The graph shows the results of evaluating the inhibitory efficacy of compound Liu-DB-1-3 against chitin synthase PsChs1. Figure 6 The figure shows the results of evaluating the inhibitory efficacy of compound Liu-DB-1-9 against chitin synthase PsChs1. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be noted that the embodiments mentioned below are only for explaining the invention and are not intended to limit the scope of the invention. The embodiments mentioned below are only some embodiments of the invention, not all embodiments. Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the objectives of the invention. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of this invention to realize and apply the technology of this invention. In the art, embodiments obtained by other those skilled in the art without creative effort are all protected by this invention.

[0018] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials, reagents, culture media and instruments used are conventional materials, reagents, culture media and instruments in the art, which can be obtained by those skilled in the art through commercial channels.

[0019] Example 1: Screening of natural product inhibitors of chitin synthase PsChs1 I. Virtual Screening of Chitin Synthase PsChs1 Inhibitors Based on the inventors' previous analysis of the three-dimensional structure of the chitin synthase PsChs1 from Phytophthora soybeanis, this invention conducted a virtual screening of PsChs1 inhibitors using a natural product compound library (approximately 400 compounds). The specific method is as follows: Based on the crystal structure information of PsChs1 (PDB accession code: 7WJO, ligand-bound state), a virtual screening study targeting chitin synthase was conducted. Protein structures were obtained from the Protein Data Bank database, standardized, and their catalytic core and substrate-binding channel regions were identified based on literature reports. Key residues included Tyr433, Val452, Pro454, Trp539, Asp496, and Glu495. Receptor models were constructed using the catalytic pocket region as target binding sites for virtual screening. A self-built natural product compound library was structurally standardized and conformationally generated, and then uniformly saved in SDF format.

[0020] Virtual screening and molecular docking analysis were performed using the deep learning-based Uni-Mol platform. By modeling and predicting the three-dimensional structure of the protein-ligand pair, the relative binding tendency of compounds in the PsChs1 active site was evaluated, and candidate compounds were ranked and screened based on prediction scores. The screening results were further analyzed by considering the conformational rationality and the interaction patterns with key catalytic residues. Compounds with good spatial matching and stable interactions with the PsChs1 active site were selected for subsequent in vitro enzyme activity verification experiments. The docking results were visualized using PyMOL to aid in interpreting the screening results and guide experimental design.

[0021] Using the virtual screening method described above, a total of 14 candidate compounds were obtained, among which compound Liu-DB-1-3 (structural formula as shown) was selected. Figure 1 (as shown) and Liu-DB-1-9 (structure as shown) Figure 2 The structures of the two compounds (shown) are highly similar, both belonging to the indolecarbazole class. Compared to Liu-DB-1-9, Liu-DB-1-3 has an additional chlorine atom substituent on the left side of the molecule. Docking results show that the two compounds are highly similar in their overall skeleton, both composed of multiple cyclic structural units, exhibiting strong conformational rigidity and similar binding modes. Both compounds are embedded within the PsChs1 product transport channel, stably bound by a hydrophobic pocket formed by multiple transmembrane α-helices. The aromatic ring forms a significant π-π stacking interaction with Trp539 and Tyr433, while the amino group on the molecule is located in a relatively hydrophilic region within the channel, which enhances binding stability. The additional chlorine atom introduced into the Liu-DB-1-3 molecule causes a slight conformational shift in its aromatic ring, thereby weakening its π-π stacking interaction with Tyr433. This conformational difference may reduce its binding stability in the transport channel, leading to a decrease in inhibitory activity. Figure 3 and Figure 4 ).

[0022] In summary, the docking model shows that compounds Liu-DB-1-3 and Liu-DB-1-9 can both occupy the chitin oligosaccharide transport channel of PsChs1, hindering the normal efflux of newly formed chitin chains, thereby interfering with the continuous catalytic process of PsChs1, and providing a structural explanation for its inhibition mechanism.

[0023] II. Determination of PsChs1 inhibitory activity of candidate compounds The in vitro inhibitory activity of chitin synthase PsChs1 was determined in the 14 candidate compounds obtained above at two concentrations of 50 μmol / L and 20 μmol / L.

[0024] The in vitro assay of the inhibitory activity of chitin synthase PsChs1 was performed using a substrate transformation product-based detection method. Recombinant PsChs1 protein was obtained through heterologous expression and purified by affinity chromatography and molecular sieve chromatography before being dissolved in a buffered salt and non-ionic detergent storage system for later use. The enzymatic reaction was carried out using UDP-GlcNAc as the substrate in a system containing Tris-HCl buffer, inorganic salts, and divalent metal ions. The reaction temperature and time were controlled to ensure the reaction was in its initial initiation phase. After the enzymatic reaction, the generated chitin or its oligomers were specifically captured using wheat germ lectin (WGA), and the product amount was detected using an enzyme-labeled colorimetric system. A standard curve was established with standards to convert the detection signal into product yield, thereby calculating the PsChs1 enzyme activity. The specific assay method is as follows: ① Preparation of recombinant PsChs1 protein: The recombinant PsChs1 protein was prepared using a eukaryotic expression system, and the specific steps are as follows: Construction of the recombinant expression vector: The PsChs1 gene sequence was obtained from the NCBI database (accession number: XM_009525864.1), and after codon optimization, it was artificially synthesized and cloned into the eukaryotic expression vector pcDNA3.1. To facilitate subsequent purification and detection, a TEV protease cleavage site and a Twin-Strep II affinity tag were sequentially fused to the C-terminus of the target gene. The constructed recombinant plasmid was verified to be correct by sequencing before use.

[0025] Plasmid amplification and preparation: The recombinant plasmid was transformed into competent *E. coli* Trelief@5α, plated on LB agar containing ampicillin, and cultured at 37°C to obtain single colonies. Single colonies were picked and inoculated into LB liquid agar containing ampicillin for amplification. After confirmation by sequencing, glycerol culture was prepared and stored at -80°C. Following further amplification, high-purity plasmid DNA was extracted using an endotoxin-free plasmid extraction kit for later use.

[0026] Eukaryotic cell expression: HEK293F cells in suspension culture were used as the expression host and cultured with shaking at 37℃ and 5% CO2. The cell density reached approximately 2 × 10⁻⁶ cells / year. 2 Transfection was performed at a concentration of [number] cells / mL. The polyethyleneimine (PEI) transfection method was used, where plasmid DNA and PEI were mixed at a mass ratio of 1:3, allowed to stand at room temperature to form a complex, and then added to the cell culture system. Cells were collected 48-72 hours after transfection.

[0027] Cell collection and preservation: After transfection, cells were collected by low-speed centrifugation, washed with PBS, weighed wet, flash-frozen in liquid nitrogen, and stored at -80°C.

[0028] Protein purification: After cell lysis, recombinant PsChs1 protein with a Twin-Strep II tag was purified by Strep-Tactin affinity chromatography. The tag could be removed using TEV protease, and the protein purity was further improved by gel filtration chromatography. The purity and molecular weight of the obtained protein were identified by SDS-PAGE or Western blot, yielding high-purity recombinant PsChs1 protein.

[0029] ② Determination of the inhibitory activity of chitin synthase PsChs1 This invention employs an enzyme-linked colorimetric method based on lectin recognition to detect the chitin synthesis activity of recombinant PsChs1. The specific steps are as follows: ELISA plate coating and blocking: The 96-well ELISA plates were coated with a coating solution containing lectin to immobilize chitin-binding molecules on the plate surface. After incubation at room temperature, the coating solution was discarded and the plates were washed. Subsequently, a blocking solution containing a protein blocking agent was added for blocking to reduce non-specific adsorption. After blocking, the plates can be used directly or stored at low temperature for later use.

[0030] Construction of in vitro enzymatic reaction system: Recombinant PsChs1 protein, substrate UDP-GlcNAc, and divalent metal ions (Mn) were added to the enzyme-labeled wells. 2+ The reaction mixture was brought to a final volume with reaction buffer and incubated with shaking at a suitable temperature to allow the enzyme to catalyze the formation of chitin. After the reaction was complete, the reaction solution was discarded and the plate was washed to remove unbound substrate and impurities.

[0031] Product verification experiment: Chitinase (such as OfChi-h derived from Asian corn borer) was added to the reaction system to verify that the product was chitin.

[0032] Binding and Detection: After the reaction is complete and the wells are cleaned, a binding solution containing HRP-labeled lectin is added to each well to allow it to specifically bind to the generated chitin. After incubation, the wells are thoroughly washed to remove any unbound components.

[0033] Color development and detection: An enzymatic colorimetric reaction was carried out by adding a colorimetric substrate solution, and the absorbance value was continuously measured at a specific wavelength. The rate of change in absorbance reflects the amount of chitin produced, thereby characterizing the enzyme activity of PsChs1.

[0034] Standard curve establishment and quantitative analysis: A standard solution of natural chitin was prepared by acid dissolution, and a series of gradient standards were prepared after concentration calibration. The standards were added to pre-coated ELISA plates and treated with the same binding and colorimetric steps to plot a standard curve, thereby achieving quantitative analysis of chitin production.

[0035] The above methods can be used to systematically evaluate the catalytic activity, substrate kinetic parameters, metal ion dependence, and regulatory mechanisms of recombinant PsChs1 protein.

[0036] The results of the determination of the inhibitory activity of chitin synthase PsChs1 are shown in Table 1. Both compounds Liu-DB-1-3 and Liu-DB-1-9 showed significant PsChs1 inhibitory activity, with inhibition rates of over 95% at a concentration of 20 μmol / L.

[0037] Table 1. Inhibition rate of PsChs1 by 14 candidate compounds

[0038] Example 2: Evaluation of the inhibitory efficacy of compounds Liu-DB-1-3 and Liu-DB-1-9 against PsChs1 Based on the PsChs1 enzyme activity assay system in Example 1, the inhibitory efficacy of Liu-DB-1-3 and Liu-DB-1-9 on PsChs1 was evaluated.

[0039] Different concentrations of Liu-DB-1-3 and Liu-DB-1-9 were pre-incubated with PsChs1 enzyme solution in a reaction buffer system to ensure full binding of the compounds to the enzyme. Substrate was then added to initiate the enzymatic reaction. After the reaction was complete, the amount of product generated was measured according to enzyme activity assays and compared with a control group without the compounds. The inhibition rates of Liu-DB-1-3 and Liu-DB-1-9 on PsChs1 were calculated.

[0040] The effects of compounds Liu-DB-1-3 and Liu-DB-1-9 on the activity of PsChs1 enzyme were determined under different substrate concentrations and at multiple concentration gradients. The experimental data were fitted using enzyme kinetic analysis to obtain the inhibition constant Ki of the inhibitors. This method can be used to quantitatively evaluate the inhibitory activity of compounds Liu-DB-1-3 and Liu-DB-1-9 on chitin synthase.

[0041] Enzyme kinetic analysis showed that the Ki values ​​of Liu-DB-1-3 and Liu-DB-1-9 were 0.26±0.06 μmol / L and 0.97±0.04 μmol / L, respectively (see [link to enzyme kinetic analysis]). Figure 5 and Figure 6 This indicates that these two natural products have a strong affinity for PsChs1.

[0042] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of indolecarbazole compounds as chitin synthase inhibitors, characterized in that, The structural formula of the indolecarbazole compound is as follows: or As shown; the chitin synthase is PsChs1.

2. The application according to claim 1, characterized in that, The effective concentration of the indolecarbazole compound is 20 μM-50 μM.

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