An antibacterial small molecule compound targeting bacterial phospholipid synthetases

Abstract

The present application belongs to the technical field of pharmaceutical chemistry, and particularly relates to an antibacterial small-molecule compound targeting bacterial phospholipid synthetase. The present application provides an antibacterial intervention strategy based on a novel mechanism of action, taking the key enzyme PlsY of bacterial specific membrane phospholipid synthesis as a target, and cracking the cross-resistance problem caused by the concentration of existing antibacterial drug targets. The lead compound T1 obtained by screening has strong inhibitory activity on PlsY, can effectively inhibit pathogenic bacteria such as Streptococcus pneumoniae and Staphylococcus aureus, and has better selective toxicity because the human body has no PlsY homologous protein. The compound has a novel structure and can be optimized, and can be rationally designed through structure-activity relationship, and also has inhibitory effect on plant pathogenic bacteria. The present application opens up a new antibacterial pathway, provides differentiated candidate molecules and reserves for coping with multi-drug resistant infections, and has medical and agricultural potential, and has significant innovation and practicality.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemistry technology, specifically relating to an antibacterial small molecule compound that targets bacterial phospholipid synthase. Background Technology

[0002] In the healthcare field, the long-term and widespread use of antibiotics leads to bacterial resistance. Currently, the first-line antibacterial drugs relied upon in clinical practice are mainly β-lactams, quinolones, macrolides, and glycopeptides, whose mechanisms of action are mostly focused on limited, conserved physiological processes such as interfering with bacterial cell wall synthesis, inhibiting nucleic acid replication, and blocking protein synthesis. The concentration and high overlap of these targets allow bacteria to develop cross-resistance to multiple structurally different drugs through relatively limited adaptive evolutionary mechanisms such as producing drug-inactivating enzymes, modifying drug targets, overexpressing efflux pump systems, or reducing membrane permeability. The direct consequence of this phenomenon is a significant reduction in effective clinical treatment options for infections caused by drug-resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), and carbapenem-resistant Enterobacteriaceae (CRE), leading to a corresponding increase in treatment failure rates.

[0003] In agricultural production, various plant pathogens, such as *Ralstonia solanacearum*, *Streptomyces scabiei*, and *Clavibacter michiganensis* subsp. *michiganensis*, can cause corresponding bacterial diseases in plants. Currently, field disease control still heavily relies on traditional chemical antimicrobial agents such as copper-based formulations and streptomycin. Their long-term and frequent application inevitably leads to a series of problems, including soil and environmental residues, the continuous evolution of pathogen resistance in the field, and toxic effects on non-target organisms and ecosystems. Therefore, developing novel agricultural antimicrobial agents with entirely new mechanisms of action, low cross-resistance with existing agents, and greater environmental friendliness has become an urgent and clear direction for technological breakthroughs in this field.

[0004] Despite the aforementioned problems, the development pipeline for novel antimicrobial drugs still exhibits a significant lack of innovation. Looking back over the past few decades, the vast majority of new antimicrobial chemical entities approved for marketing globally are essentially derivatives of known drug skeletons or advantageous structures, chemically modified and optimized (e.g., next-generation cephalosporins, novel oxazolidinones). Truly first-in-class drugs targeting entirely new biochemical targets and possessing original, innovative chemical structures are extremely rare. This "derivative"-dominated R&D model, unable to fundamentally circumvent the widespread inherent resistance mechanisms and increasingly prevalent acquired resistance mechanisms in bacteria, cannot provide a long-term solution to address the rapidly evolving and diverse threat of drug resistance.

[0005] Therefore, exploring and validating novel drug targets specific to bacteria and essential for their survival and proliferation is considered a key strategy to break the current deadlock in antibacterial research and development and open up new pathways. The biosynthetic pathway of bacterial cell membrane phospholipids, especially its initiation steps, is highly conserved in bacteria but completely absent in humans and other eukaryotes, making it a highly attractive source of novel targets. Glycerol-3-phosphoacyltransferase (PlsY), which catalyzes the first step of the bacterial phospholipid synthesis pathway, is an enzyme essential for maintaining cell membrane integrity, homeostasis, and survival in many important Gram-positive pathogens (such as Streptococcus pneumoniae and Staphylococcus aureus) and some Gram-negative bacteria. Humans and higher eukaryotes use a completely different cytoplasmic acetyl-CoA-dependent pathway for phospholipid synthesis, and their genomes lack functional homologs of PlsY. This fundamental difference in biochemical pathways between species theoretically provides a clear window of opportunity for developing innovative antibacterial agents with high selectivity and low expected host toxicity.

[0006] Based on the above understanding, developing small-molecule inhibitors with solid structures and defined activities targeting the PlsY enzyme has become a research direction with both significant scientific value and promising applications. Selecting *Streptococcus pneumoniae* and its PlsY enzyme as an initial discovery and validation model is a reasonable research path. This bacterium is an important human pathogen, and the function and importance of its PlsY enzyme have been extensively studied. By conducting dual screening on the biochemical inhibitory activity of *Streptococcus pneumoniae* PlsY and its growth-inhibiting effect on the bacterium, it is hoped that lead compounds with substantial activity can be discovered. The resulting active compounds can then be further systematically evaluated for their inhibitory effects on other important human pathogenic Gram-positive bacteria, such as *Staphylococcus aureus* (including MRSA strains), thereby exploring their broad-spectrum antibacterial potential.

[0007] Furthermore, considering that PlsY homologs are also found in sexually active plant pathogens (such as Tomato Ulcer Bacterium), such inhibitors based on PlsY from Streptococcus pneumoniae also have the potential value of evaluating their inhibitory activity against pathogens of important crops, which also provides the possibility for the development of new agricultural antimicrobial agents.

[0008] However, despite the significant theoretical advantages of PlsY as an antibacterial target, its translation into solid inhibitors faces substantial obstacles and gaps in research. This field has long lacked a class of lead compounds—obtained based on a clear understanding of the pathogen and its PlsY target enzyme as a common direct screening basis, possessing potent inhibitory activity, well-defined antibacterial effects, and good optimizability. The absence of such high-quality solid molecules severely restricts systematic structure-activity relationship studies, medicinal chemistry optimization, and subsequent development of this target, preventing the enormous potential of the PlsY target from remaining at the academic conceptual level and failing to translate into realistic candidates for pharmaceutical or agricultural applications.

[0009] Therefore, developing a novel small molecule inhibitor that targets Streptococcus pneumoniae PlsY as a specific molecular target, uses the inhibition of the bacterium's growth as a phenotypic screening criterion, and obtains it through experimental verification is not only a key experimental verification of the drug-likeness of PlsY as a novel target, but also provides an innovative R&D path that is both substantial and clearly targeted in order to address the current drug resistance crisis in the clinical and agricultural fields. Summary of the Invention

[0010] To overcome the shortcomings of the existing technologies, and addressing the systemic technical bottlenecks caused by a narrow target spectrum, a lack of prevention and control methods, a lack of original innovation, and stagnation in the transformation of new targets, the present invention aims to provide a physical compound based on the novel target of PlsY, a key enzyme in bacterial phospholipid synthesis. The specific objectives are as follows: (1) The primary objective of this invention is to provide a small molecule compound with a clearly defined and novel chemical structure, named T1. This compound is studied using *Streptococcus pneumoniae*, an important human pathogen. The inhibition of the in vitro catalytic activity of the PlsY enzyme, essential for the survival of this bacterium, and the inhibition of in vitro bacterial growth are used as parallel and mutually verifiable initial screening and core optimization indicators. Through systematic experimental screening, followed by subsequent chemical synthesis and structural confirmation, the physical substance was finally obtained. Experimental results show that compound T1 can efficiently and specifically inhibit the biocatalytic function of *Streptococcus pneumoniae* PlsY, and its mechanism of action is completely different from all currently marketed or investigational clinical antibiotics and agricultural fungicides.

[0011] (2) A second objective of this invention is to provide the application of compound T1 in inhibiting human pathogenic bacteria. Based on the confirmed potent inhibitory effect of compound T1 against Streptococcus pneumoniae PlysY and its cells, this invention, through standardized in vitro antibacterial activity testing, confirms that compound T1 exhibits significant growth-inhibiting activity against various important Gram-positive pathogens, including Staphylococcus aureus (including methicillin-resistant MRSA strains) and Streptococcus pyogenes. This result provides direct experimental evidence for developing compound T1 as a novel therapeutic candidate drug with a novel mechanism of action against infections caused by multidrug-resistant Gram-positive bacteria.

[0012] (3) The third objective of this invention is to provide the application of compound T1 in inhibiting plant pathogenic bacteria. Given that PlsY homologs are also present in some plant pathogens, this invention experimentally verified that compound T1 also has an inhibitory effect on plant pathogens (such as *Streptomyces scabiei* and *Clavibactermichiganensis* subsp. *Michiganensis*). This provides a practical research direction and experimental support for developing compound T1 and its structurally optimized derivatives into novel agricultural fungicides with a completely new mechanism of action that can be used to control bacterial diseases in crops.

[0013] By achieving the three specific objectives mentioned above, this invention has, for the first time, successfully obtained a solid small molecule compound, T1, with *Streptococcus pneumoniae* PlsY as a clearly defined molecular target, and verified by both target inhibition activity and phenotypic antibacterial activity. This achievement not only realizes the crucial experimental transformation and verification of the high-value novel target PlsY from a theoretical concept to a solid inhibitor with clearly defined activity, but also directly addresses and attempts to overcome the research and development dilemma in existing technologies caused by the lack of novel antibacterial agents with novel mechanisms of action. The successful acquisition of compound T1 provides a substantial chemical starting point with a well-defined structure, clear activity, and the potential for systematic chemical optimization in developing novel human anti-infective drugs and agricultural antibacterial agents unaffected by existing cross-resistance mechanisms. Furthermore, it opens up a new technical path based on a solid experimental foundation for addressing the increasingly severe challenges of bacterial resistance in the pharmaceutical and agricultural fields.

[0014] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention provides a PlsY inhibitor, wherein the PlsY inhibitor is compound T1, and its structure is shown below: .

[0015] The second aspect of the present invention also provides the use of the Plsy inhibitor described in the first aspect in the preparation of a medicament for inhibiting human pathogenic bacteria.

[0016] Preferably, the human pathogenic bacteria include Streptococcus pneumoniae or Staphylococcus aureus.

[0017] Preferably, the drug further comprises pharmaceutically acceptable excipients selected from at least one of the following: solubilizers, emulsifiers, colorants, binders, disintegrants, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, coating materials, pH adjusters, absorbents, and antioxidants. Various excipients can be rationally combined according to the drug's route of administration, dosage form characteristics, and clinical application needs, respectively playing roles such as improving drug solubility and dispersibility, ensuring formulation stability, adjusting formulation osmotic pressure and pH, improving medication compliance, delaying drug degradation and deterioration, and promoting drug absorption and utilization.

[0018] Preferably, the dosage form of the drug includes oral preparations, injectable preparations, inhaled preparations, or transdermal preparations. Different dosage forms can be specifically adapted to different clinical administration scenarios to meet the medication needs of different patient groups.

[0019] The third aspect of the present invention also provides the application of the PlsY inhibitor described in the first aspect in the preparation of agricultural fungicides that inhibit plant pathogens.

[0020] Preferably, the plant pathogen includes Streptomyces scabiei or Clavibacter michiganensis subsp. Michiganensis.

[0021] Preferably, the agricultural fungicide further includes pesticide-acceptable excipients, including water, dispersants, wetting agents, fillers, stabilizers, co-solvents, adhesives, and defoamers. Water, as a green and environmentally friendly solvent, can dissolve water-soluble active ingredients and adjust the formulation concentration; dispersants (such as lignin sulfonates and alkylnaphthalene sulfonates) and wetting agents (such as alkylbenzene sulfonates and soap powder) can reduce the surface tension of the pesticide solution and improve the adhesion and spreading ability of the pesticide on the crop surface; fillers are mostly inert powders such as kaolin, talc, diatomaceous earth, and corn starch, used to dilute highly active active ingredients and ensure the stability of the formulation; stabilizers can inhibit the decomposition of active ingredients and extend the shelf life of the formulation; co-solvents can improve the solubility of poorly soluble active ingredients; adhesives enhance the pesticide solution's resistance to rain erosion; and defoamers prevent foaming during formulation processing or use.

[0022] Preferably, the formulation of the agricultural fungicide is a powder, suspension concentrate, emulsifiable concentrate, water-dispersible granule, granule, or water-emulsion. Powders use solid fillers as a carrier and are prepared by mixing and pulverizing; they can be directly sprayed or mixed into the soil for application, suitable for field pest and disease control. Suspensions are heterogeneous liquid formulations with stable dispersion of active ingredients and low risk of phytotoxicity to crops. Emulsifiable concentrates use organic solvents as a matrix and are easily dispersed in water after being combined with emulsifiers, resulting in rapid efficacy. Water-dispersible granules combine the storage and transportation stability of granules with the ease of use of suspension concentrates, exhibiting good solubility and dispersibility. Granules have a larger particle size, making them suitable for soil treatment and allowing for targeted control of underground pests. Water-emulsions use water as a continuous phase, making them environmentally friendly and safe.

[0023] Compared with the prior art, the beneficial effects of the present invention are: (1) It provides an antimicrobial intervention strategy based on a novel mechanism of action, offering a practical possibility for overcoming existing cross-resistance problems: Currently, most antimicrobial agents widely used in clinical and agricultural fields focus on limited and well-developed targets such as interfering with bacterial cell wall synthesis, inhibiting protein synthesis, or blocking nucleic acid replication. Long-term selective pressure has led bacteria to evolve diverse and efficient resistance mechanisms against these targets, such as producing various β-lactamases to hydrolyze drugs, modifying ribosomes or DNA gyrases to reduce drug affinity, and continuously expressing broad-spectrum drug efflux pumps. These mechanisms often result in cross-resistance to drugs with different structures but similar mechanisms of action. In contrast, the compound T1 developed in this invention targets glycerol-3-phosphoacyltransferase (PlsY), a key initiating enzyme in the bacterial membrane phospholipid biosynthesis pathway. This target represents a novel metabolic pathway essential for bacterial survival but not utilized by any currently marketed antimicrobial agents. Because compound T1 has a fundamentally different molecular target from existing antibacterial agents, the main drug resistance mechanisms that bacteria have evolved against traditional drugs are expected to have little or no effect on T1. This provides a new chemical entity and a clear mechanism of action to support the solution to the increasingly serious problem of cross-resistance.

[0024] (2) Based on the precise inhibition of essential bacterial targets, it exhibits superior potential selective toxicity characteristics: Many existing antibiotics may cause unavoidable off-target effects and toxic side effects because their bacterial targets have functional analogs or homologous domains in human cells. For example, some antibiotics that inhibit bacterial protein synthesis may inhibit protein synthesis in mammalian mitochondria. The PlsY target of this invention, however, has no biochemical function in humans or other higher eukaryotes. Human cells use a completely different cytoplasmic acetyl-CoA-dependent pathway for phospholipid synthesis, and there is no PlsY homolog in the genome. This fundamental difference in biochemical pathways between species theoretically lays the foundation for the superior selectivity of PlsY inhibitors. In vitro experimental data also support this judgment: given that compound T1 exhibits effective inhibitory concentrations (MIC values ​​typically in the single-digit µg / mL range) against several representative human pathogens (such as Streptococcus pneumoniae and Staphylococcus aureus), further optimization starting from T1 holds promise for developing novel antibacterial drugs with a wider therapeutic window and better expected safety profiles.

[0025] (3) A lead compound with well-defined activity and novel structure was obtained, laying a solid experimental foundation for systematic medicinal chemistry optimization targeting this novel target: The core bottleneck in the long-term research and development of PlsY, a highly promising new target, has been the lack of lead compounds that possess both potent enzyme inhibitory activity and a clear antibacterial phenotype, while also being optimizable. This invention successfully screened compound T1 by constructing a dual-guided physical screening system targeting both the inhibition of Streptococcus pneumoniae PlsY enzyme activity and the inhibition of the bacterium's growth. This compound not only exhibits potent inhibitory activity (IC50) on purified Streptococcus pneumoniae PlsY enzyme at the nanomolar to micromolar levels, but also… 50 More importantly, it exhibits a clear and reproducible minimum inhibitory concentration (MIC) against both standard strains and clinical isolates of Streptococcus pneumoniae at the cellular level. Based on the clear chemical structure of T1, subsequent research can systematically conduct structure-activity relationship analysis: by designing and synthesizing a series of derivatives or analogs focusing on different functional groups and molecular backbone regions, and simultaneously measuring their PlsY enzyme inhibitory activity and antibacterial activity against various pathogens, key pharmacophores affecting activity can be quickly identified, and modifiable sites that can be used to improve physicochemical properties or pharmacokinetic behavior can be identified. This will transform subsequent optimization work from blind and extensive screening to targeted and rational design, significantly improving research and development efficiency and the probability of success.

[0026] (4) It expands the potential application range of antimicrobial agents and provides new reserves for addressing the challenge of widespread drug resistance: The evolution of bacterial resistance is continuous and irreversible, and limited improvements to existing drug classes alone cannot meet long-term prevention and control needs. The significance of this invention lies in its provision of not only an active compound but also the successful validation of the feasibility of a novel antibacterial strategy targeting essential bacterial phospholipid synthesis pathways. The discovery of compound T1 and its in vitro inhibitory effects on important human and plant pathogens, including Streptococcus pneumoniae, Staphylococcus aureus, and rice glume blight fungus, demonstrate that PlsY is a "drug-worthy" target with broad-spectrum development potential. This adds a novel and unexplored mechanism of action to the anti-infective drug development pipeline, enriching the options for combating drug-resistant bacteria. From an overall perspective, drugs developed based on this novel mechanism will complement existing drugs in terms of mechanism, providing valuable additional options and reserves for addressing current and future complex drug-resistant bacterial infections in clinical and agricultural fields.

[0027] In summary, the core benefits of this invention are manifested in its fundamental innovation (opening up a completely new mechanism of action), clear practicality (effective against both sensitive and drug-resistant pathogens), broad forward-looking perspective (exploring new research and development pathways and control strategies), and sustainable research and development (providing high-quality lead compounds that can be systematically optimized). Compared to existing antibacterial technologies facing severe challenges from drug resistance, this invention provides a substantially differentiated solution and clear application prospects for the development of next-generation antibacterial drugs and agricultural fungicides. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the pETSG vector; Note: Italic text represents the promoter, the dashed box represents the 3C restriction site, TGP is a thermostable green fluorescent protein, and the background is green; from left to right, it represents the N-terminus to the C-terminus of the expressed protein.

[0029] Figure 2 This is the plsY gene of Streptococcus pneumoniae synthesized through codon optimization.

[0030] Figure 3 The results show the purification of spPlsY-TGP-Twin Strep; Note: Broad_M is a commercially available protein standard.

[0031] Figure 4 The reaction curve for spPlsY enzyme activity assay; Note: The product amount (inorganic phosphate Pi) of spPlsY was calculated using self-made fluorescent reagents and a standard curve. The enzyme concentration was 0.02 μg / mL, and the reaction temperature was 26℃.

[0032] Figure 5The enzymatic characterization results of the T1 inhibitor are shown. Note: (A, B) Reaction curves (A) and initial rate (B) of spPlsY at different T1 inhibitor concentrations; In the absence of T1, the activity of spPlsY (1.34 ± 0.17 μmol / min / mg, mean ± standard deviation) was obtained from three independent experiments, and the mean was set as 100%; The amount of product (Pi) was calculated using a self-made fluorescent reagent and a standard curve, with an enzyme concentration of 0.02 μg / mL and a reaction temperature of 26℃. The T1 concentration (μM) is marked in the figure.

[0033] Figure 6 The results show the microbiological characterization of the T1 inhibitor; Note: The concentration-dependent effect of T1 on the growth of Streptococcus pneumoniae PSSP is shown, and the MIC values ​​were obtained by fitting the sigmoidal equation in OriginPro software.

[0034] Figure 7 The growth inhibition effect of T1 inhibitor on methicillin-resistant Staphylococcus aureus MRSA-USA300 was obtained by fitting the curve using the sigmoidal equation in OriginPro software.

[0035] Figure 8 The growth inhibition effect of T1 inhibitor on Streptomyces scabiei of potato was obtained by fitting the curve using the sigmoidal equation in OriginPro software.

[0036] Figure 9 The T1 inhibitor represents the growth inhibition effect of Clavibacter michiganensis subsp. michiganensis on tomato. NA indicates that no dimethyl sulfoxide (DMSO) or T1 inhibitor was added. Detailed Implementation

[0037] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0038] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0039] The extremely limited and highly overlapping target range of existing antimicrobial drugs directly leads to the widespread occurrence and rapid spread of cross-resistance. Currently, the vast majority of antimicrobial agents used in clinical treatment and agricultural production operate on a few highly conserved physiological pathways, such as interfering with bacterial cell wall biosynthesis, inhibiting protein synthesis, blocking nucleic acid replication, or disrupting folic acid metabolism. This severe lack of target diversity allows bacteria to develop resistance to a large class of drugs with different chemical structures but similar mechanisms of action through relatively limited genetic evolutionary strategies (e.g., acquiring genes encoding drug-inactivating enzymes such as β-lactamases and aminoglycoside-modifying enzymes; modifying target proteins such as ribosomes and RNA polymerases; and continuously overexpressing multiple drug efflux pump systems). The direct consequence is that effective alternative treatments available clinically and in the field for infections caused by drug-resistant strains are rapidly depleted, and the entire antimicrobial drug research and application is trapped in a vicious cycle of "new drug development - deployment - resistance development," significantly compressing the return on investment and the effective lifespan of drugs.

[0040] Meanwhile, facing multidrug-resistant "superbugs" and other plant pathogens, existing technologies lack efficient, safe, and sustainable control methods. In clinical medicine, the selection of drugs for treating infections caused by methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), and carbapenem-resistant Enterobacteriaceae (CRE) is limited. These few drugs often come with significant toxic side effects (such as vancomycin's nephrotoxicity and linezolid's bone marrow suppression) or extremely high treatment costs, limiting their widespread application. In agricultural production, disease control has long relied on copper-based antibiotics and streptomycin, which not only leads to decreased control efficacy due to the development of pathogen resistance but also causes other problems such as soil heavy metal pollution, microbial community imbalance, and excessive chemical residues in agricultural products. Therefore, both the pharmaceutical and agricultural sectors face the dual dilemma of "limited drug options with significant side effects" or "declining efficacy of existing agents and environmental unsustainability."

[0041] Furthermore, the discovery of new antibacterial chemical entities heavily relies on the modification and derivation of known core frameworks, making truly novel structural compounds with original and innovative mechanisms of action extremely rare. Statistics show that over 80% of the new antibacterial molecular entities approved for marketing globally in the past two decades are derivatives or analogues obtained by chemically modifying existing drug classes (such as fluoroquinolones, glycopeptides, and oxazolidinones). These "improved" drugs are highly homologous to their predecessors in core chemical structure, and their molecular targets are the same or closely related. Therefore, they are unlikely to effectively circumvent the inherent resistance mechanisms or widely acquired resistance mechanisms that bacteria have developed against this target class. This research paradigm, dominated by "incremental improvement" rather than "breakthrough innovation," cannot fundamentally address the rapidly evolving and increasingly diverse drug resistance threats of bacteria, and is unlikely to provide entirely new solutions with long-term efficacy guarantees.

[0042] To address the systemic technical bottlenecks in existing technologies, namely narrow target spectrum, shortage of prevention and control methods, lack of original innovation, and stagnation in the transformation of new targets, this invention provides a research method for obtaining lead compounds with clearly defined antibacterial activity targeting the essential bacterial target PlsY through systematic experimental screening and verification. The core of this method lies in constructing a physical screening system guided by both the inhibition of the target enzyme's biochemical function and the inhibition of the target bacterial growth. This effectively overcomes the research stagnation caused by the scarcity of high-quality inhibitors for this target, ultimately achieving the crucial transformation from theoretical verification of the target to empirical evidence of active compounds. Specific implementation methods are as follows: (1) Preparation and biochemical characterization of PlsY protein of target pathogen: To establish a reliable target inhibition screening system, it is first necessary to obtain target proteins with catalytic activity. The *Streptococcus pneumoniae* PlsY gene was selected as the research object, and its coding sequence was constructed into a prokaryotic expression vector using gene cloning technology. The recombinant plasmid was transformed into an *E. coli* expression strain, and after induction of expression, high-purity, soluble recombinant PlsY protein was obtained by purification using affinity chromatography and molecular sieve chromatography. Subsequently, an in vitro enzyme activity detection system was established and optimized. By detecting the formation of inorganic phosphate in the reaction, it was confirmed that the catalytic activity of the recombinant protein was consistent with its physiological function. This step laid the necessary standardized biochemical experimental foundation for subsequent high-throughput enzyme inhibition screening.

[0043] (2) High-throughput screening and lead compound discovery based on target enzyme inhibition: Using the recombinant PlsY protein prepared and validated in Part (1) as the core, a miniaturized in vitro enzyme inhibition detection method suitable for high-throughput operation was established. A systematic screening was conducted on physical libraries or designed synthetic libraries containing tens to hundreds of thousands of structurally diverse small molecule compounds. At a uniform initial test concentration, each compound was co-incubated with PlsY protein and reaction substrates, and the inhibitory effect of each compound on enzyme activity was quantitatively determined using the established enzyme activity detection system. Based on a systematic analysis of all test data, "hit compounds" that significantly reduce PlsY catalytic activity were rapidly screened and identified.

[0044] (3) Validation and optimization of the in vitro activity of the lead compound: For the lead compounds obtained from Part (2), in-depth dose-response studies were conducted. The inhibition rates of different concentrations of compounds on Plsyzyme activity were measured, and the precise half-maximal inhibitory concentration (IC50) was calculated using nonlinear fitting. 50 The IC50 value was used to quantitatively assess its inhibitory efficacy. Based on this, preliminary chemical modifications and structure-activity relationship explorations were conducted around the most promising core structure, resulting in the synthesis of a series of structural analogs and the determination of their IC50 values. 50 The aim was to obtain a lead compound with superior inhibitory activity and higher selectivity. Through this step, a compound with potent Plsy inhibitory activity was successfully identified and optimized, and it was established as the lead compound for this study, named T1.

[0045] (4) Systematic evaluation of the antibacterial activity spectrum of compounds After confirming activity at the target level, this step aims to evaluate the actual function and potential application breadth of the lead compound T1 in inhibiting bacterial growth from multiple dimensions. The specific implementation details are as follows: 1) Validation of the activity of the target strain: First, the minimum inhibitory concentration (MIC) of compound T1 against the screening model strain Streptococcus pneumoniae (including standard sensitive strains and clinical isolates) was determined by the internationally standardized broth microdilution method to clarify its in vitro antibacterial effect.

[0046] 2) Antibacterial spectrum expansion study: The evaluation scope is extended to other important human pathogens, with a focus on Gram-positive pathogens such as Staphylococcus aureus and Streptococcus pyogenes. The broad-spectrum antibacterial potential of these strains is evaluated by measuring the MIC value of T1 against them.

[0047] 3) Evaluation of inhibitory activity against plant pathogens: Based on the conserved characteristics of PlsY homologs in some plant pathogens, the MIC values ​​of compound T1 against important crop pathogens such as Streptomyces scabiei and Clavibacter michiganensis subsp. Michiganensis were further determined to explore its feasibility for application in the field of agricultural disease control. Through the progressive experimental process described above, from "target enzyme preparation and characterization" to "high-throughput biochemical screening," then to "lead compound identification and optimization," and finally to "multi-level antibacterial function evaluation," this invention establishes a lead compound discovery and verification pathway centered on physical biochemical and phenotypic screening, independent of the target's three-dimensional structural information. Using this method, compound T1 was successfully obtained, providing a key lead molecular entity validated through multi-level experiments for developing antibacterial drugs targeting novel PlsY targets with potential for both human medical and agricultural applications.

[0048] To fully and clearly present the technical solution and significant advantages of the present invention, the present invention will be described in detail below with reference to specific embodiments.

[0049] Example 1: Expression and purification of spPlsY protein (1) Experimental materials 1) Strains, plasmids, and reagents: E. coli strain: BL21(DE3).

[0050] Expression vector: pETSG vector constructed in the laboratory, such as Figure 1 As shown (Construction method reference: Cai H, Yao H, Li T, Hutter CAJ, Li Y, Tang Y, Seeger MA, Li D. An improved fluorescent tag and its nanobodies for membrane protein expression, stability assay, and purification. Commun Biol. 2020 Dec 10;3(1):753. doi: 10.1038 / s42003-020-01478-z. PMID: 33303987; PMCID: PMC7729955.).

[0051] Target gene: The plsY gene of Streptococcus pneumoniae, synthesized through codon optimization, such as... Figure 2 As shown. The specific sequence is as follows: plsY sequence before optimization: Atgattacaatagttttattaatcctagcctatctgctgggttcgattccatctggtctctggattggacaagtattctttcaaatcaatctacgcgagcatggttctggtaacactggaacgaccaataccttccgcattttaggtaagaaagctggtatggcaacctttgtgattgactttttcaaaggaaccctagcaacgctgcttccgattatttttcatctacaaggcgtttctcctctcatctttggacttttggctgttatcggccataccttccctatctttgcaggatttaaaggtggtaaggctgtcgcaaccagtgctggagtaattttcggatttgcgcctatcttctgtctctaccttgcgattatcttctttggagctctctatcttggcagtatgatttcactgtctagtgtcacagcatcgatcgcggctgttatcggggttctgctctttccactttttggttttatcctgagtaactatgactctctcttcatcgctattatcttagcacttgctagtttgattatcattcgtcataaggacaatatagctcgtatcaaaaataaaactgaaaatttggtcccttggggattgaacctaacccatcaagatcctaaaaaataa。

[0052] Optimized plsY sequence: .

[0053] Culture medium: M9 medium (containing 50 μg / mL kanamycin).

[0054] Inducer: Isopropyl-β-D-thiogalactoside (IPTG).

[0055] Buffer A (resuspending / lysis buffer): 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM MPMSF, 10 μg / mL DNase I.

[0056] Buffer B (Solubilization / Binding Buffer): 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% (w / v) n-dodecyl-β-D-maltodextrin (DDM), 1 mM PMSF.

[0057] Buffer C (wash / storage buffer): 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.005% (w / v) lauryl-β-D-maltose neopentyl glycol (LMNG), 0.2 mM TCEP.

[0058] Eluent: Buffer C supplemented with 5 mM d-desulfobiotin.

[0059] Affinity chromatography medium: Strep-Tactin resin.

[0060] 2) Instruments and Consumables: Constant temperature shaker; high pressure homogenizer; high speed centrifuge and ultracentrifuge (Beckman Coulter); chromatography column and protein purification system (BIO-RAD); micro UV-Vis spectrophotometer (Nanodrop); protein concentration centrifuge tubes (MerckMillipore Amicon® Ultra).

[0061] (2) Experimental methods 1) Construction of expression strain and induction of protein expression: The synthesized plsY gene was cloned into the multiple cloning site of the pETSG vector to construct the recombinant expression plasmid pETSG-spPlsY, which is fused to the C-terminus with a TGP (modified GFP, with higher thermostability)-Twin Strep tag. The verified plasmid was transformed into E. coli BL21(DE3) competent cells. Single colonies were picked and inoculated into LB medium containing kanamycin and cultured overnight at 37°C. Then, the cells were transferred to M9 basal medium at a 1:100 inoculum and cultured at 37°C with shaking at 220 rpm until OD500. 600 The concentration was 0.6-0.8. Then IPTG was added to a final concentration of 0.1 mM, the culture temperature was lowered to 20℃, and expression was induced for another 18 hours.

[0062] 2) Cell Harvesting and Membrane Component Preparation: Collect bacterial cells by centrifugation at 4500 ×g for 15 minutes at room temperature. Resuspend the cells in pre-chilled buffer A on ice and lyse them using an autoclave at 4°C and 600 bar. Centrifuge the resulting lysate at 20,000 ×g for 30 minutes at 4°C to remove cell debris. Collect the supernatant and ultracentrifuge at 48,000 ×g for 2 hours at 4°C. The resulting precipitate is the cell membrane component.

[0063] 3) Membrane protein solubilization and affinity purification: The membrane precipitate was resuspended in buffer B and incubated with gentle stirring at 4°C for 2 hours to solubilize the membrane protein. Subsequently, the mixture was centrifuged at 48,000 ×g for 1 hour at 4°C, and the supernatant containing soluble spPlsY protein was collected. The supernatant was mixed with equilibrated Strep-Tactin resin and incubated at 4°C for 1.5 hours. The mixture was packed into a chromatography column and washed with 15 column volumes of buffer C to fully displace the detergent and remove non-specifically bound impurities.

[0064] 4) Protein elution and concentration: Elute the target protein with 5 column volumes of elution buffer and collect the elution peak. Evaluate the purity and yield of the purified protein using SDS-PAGE and Nanodrop.

[0065] (3) Experimental results SDS-PAGE analysis showed that after purification using the above steps, a spPlsY-TGP-TwinStrep band with a purity of approximately 95% was obtained at a molecular weight of approximately 40 kDa. Figure 3 This protein product can be used for subsequent enzyme activity assays, inhibitor screening, etc.

[0066] Example 2: Enzyme activity assay of spPlsY in detergent environment (1) Experimental materials 1) Proteins and reagents: Purified Streptococcus pneumoniae spPlsY protein (purified in Example 1).

[0067] The experimental system buffer (Assay buffer) consists of 150 mM NaCl, 100 mM Tris-HCl 8.5, and 0.03% (w / v) LMNG.

[0068] Substrate: hexadecyl phosphate (Acyl-P, diluted in Assay buffer, 25 μM).

[0069] Substrate: Glycerol-3-phosphate (G3P, diluted in Assay buffer, 20 mM).

[0070] Fluorescent probe: MDCC-labeled E. coli phosphate-binding protein (MDCC-ecPBP, self-made, responsive to changes in inorganic phosphate concentration). The preparation method of MDCC-ecPBP is described in the following reference: Brune, M., Hunter, JL, Corrie, JET & Webb, MR Direct, real-time measurement of rapid inorganic phosphate release using a novel fluorescent probe and its application to actomyosin subfragment 1 ATPase. Biochemistry 33, 8262–8271 (1994). Purification of phosphate-binding protein: The coding sequence of phosphate-binding protein (PBP) from glutamate 26 to tyrosine 346 was amplified from the genome of *E. coli* BL21(DE3) and cloned into the p3EC vector. The A197C mutant was constructed using site-directed mutagenesis (Reference: Brune, M., Hunter, JL, Corrie, JET & Webb, MR Direct, real-time measurement of rapid inorganic phosphate release using a novel fluorescent probe and its application to actomyosinsubfragment 1 ATPase. Biochemistry 33, 8262–8271 (1994)). The *E. coli* BL21(DE3) strain carrying this plasmid was then cultured to OD200. 600 The value was equal to 0.6–0.8, and expression was induced again with 1 mM IPTG. The resulting cells were placed in buffer G (containing 0.5 mM TECP, 1 mM PMSF, 8 mM MgCl2, 10 μg / mL). -1Cells were lysed in DNase I (20 mM Tris-HCl, pH 8.0) using a cell disruptor. After centrifugation to remove debris, the lysate was incubated with 10 mL Ni-NTA resin for 2 hours. The resin was then washed sequentially with 1 L of buffer H (10 mM Tris-HCl, pH 8.0) and 150 mL of buffer H containing 40 mM imidazole, followed by elution of proteins with buffer H containing 250 mM imidazole. Desalting was then performed (using an Econo-Pac10DG Desalting Columns #7322010: ① Remove the top cap and discard any excess storage buffer; ② Add 20 mL of buffer H (to the 30 mL mark), and cut the snap-off tip to start flow; ③ Allow the buffer to flow naturally to the upper filter membrane (frit); the column will not dry out, and the flow will automatically stop when the liquid level reaches the filter membrane; ④ Add 3.0 mL of sample; if the sample volume is < 3.0 mL, add buffer to bring the total volume to 3.0 mL; ⑤ After the sample has completely entered the column bed, add 4 mL of buffer to elute the proteins, and collect the eluent directly to complete the desalting step). The sample was then loaded onto 5 mL of HiTrap Q Sepharose pre-equilibrated with buffer H. In an FF column, at 1 mL·min -1 The PBP was collected by gradient elution with 40 mL of buffer containing 0–100 mM NaCl (performed in a Bio-Rad NGC protein purification system; pump A: connected to Buffer H (10 mM Tris-HCl pH 8.0, 0 mM NaCl), pump B: connected to Buffer H + 100 mM NaCl (10 mM Tris-HCl pH 8.0 + 100 mM NaCl); flow rate: constant 1 mL / min throughout (including loading, elution, and gradient); gradient steps (Linear Gradient module): initial %B = 0% (i.e., 100% A, NaCl = 0 mM); ending %B = 100% (i.e., 0% A, NaCl = 100 mM); gradient volume = 40 mL (or gradient duration = 40 min, flow rate 1 mL / min); gradient type: linear); finally concentrated to 32.5 mg / mL. -1 After being flash-frozen in liquid nitrogen at -80°C o Save as C.

[0071] Fluorescent labeling of PBP: The following steps were used to label the PBP A197C mutant with the thiol-reactive fluorescent probe N-[2-(1-maleimino)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC): First, impurity phosphate was removed from the 4 mL reaction system containing 13 mg PBP and 0.05 mg·mL⁻¹. -1 Purine nucleoside phosphorylase (PNPase), 0.2 mM 7-methylguanosine (MEG), and 10 mM Tris-HCl (pH 8.0) were added, and the mixture was treated at room temperature for 30 minutes. MDCC was then added to a final concentration of 0.15 mM, and the mixture was labeled at room temperature for 2 hours. Unreacted MDCC was removed using a Superdex 20010 / 300 GL column pre-equilibrated with 10 mM Tris-HCl (pH 8.0), and labeled and unlabeled PBP were separated using a 5 mL HiTrap Q Sepharose FF column. Elution was then performed using a 200 mL 0–250 mM NaCl gradient (using the same gradient elution program as described above) at a flow rate of 1 mL / min. -1 The concentration of MDCC-PBP was then calculated by correcting the absorbance of MDCC to A280, using the formula referenced in "Brune, M., Hunter, JL, Corrie, JET & Webb, MR Direct, real-time measurement of rapid inorganic phosphate release using a novel fluorescent probe and its application to actomyosin subfragment 1 ATPase. Biochemistry 33, 8262–8271 (1994)": [(A280, 1cm - A430, 1cm × 0.164) / 61,656 M -1 Finally, MDCC-PBP was concentrated to 3.7 mg / mL. -1 After being flash-frozen in liquid nitrogen at -80°C o Save as C.

[0072] Standard: Sodium dihydrogen phosphate (NaH2PO4, 25 μM aqueous solution, used to standardize the response of MDCC-ecPBP to inorganic phosphoric acid).

[0073] Control solvent: dimethyl sulfoxide (DMSO).

[0074] 2) Instruments and Consumables: The multi-functional microplate reader (Molecular Devices SpectraMax series) is equipped with a temperature control system and an optical module for bottom reading, SpectraMax M2; a black-bottomed, transparent 96-well half-plate (Corning 3880); a multi-channel pipette and corresponding tips.

[0075] (2) Experimental methods Add an appropriate amount of Assay buffer to a 96-well half-well plate, then add purified spPlsY to a concentration of 0.02 μg / mL, acly-P to 2.5 μM, and MDCC-ecPBP to 8 μM. Set the microplate reader temperature to 26℃ and read the fluorescence (FL425 / 466) using the "bottom-read" method. Calculate the corresponding ratio of product formation and fluorescence value change using 1 μM NaH2PO4. Add 5 μL of 20 mM G3P to start the reaction, bringing the final reaction volume to 45 μL. Read every 30 seconds for a total of 40 min.

[0076] (3) Data Analysis After completing the readings, a scatter plot of the FL425 / 466 fluorescence value over time was plotted and linear fitting was performed. Based on the relationship between 1 μM NaH2PO4 and the fluorescence value changes obtained in the experiment, the actual enzyme-catalyzed reaction rate was calculated.

[0077] (4) Experimental results Enzyme activity assay results showed that the reaction rate of spPlsY slowed down over time, exhibiting good linearity within 15 minutes. Figure 4 The enzyme activity conforms to the characteristics of classical enzyme kinetics. Based on the reaction curve, using the slope data within 10 minutes as the initial rate, the enzyme activity was calculated to be 1.34 ± 0.17 μmol / min / mg (mean ± standard deviation from three independent experiments).

[0078] Example 3: Half-maximal inhibitory concentration (IC50) of compound T1 50 ) Measurement To characterize the enzymatic inhibitory effect of T1, the IC50 of this compound was measured. 50 The value was measured.

[0079] (1) Experimental materials 1) Reagents: Concentration gradient solutions for the test compounds: 50 mM compound T1 (DMSO stock solution) was diluted with Assay buffer to prepare test solutions with final concentrations of 0, 6.25, 12.5, 25, 50, and 100 μM (the final DMSO concentration in each well was kept consistent). The remaining reagents were exactly the same as those listed in Part (1) of Example 2 (including spPlsY protein, Acyl-P, G3P, MDCC-ecPBP, DMSO-containing solvent control, etc.).

[0080] The molecular formula of compound T1 is: C 22 H 16 N4O3.

[0081] The structural formula is: O=C2 / C(=N\NC=1C=CC(=CC=1)C(=O)O)C(=NN2C=3C=CC=CC=3)C=4C=CC=CC=4 .

[0082] 2) Instruments and Consumables: Completely consistent with the description in Part (1) of Example 2 (including multifunctional microplate reader, black 96-well half-well plate, multichannel pipette, etc.).

[0083] (2) Experimental methods Add the following sequentially to the 96-well semi-perforated plate: Assay buffer, acyl-P (final concentration 2.5 μM), MDCC-ecPBP (final concentration 8 μM), analyte compounds (final concentrations of 0, 6.25, 12.5, 25, 50, and 100 μM), and spPlsY protein (final concentration 0.02 μg / mL). Each concentration was tested in duplicate, and the solvent control wells contained an equal volume of the corresponding DMSO solution without the compound. The microplate reader was set to 26°C, and fluorescence data were collected using bottom-reading fluorescence (excitation / emission wavelengths 425 / 466 nm). G3P (final concentration 2 mM) was added to start the reaction, with a final system volume of 50 μL. Fluorescence was read every 30 seconds for 40 min.

[0084] (3) Data Analysis Calculate the reaction rate of compound T1 at different concentrations. Define the enzyme activity at 0 μM as 100%, and normalize the enzyme activity at other concentrations based on this (calculate the relative percentage of enzyme activity).

[0085] Using OriginPro software, a scatter plot was created with compound concentration on the x-axis (logarithmic scale) and normalized relative enzyme activity on the y-axis. The Sigmoidal equation was used for nonlinear fitting, and the IC50 was calculated by the software. 50 value.

[0086] (4) Experimental results The results showed that the reaction rate of spPlsY decreased with increasing T1 concentration. Figure 5 (A) Plotting and fitting the reaction rate against concentration revealed that the half-inhibition concentration (IC50) at T1 was... 50 10.4 μM ( Figure 5 (B in the text) has value for further research and modification.

[0087] Example 4: Determination of the minimum inhibitory concentration (MIC) of compound T1 - Streptococcus pneumoniae To investigate the antibacterial effect of compound T1, the growth capacity of Streptococcus pneumoniae under different concentrations of this compound was tested. Because the pathogenic Streptococcus pneumoniae was involved, all the following procedures were performed in a P2 laboratory.

[0088] (1) Experimental materials 1) Strains and reagents: Streptococcus pneumoniae PSSP glycerol-preserved strain (strain D39, ATCC BAA-255); source: purchased from ATCC.

[0089] THYE liquid culture medium.

[0090] Compound T1 to be tested: 50 mM stock solution prepared with DMSO.

[0091] MTT solution (5 mg / mL, dissolved in PBS or deionized water, diluted to the required concentration before use).

[0092] Negative control: THYE medium containing 0.2% DMSO.

[0093] Sterile physiological saline or THYE medium (for dilution of bacterial culture).

[0094] 2) Instruments and Consumables: 37°C constant temperature incubator (containing 5% CO2); ELISA reader; sterile 96-well cell culture plate (full-well plate); biosafety cabinet; constant temperature shaker; pipettes and sterile pipette tips.

[0095] (2) Experimental methods 1) Preparation of bacterial culture: Take out the PSSP glycerol bacteria stored at -80°C and thaw at room temperature. Transfer to fresh THYE medium at a volume ratio of 1:50 and incubate at 37°C, 5% CO2 for about 14 hours, until OD reaches the target value. 600 ≈ 0.3. Then dilute the bacterial culture to OD using THYE medium. 600 = 0.002, reserved.

[0096] 2) Compound dilution and plate preparation: Dilute the 50 mM DMSO stock solution of compound T1 to 100 μM using THYE medium. Then, mix this intermediate solution with THYE medium containing 0.2% DMSO at different ratios to prepare a series of dilutions at concentrations of 0, 6.25, 12.5, 25, and 50 μM (the final volume of each concentration must meet the requirements for subsequent sample loading). Add 50 μL of each of the above-mentioned compound dilutions to each well of a sterile 96-well plate.

[0097] 3) Inoculation and culture: Add 50 μL OD to each well 600 =0.002 PSSP bacterial suspension, to make a final volume of 100 μL per well, with final compound concentrations of 0, 3.125, 6.25, 12.5, and 25 μM. Simultaneously, wells containing no compound, but only 0.5% DMSO and bacterial suspension, were set up as growth controls. After sealing the plates with breathable sealing film, they were incubated at 37°C in a 5% CO2 incubator for approximately 20 hours.

[0098] 4) Activity detection and reading: After incubation, add 10 μL of MTT solution to each well (to bring the final MTT concentration to approximately 10 μM). After incubation for another 30 minutes, measure the absorbance (OD) of each well at 595 nm using a microplate reader. 595 Record the OD values ​​of each compound concentration well and the control well. 595 The values ​​were calculated using OriginPro software, with the final concentration of the compound on the x-axis, and the corresponding OD values. 595 The values ​​are plotted as ordinates to create a scatter plot, and the Sigmoidal equation is used to perform nonlinear fitting on the data.

[0099] (3) Experimental results The results showed that as the concentration of compound T1 increased (greater than 8 μM), the growth of PSSP was gradually inhibited. Figure 6 The concentrations of the bacterial culture and the corresponding compound concentrations were plotted as a scatter plot and fitted to determine that the minimum inhibitory concentration (MIC) for T1 was 23.0 μM. Figure 6 ).

[0100] Example 5: Determination of the minimum inhibitory concentration of compound T1 - MRSA - USA300 (1) Experimental materials 1) Strains and reagents: Methicillin-resistant Staphylococcus aureus MRSA-USA300 glycerol-preserved strain, source: Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

[0101] TSB liquid medium.

[0102] Compound T1 to be tested: 50 mM stock solution prepared with DMSO.

[0103] MTT solution (5 mg / mL, dissolved in PBS or deionized water, diluted to the required concentration before use).

[0104] Negative control: TSB medium containing 0.2% DMSO.

[0105] 2) Instruments and Consumables: 37°C constant temperature incubator; ELISA reader; sterile 96-well cell culture plate (full-well plate); biosafety cabinet; 37°C constant temperature shaker; pipettes and sterile pipette tips.

[0106] (2) Experimental methods 1) Bacterial culture preparation: Remove the MRSA-USA300 glycerol bacteria stored at -80°C and streak them onto a TSA plate. Incubate overnight at 37°C. The next day, pick single colonies and transfer them to 3 mL of TSB medium. Incubate overnight at 37°C using a shaker. The following day, transfer the colonies to fresh TSB medium at a 1:100 volume ratio and incubate at 37°C for approximately 1.5 hours, until OD (dose elapsed). 600 ≈0.3-0.4, then dilute the bacterial culture to OD using TSB medium. 600 = 0.002, reserved.

[0107] 2) Compound Dilution and Plate Preparation: Dilute the 50 mM DMSO stock solution of compound T1 to 38.4 μg / mL using TSB medium. Then, mix this intermediate solution with TSB medium containing 0.2% DMSO at different ratios to prepare a series of dilutions with concentrations of 0, 3.84, 7.69, 15.37, 19.22, 26.9, 30.7, and 38.4 μg / mL (the final volume of each concentration must meet the requirements for subsequent sample loading). Add 50 μL of each of the above-mentioned compound dilutions to each well of a sterile 96-well plate.

[0108] 3) Inoculation and culture: Add 50 μL OD to each well 600=0.002 USA300 bacterial suspension, making the final volume of each well 100 μL, with final compound concentrations of 0, 1.92, 3.84, 7.69, 9.61, 13.45, 15.37, and 19.22 μg / mL. Simultaneously, wells containing only 0.1% DMSO and bacterial suspension without the compound were set up as growth controls. After sealing the plate with breathable sealing film, it was incubated at 37°C for approximately 20 hours.

[0109] 4) Activity detection and reading: After incubation, add 10 μL of MTT solution to each well (to bring the final MTT concentration to approximately 10 μM). After incubation for another 30 minutes, measure the absorbance (OD) of each well at 595 nm using a microplate reader. 595 Record the OD values ​​of each compound concentration well and the control well. 595 The values ​​were calculated using OriginPro software, with the final concentration of the compound on the x-axis, and the corresponding OD values. 595 The values ​​are plotted as ordinates to create a scatter plot, and the Sigmoidal equation is used to perform nonlinear fitting on the data.

[0110] (3) Experimental results The results showed that as the concentration of compound T1 increased, the growth of USA300 was gradually inhibited, with a MIC value of 12 μg / mL. Figure 7 ).

[0111] Example 6: Inhibitory effect of T1 on plant pathogen Streptomyces scabiei (1) Experimental materials 1) Strains and reagents: Glycerin-preserved strain of *Streptomyces scabiei*, *Potato scabies*. Source: Research group of Zhao Pan, Institute of Microbiology, Chinese Academy of Sciences. TSB liquid medium.

[0112] Compound T1 to be tested: 50 mM stock solution prepared with DMSO.

[0113] MTT solution (5 mg / mL, dissolved in PBS or deionized water, diluted to the required concentration before use).

[0114] Negative control: TSB medium containing 0.1% DMSO.

[0115] 2) Instruments and Consumables: 28°C constant temperature incubator; ELISA reader; sterile 96-well cell culture plate (full-well plate); biosafety cabinet; 28°C constant temperature shaker; pipettes and sterile pipette tips.

[0116] (2) Experimental methods 1) Bacterial culture preparation: Remove the *Streptomyces scabiei* glycerol bacteria stored at -80 °C and streak them onto a TSA plate. Incubate overnight at 28 °C. The next day, pick single colonies and transfer them to 3 mL of TSB medium. Incubate overnight at 28 °C using a shaker. The following day, transfer the colonies to fresh TSB medium at a 1:100 volume ratio and incubate at 28 °C for approximately 1.5 hours until OD (dose elapsed). 600 ≈ 0.3-0.4, then dilute the bacterial culture to OD using TSB medium. 600 = 0.002, reserved.

[0117] 2) Compound Dilution and Plate Preparation: Dilute the 50 mM DMSO stock solution of compound T1 to 100 μM using TSB medium. Then, mix this intermediate solution with TSB medium containing 0.2% DMSO at different ratios to prepare a series of dilutions with concentrations of 0, 1.57, 3.125, 6.25, 12.5, 25, 50, and 100 μM (the final volume of each concentration must meet the requirements for subsequent sample loading). Add 50 μL of each of the above-mentioned compound dilutions to each well of a sterile 96-well plate.

[0118] 3) Inoculation and culture: Add 50 μL OD to each well 600 =0.002 Streptomyces scabiei bacterial suspension to make the final volume of each well 100 μL, with final compound concentrations of 0, 0.79, 1.57, 3.125, 6.25, 12.5, 25, and 50 μM. Simultaneously, wells containing no compound, but only 0.1% DMSO and bacterial suspension, were set up as growth controls. After sealing the plate with breathable sealing film, it was incubated at 28 °C for approximately 20 hours.

[0119] 4) Activity detection and reading: After incubation, add 10 μL of MTT solution to each well (to bring the final MTT concentration to approximately 10 μM). After incubation for another 30 minutes, measure the absorbance (OD) of each well at 595 nm using a microplate reader. 595 Record the OD values ​​of each compound concentration well and the control well. 595 The values ​​were calculated using OriginPro software, with the final concentration of the compound on the x-axis, and the corresponding OD values. 595 The values ​​are plotted as ordinates to create a scatter plot, and the Sigmoidal equation is used to perform nonlinear fitting on the data.

[0120] (3) Experimental results The results showed that T1 inhibited the growth of Streptomyces scabiei by approximately 7 μM ( ). Figure 8 ).

[0121] Example 7: Inhibitory effect of T1 on plant pathogen Clavibacter michiganensis subsp. michiganensis (1) Experimental materials 1) Strains and reagents: Clavibacter michiganensis subsp. michiganensis (Smith 1910) ATCC 492 glycerol-preserved strain. Source: Purchased from Taisto Biotechnology Co., Ltd.

[0122] Other reagents are the same as in Example 6.

[0123] 2) Instruments and consumables: Same as in Example 6.

[0124] (2) Experimental methods 1) Bacterial culture preparation: The glycerol-containing Clavibacter michiganensis subsp. michiganensis, stored at -80 °C, was removed and streaked onto a TSA plate. It was then incubated overnight at 28 °C. The next day, single colonies were picked and transferred to 3 mL of TSB medium and incubated overnight at 28 °C using a shaker. The following day, the colonies were transferred to fresh TSB medium at a 1:100 volume ratio and incubated at 28 °C for approximately 1.5 hours until OD (dose elapsed). 600 ≈ 0.3-0.4, then dilute the bacterial culture to OD using TSB medium. 600 = 0.002, reserved.

[0125] 2) Compound dilution and plate preparation: Dilute the 50 mM DMSO stock solution of compound T1 to 100 μM using TSB medium, and mix 50 μL with an equal volume of bacterial suspension in a well plate; use TSB containing 0.2% DMSO as a control.

[0126] 4) Activity detection and reading: After the culture was completed, MTT staining was performed according to the procedure in Example 6. (3) Experimental results The results showed that, compared with the control group, 50 μM T1 completely inhibited the growth of Clavibacter michiganensis subsp. michiganensis. Figure 9 ).

[0127] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A PlsY inhibitor, characterized in that, The PlsY inhibitor is compound T1, whose structure is shown below: 。 2. The use of the Plsy inhibitor of claim 1 in the preparation of a drug for inhibiting human pathogenic bacteria.

3. The application according to claim 2, characterized in that, The human pathogenic bacteria include Streptococcus pneumoniae or Staphylococcus aureus.

4. The application according to claim 2, characterized in that, The drug further comprises pharmaceutically acceptable excipients selected from at least one of solubilizers, emulsifiers, colorants, binders, disintegrants, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, coating materials, pH adjusters, absorbents, and antioxidants.

5. The application according to claim 4, characterized in that, The dosage forms of the drug include oral formulations, injectable formulations, inhaled formulations, or transdermal formulations.

6. The use of the PlsY inhibitor according to claim 1 in the preparation of agricultural fungicides that inhibit plant pathogens.

7. The application according to claim 6, characterized in that, The plant pathogens include Streptomyces tumefaciens (potato scab) or Streptomyces chlamydiides (tomato ulcer).

8. The application according to claim 6, characterized in that, The agricultural fungicide also includes pesticide-acceptable excipients, including water, dispersants, wetting agents, fillers, stabilizers, solubilizers, adhesives, and defoamers.

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