Inhibitor compounds that interfere with the biosynthesis of bacterial membrane phospholipids and uses thereof

Abstract

The present application belongs to the technical field of pharmaceutical chemistry, and particularly relates to an inhibitor compound interfering with the biosynthesis of bacterial membrane phospholipids and uses thereof. The present application provides an antibacterial research and development scheme targeting a novel target site, taking the key enzyme PlsY of bacterial phospholipid synthesis as the action site, and avoiding the cross-resistance caused by the homogenization of existing antibacterial drug target sites. The screened compound T5 can strongly inhibit the biological activity of PlsY, and significantly antagonize pathogenic strains such as Streptococcus pneumoniae and Staphylococcus aureus. Since the human body does not contain PlsY homologous proteins, the drug safety window is better. The chemical structure of the molecule is different from the known antibiotics and substrate analogs, which is convenient for structure optimization based on the structure-activity relationship, and also shows inhibitory activity to various crop pathogenic bacteria. The present application creates a new path for PlsY-targeted antibacterial research and development, reserves a differentiated lead skeleton for the prevention and treatment of drug-resistant bacterial infections, and has good development prospects in the fields of human anti-infective drugs and agricultural bactericides.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemistry technology, specifically relating to an inhibitory compound that interferes with bacterial membrane phospholipid biosynthesis and its uses. 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 approved new antimicrobial chemical entities are essentially derivatives of known drug skeletons or advantageous structures, chemically modified and optimized (e.g., next-generation cephalosporins, novel oxazolidinones). Truly original drugs targeting entirely new biochemical targets and possessing original, innovative chemical structures are extremely rare. This "derivative"-dominated development 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 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] In summary, developing a novel small molecule inhibitor targeting Streptococcus pneumoniae PlsY as a specific molecular target, using the inhibition of this bacterium's growth as the phenotypic screening criterion, and obtaining it through experimental verification is not only a crucial experimental verification of the druggability of this novel target PlsY, 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 prior art, this invention addresses the systemic technical bottlenecks in the prior art, which are 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 purpose of this invention is to provide a physical compound based on the novel action site of PlsY, a key enzyme in bacterial phospholipid synthesis, and its applications. Specific objectives are as follows: (1) The primary objective of this invention is to provide a small molecule compound with a novel and precise chemical structure, named T5. This compound was 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 were 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 T5 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 T5 in inhibiting human pathogenic bacteria. Based on the confirmed potent inhibitory effect of compound T5 on Streptococcus pneumoniae PlysY and its cells, standardized in vitro antibacterial activity tests further demonstrated that compound T5 exhibits significant growth-inhibiting activity against a variety of important Gram-positive pathogens, including Staphylococcus aureus (including methicillin-resistant MRSA strains) and Streptococcus pyogenes. This result provides direct experimental evidence for developing compound T5 as a novel therapeutic candidate drug with a novel mechanism of action against infections caused by multidrug-resistant Gram-positive bacteria.

[0012] (3) A third objective of this invention is to provide the application of compound T5 in inhibiting plant pathogenic bacteria. Given that PlsY homologs also exist in some plant pathogens, this invention experimentally verified that compound T5 also has a potential inhibitory effect on potato-related pathogens. This provides a practical research direction and experimental support for developing compound T5 and its structurally optimized derivatives into novel agricultural fungicides with a completely new mechanism of action that can be used for the control of 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, T5, 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 completes the crucial experimental transformation and verification of the high-value novel target PlsY from theoretical conception to a solid active inhibitor, but also breaks through the technical bottleneck in existing antibacterial drug development caused by the lack of novel mechanism-leading entities. The successful acquisition of compound T5 provides a substantial chemical starting point with a well-defined structure, clear activity, and the potential for systematic chemical optimization for 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 the use of a small molecule compound T5 in the preparation of a PlsY inhibitor, wherein the PlsY inhibitor is used to prepare a drug for inhibiting human pathogenic bacteria, and the structure of the small molecule compound T5 is shown below: .

[0015] Preferably, the human pathogenic bacteria include methicillin-sensitive Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, or Streptococcus pyogenes.

[0016] Preferably, the drug further comprises pharmaceutically acceptable excipients, including solubilizers, emulsifiers, colorants, binders, disintegrants, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, coating materials, pH adjusters, absorbents, or antioxidants. These 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 osmotic pressure and pH, improving medication compliance, delaying drug degradation and deterioration, and promoting drug absorption and utilization.

[0017] More preferably, the dosage form of the drug includes oral formulations, injectable formulations, inhaled formulations, or transdermal formulations. Different dosage forms can be specifically adapted to different clinical administration scenarios to meet the medication needs of different patient groups.

[0018] A second aspect of the present invention also provides the application of the small molecule compound T5 in the preparation of a PlsY inhibitor, wherein the PlsY inhibitor is used to prepare an agricultural fungicide for inhibiting plant pathogens, and the structure of the small molecule compound T5 is shown below: .

[0019] Preferably, the plant pathogen includes *Tomato Ulcer Bacterium*.

[0020] Preferably, the agricultural fungicide further includes pesticide-acceptable excipients, including water, dispersants, wetting agents, fillers, stabilizers, co-solvents, adhesives, or 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, alkylnaphthalene sulfonates) and wetting agents (such as alkylbenzene sulfonates, soapberry 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.

[0021] Preferably, the formulation of the agricultural fungicide includes aqueous solutions, suspensions, wettable powders, granules, or emulsifiable concentrates. Aqueous solutions are liquid preparations in which the active ingredient is dissolved in water, with water as the main carrier. Suspensions are uniform suspensions formed by stabilizing solid active ingredients in water with the aid of dispersing and wetting agents. Wettable powders are made by mixing and pulverizing pesticide technicals with fillers and wetting / dispersing agents, and can form a stable spray solution after adding water and stirring. Granules are processed into solid granules with fillers as carriers, and can be directly applied. Emulsifiable concentrates rely on cosolvents to dissolve the technicals and are then combined with emulsifying agents; dilution with water forms a uniform emulsion spray.

[0022] Compared with the prior art, the beneficial effects of the present invention are: Compared to various antibacterial drugs currently used in clinical and agricultural fields, the compound T5 and its representative PlsY-targeting inhibitor provided by this invention have the following outstanding beneficial effects verified by experiments: (1) It introduces an antibacterial mechanism based on novel molecular targets, providing a practical approach to overcome the cross-resistance dilemma: Existing antibacterial agents primarily target a few well-developed physiological pathways, such as cell wall synthesis, protein synthesis, and nucleic acid replication. Long-term selective pressure has led bacteria to evolve diverse and efficient resistance systems, including expressing various β-lactamases to inactivate drugs, modifying ribosomes or DNA gyrases to reduce drug binding affinity, and continuously overexpressing various efflux pumps, thus generating cross-resistance to drugs with different chemical structures but the same mechanism of action. The compound T5 obtained in this invention targets glycerol-3-phosphoacyltransferase (PlsY), a key initiating enzyme in bacterial membrane phospholipid biosynthesis. This target represents a novel metabolic pathway essential for bacterial survival but completely untapped by existing marketed antibacterial drugs. Because the molecular site of action of T5 differs fundamentally from all marketed antibacterial agents, the aforementioned major resistance mechanisms evolved by bacteria against traditional drugs are expected to be ineffective or have extremely limited effect on it. This provides a novel chemical molecule and a precise mechanism of action for addressing the increasingly severe problem of cross-resistance.

[0023] (2) Based on essential targets specific to bacteria and lacking human origin, it exhibits superior potential selective toxicity characteristics: Many existing antibiotics, due to the presence of functional analogs or highly homologous domains of their bacterial target proteins in human cells, are prone to unpredictable off-target effects and toxic side effects. For example, some bacterial protein synthesis inhibitors can simultaneously interfere with protein synthesis in mammalian mitochondria. However, the PlsY target of this invention catalyzes a biochemical reaction that is completely absent in humans and higher eukaryotes. Human cells synthesize phospholipids through a distinctly different cytoplasmic acetyl-CoA-dependent pathway, and there are no functional homologous genes for PlsY in the human genome. This fundamental difference in phospholipid metabolism pathways between species provides the theoretical basis for the high selective toxicity of PlsY inhibitors. In vitro experimental results also confirm this characteristic: compound T5 exhibits good antibacterial activity against various pathogenic bacteria such as Streptococcus pneumoniae and Staphylococcus aureus, with minimum inhibitory concentrations (MICs) generally as low as single-digit μg / mL. Structural optimization using T5 as a lead compound holds promise for developing novel antibacterial drugs with a wider therapeutic window and better safety profiles.

[0024] (3) A novel and validated lead compound with potential for optimization was obtained, paving the way for systematic medicinal chemistry optimization work around novel targets: While the PlsY target holds immense potential, its long-standing limitation has been the lack of an optimizable lead molecule possessing both strong enzyme activity inhibition and a definitive antibacterial phenotype. This study established a physical compound screening system using both Streptococcus pneumoniae PlsY enzyme activity inhibition and strain growth inhibition as dual evaluation indicators, ultimately identifying the lead compound T5. This compound not only exhibits micromolar-level inhibitory activity (with a clearly defined IC50 value) against purified Streptococcus pneumoniae PlsY enzyme at the biochemical level, but also demonstrates a stable and reproducible minimum inhibitory concentration (MIC) against standard Streptococcus pneumoniae strains at the cellular level. Based on the well-defined chemical structure of compound T5, subsequent systematic structure-activity relationship studies can be conducted: designing and synthesizing a series of derivatives and analogs targeting its molecular skeleton and various functional groups, simultaneously evaluating the compound's inhibitory activity against PlsY enzyme and its antibacterial effects against various pathogens, thereby identifying the core pharmacophore and screening modification sites for optimizing physicochemical properties and pharmacokinetic characteristics. This approach can shift the research and development model from traditional experience-based screening to rational drug design based on molecular mechanisms, significantly improving the efficiency of structure optimization and the success rate of research and development.

[0025] (4) It broadens the potential application spectrum of antibacterial agents and provides new reserves for dealing with the serious threat of pan-drug resistance: Bacterial resistance evolves continuously and is difficult to reverse; simply modifying the structure of existing drugs cannot meet the long-term control needs in the field of anti-infection. Therefore, the core value of this invention lies not only in obtaining a highly active lead compound, but also in experimentally verifying the practical application potential of the novel antibacterial strategy of "targeting the essential phospholipid synthesis pathway in bacteria." The lead compound T5 exhibited good in vitro antibacterial activity against various human and plant pathogens, including Streptococcus pneumoniae, Staphylococcus aureus, and Rice glume blight pathogens, confirming that PlsY is a high-quality drug target with broad-spectrum research and development prospects. This research opens up a completely new antibacterial mechanism of action and expands the research direction for combating drug-resistant bacteria. Drugs developed based on this novel mechanism can complement the mechanisms of action of existing antibacterial drugs, providing new drug options and long-term technical reserves for the medical, clinical, and agricultural fields to cope with the increasingly complex drug-resistant bacterial infections of today and the future.

[0026] In summary, the core advantages of this invention are mainly reflected in four aspects: First, it achieves fundamental innovation in mechanism, opening up entirely new drug targets and metabolic pathways; second, it has outstanding application value, exhibiting good inhibitory effects on both sensitive bacteria and multidrug-resistant pathogens; third, it possesses forward-looking value, creating a new research direction and disease control strategy for the antibacterial field; and fourth, it has a solid research foundation, providing high-quality lead compounds for systematic structure-activity relationship optimization. Compared to traditional antibacterial technologies currently constrained by drug resistance issues, this invention provides a completely new solution for the development of next-generation differentiated human antibacterial drugs and agricultural fungicides, with broad application prospects. Attached Figure Description

[0027] 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.

[0028] Figure 2 This is the plsY gene of Streptococcus pneumoniae synthesized through codon optimization.

[0029] Figure 3 The results show the purification of spPlsY-TGP-Twin Strep; Note: Broad_M is a commercially available protein standard.

[0030] 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℃.

[0031] Figure 5 The results show the enzymatic characterization of the T5 inhibitor; Note: In the absence of T5, the activity of spPlsY was set to 100%; the enzyme concentration was 0.02 μg / mL, the reaction temperature was 26℃, and the T5 concentration (μM) is labeled in the figure.

[0032] Figure 6 The results show the growth inhibition of human pathogenic bacteria by the T5 inhibitor; Note: SP represents Streptococcus pneumoniae; SPE represents Staphylococcus epidermidis; SPY represents Streptococcus pyogenes; RN4220 represents methicillin-sensitive Staphylococcus aureus; MRSA represents methicillin-resistant Staphylococcus aureus.

[0033] Figure 7The results show the growth inhibition of the T5 inhibitor on Clavibacter michiganensis subsp. Michiganensis; Note: The dimethyl sulfoxide (DMSO) group is the control of T5 to exclude the influence of DMSO. Detailed Implementation

[0034] 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.

[0035] 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.

[0036] The current spectrum of antimicrobial drug targets is extremely narrow and highly overlapping, leading to widespread and rapidly spreading cross-resistance. The vast majority of antimicrobial agents used in clinical and agricultural fields operate through mechanisms concentrated in a few highly conserved physiological pathways, such as interfering with cell wall synthesis, blocking protein synthesis, inhibiting nucleic acid replication, or disrupting folic acid metabolism. This severe lack of target diversity allows bacteria to develop resistance to a large number of drugs with different chemical structures but similar mechanisms of action using only very limited genetic evolutionary strategies. For example, bacteria can acquire genes encoding inactivating enzymes such as β-lactamases and aminoglycoside-modifying enzymes, modify the structure of drug targets such as ribosomes and RNA polymerases, and overexpress various drug efflux pumps, thus resisting the effects of multiple antimicrobial drugs through any of these pathways. Consequently, when faced with infections caused by drug-resistant strains, effective alternative treatments available in clinical and field settings are quickly exhausted, trapping the entire antimicrobial drug research and application in a vicious cycle of "new drug development - deployment - resistance development," significantly compressing the return on investment and the effective lifespan of drugs.

[0037] Meanwhile, facing multidrug-resistant "superbugs" and other plant pathogens, the existing technological system lacks efficient, safe, and sustainable control tools. In the field of 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 the nephrotoxicity of vancomycin and the bone marrow suppression of linezolid) 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 fields face the dual dilemma of "limited drug options with significant side effects" or "declining efficacy of existing agents and environmental unsustainability."

[0038] 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 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.

[0039] To this end, this invention has constructed a systematic discovery and verification system for lead compounds based on the essential bacterial target PlsY and guided by dual screening of enzyme activity inhibition and bacterial phenotypic antibacterial activity. This system has overcome the long-standing research and development barrier of lacking high-quality inhibitors for this target and has completed the key transformation from target theory research to the verification of active small molecule entities.

[0040] The technical solution of the present invention is implemented step by step according to the following progressive steps: Step 1: Preparation and functional confirmation of Streptococcus pneumoniae PlsY protein: To construct a stable and reliable in vitro enzyme inhibition screening platform, it is first necessary to obtain recombinant target proteins with natural catalytic capabilities. Therefore, the PlsY encoding gene of *Streptococcus pneumoniae* was selected, and its open reading frame was cloned into a prokaryotic expression vector using gene recombination technology. This vector was then transformed into *Escherichia coli* as the expression host, and after optimized induction conditions, the target protein was expressed in large quantities. Subsequently, a combination of affinity chromatography and gel filtration chromatography was used for purification to obtain high-purity, homogeneous, soluble recombinant PlsY protein. Based on this, an in vitro enzyme activity assay method based on inorganic phosphate release detection was established, verifying that the obtained protein possesses definite catalytic activity, and its reaction characteristics are consistent with physiological functions. This step provides a standardized and quantitative biochemical detection basis for subsequent large-scale enzyme inhibition screening.

[0041] Step 2: High-throughput screening and lead compound identification based on target enzyme activity: Based on the recombinant PlsY protein prepared and validated in the first step, the original enzyme activity detection system was optimized and adapted to a microplate high-throughput detection mode, establishing a stable and reproducible enzyme activity inhibition screening process. Using this screening system, a comprehensive systematic screening was conducted on libraries of physical compounds and combinatorial synthesis libraries containing tens of thousands to hundreds of thousands of structurally diverse small molecules. Under uniformly set initial screening concentrations, the test compounds were co-incubated with PlsY protein and reaction substrates, respectively. Enzyme activity fluctuations were quantitatively measured, and the relative inhibition rates of each compound were calculated. All experimental data were summarized and statistically analyzed to rapidly screen candidate molecules that significantly inhibited PlsY catalytic activity, i.e., lead compounds.

[0042] Step 3: Validation of the dose-effect relationship of the lead compound and optimization of the lead compound: For each of the lead compounds obtained in the previous step, a complete concentration gradient-enzyme activity inhibition experiment was performed to plot dose-response curves. The half-maximal inhibitory concentration (IC50) was then determined by nonlinear regression fitting. 50 The inhibitory efficacy was precisely quantified. Based on this, preliminary structure-derived and structure-activity relationship analyses were conducted focusing on the core framework with the most prominent inhibitory activity. A series of structural analogs were designed and synthesized, and their IC50 values ​​were determined. 50 The value was used to assess the impact of structural changes on inhibitory activity. Through this round of guided optimization, a compound exhibiting potent inhibitory activity against Streptococcus pneumoniae PlsY was successfully obtained and established as the lead compound for this study, named T5.

[0043] Step 4: Multidimensional systematic evaluation of the antibacterial activity of compound T5: After completing target-level activity validation, this step systematically evaluates the antibacterial activity and application potential of the lead compound T5 at the bacterial level. First, using standard sensitive strains of Streptococcus pneumoniae and representative clinical isolates as test subjects, the minimum inhibitory concentration (MIC) of T5 in vitro was determined using the microbroth dilution method to clarify its antibacterial efficacy against this pathogen. Subsequently, the test species were broadened to include typical Gram-positive pathogens such as Staphylococcus aureus (including MRSA-resistant strains) and Streptococcus pyogenes for MIC determination, assessing its research and development value as a broad-spectrum anti-Gram-positive bacterial drug. Given the high conservation of the PlsY homologous sequence in various plant pathogenic bacteria, antibacterial tests were further conducted using important crop pathogenic bacteria such as Potato scab pathogen and Tomato canker pathogen to explore the application prospects of this compound in the development of novel agricultural antibacterial agents.

[0044] Through the progressive experimental pathway described above—"target enzyme preparation and characterization—high-throughput biochemical screening—lead compound identification and optimization—multi-level antibacterial function verification"—this invention constructs a lead compound discovery technology system that requires no target protein three-dimensional structure data and focuses on dual verification of enzyme activity inhibition and bacterial antibacterial phenotype. Based on this technology strategy, a novel small molecule compound, T5, with a novel mechanism of action, was screened, providing a high-quality lead entity supported by multiple rounds of experimental data for the development of novel antibacterial agents targeting PlsY that possess both human pharmaceutical and agricultural plant protection value.

[0045] 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.

[0046] Example 1: Expression and purification of spPlsY protein 1. Experimental Materials 1.1 Strains, Plasmids, and Reagents: (1) E. coli strain: BL21(DE3).

[0047] (2) Expression vector: pETSG vector constructed in the laboratory, such as Figure 1As shown, the construction method refers to the literature "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, stabilityassay, and purification. Commun Biol. 2020 Dec 10;3(1):753. doi: 10.1038 / s42003-020-01478-z. PMID: 33303987; PMCID: PMC7729955.".

[0048] (3) Target gene: The Streptococcus pneumoniae plsY gene (i.e., spPlsY gene) synthesized through codon optimization, such as Figure 2 As shown. The specific sequence is as follows: plsY sequence before optimization: Atgattacaatagttttattaatcctagcctatctgctgggttcgattccatctggtctctggattggacaagtattctttcaaatcaatctacgcgagcatggttctggtaacactggaacgaccaataccttccgcattttaggtaagaaagctggtatggcaacctttgtgattgactttttcaaaggaaccctagcaacgctgcttccgattatttttcatctacaaggcgtttctcctctcatctttggacttttggctgttatcggccataccttccctatctttgcaggatttaaaggtggtaaggctgtcgcaaccagtgctggagtaattttcggatttgcgcctatcttctgtctctaccttgcgattatcttctttggagctctctatcttggcagtatgatttcactgtctagtgtcacagcatcgatcgcggctgttatcggggttctgctctttccactttttggttttatcctgagtaactatgactctctcttcatcgctattatcttagcacttgctagtttgattatcattcgtcataaggacaatatagctcgtatcaaaaataaaactgaaaatttggtcccttggggattgaacctaacccatcaagatcctaaaaaataa.

[0049] Optimized plsY sequence: .

[0050] (4) Culture medium: M9 medium (containing 50 μg / mL kanamycin).

[0051] (5) Inducer: Isopropyl-β-D-thiogalactoside (IPTG).

[0052] (6) Buffer A (resuspending / lysis buffer): 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM MPMSF, 10 μg / mL DNase I.

[0053] (7) Buffer B (solubilizing / binding buffer): 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% (w / v) n-dodecyl-β-D-maltodextrin (DDM), 1 mM PMSF.

[0054] (8) 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.

[0055] (9) Eluent: Buffer C is supplemented with 5 mM d-desulfobiotin.

[0056] (10) Affinity chromatography medium: Strep-Tactin resin.

[0057] 1.2 Instruments and Consumables: Constant temperature shaker; high pressure homogenizer; high speed centrifuge and ultracentrifuge (Beckman Coulter); chromatography column and protein purification system (BIO-RAD); Nanodrop; protein concentration centrifuge tubes (Merck Millipore Amicon® Ultra).

[0058] 2. Experimental Methods 2.1 Construction of Expression Strains 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. The inoculum was then transferred to M9 basal medium at a 1:100 inoculum ratio 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.

[0059] 2.2 Cell Harvesting and Membrane Component Preparation: Cells were collected by centrifugation at 4500 ×g for 15 minutes at room temperature and resuspended in pre-cooled buffer A. Cells were then homogenized using an autoclave at 4°C and 600 bar. The resulting lysate was then centrifuged at 20,000 ×g for 30 minutes at 4°C to remove cell debris. Finally, the supernatant was collected and ultracentrifuged at 48,000 ×g for 2 hours at 4°C. The resulting precipitate was the cell membrane component.

[0060] 2.3 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 then 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.

[0061] 2.4 Protein Elution and Concentration: Elute the target protein with 5 column volumes of eluent and collect the elution peak. Evaluate the purity and yield of the purified protein using SDS-PAGE and Nanodrop.

[0062] 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.

[0063] Example 2: Enzyme activity assay of spPlsY in detergent environment 1. Experimental Materials 1.1 Proteins and Reagents: (1) Purified Streptococcus pneumoniae spPlsY protein (purified in Example 1).

[0064] (2) Experimental system buffer (Assay buffer): 150 mM NaCl, 100 mM Tris-HCl 8.5, 0.03% LMNG.

[0065] (3) Substrate: Acyl-P (diluted in Assay buffer, 25 μM).

[0066] (4) Substrate: Glycerol-3-phosphate (G3P, diluted in Assay buffer, 20 mM).

[0067] (5) Fluorescent probe: MDCC-labeled E. coli phosphate-binding protein (MDCC-ecPBP, which responds to changes in inorganic phosphate concentration); its preparation method is described in the literature: 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 ATPase. Biochemistry 33, 8262–8271 (1994). Specifically, it includes the following steps: ① Purification of phosphate-binding proteins: The coding sequence of phosphate-binding protein (PBP) from glutamate position 26 to tyrosine position 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 actomyosin subfragment 1 ATPase. Biochemistry 33, 8262–8271 (1994).). The *E. coli* BL21(DE3) strain carrying this plasmid was tested at OD... 600 When the pH value reached 0.6–0.8, expression was induced using 1 mM IPTG, and then the cells were placed in buffer G (containing 0.5 mM TECP, 1 mM PMSF, 8 mM MgCl2, 10 μg / mL). -1DNase I (20 mM Tris-HCl, pH 8.0) was used for cell lysis using a cell disruptor. The resulting lysate was centrifuged to remove debris and then incubated with 10 mL Ni-NTA resin for 2 hours. The resin was then washed with 1 L of buffer H (10 mM Tris-HCl, pH 8.0) and 150 mL of buffer H containing 40 mM imidazole. Proteins were then eluted with buffer H containing 250 mM imidazole, desalted, and loaded onto a 5 mL HiTrap QSepharose FF column pre-equilibrated with buffer H, at a flow rate of 1 mL / min. -1 At a flow rate of [value missing], gradient elution was performed using 40 mL of buffer H containing 0-100 mM NaCl, and the PBP was collected and concentrated to 32.5 mg / mL. -1 Then, it was flash-frozen with liquid nitrogen and stored at -80 °C.

[0068] ②Fluorescent labeling of PBP: The following steps were used to label the thiol-reactive fluorescent probe N-[2-(1-maleimino)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC) to the PBP A197C mutant: First, impurity phosphate was removed from a 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 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. After the reaction, unreacted MDCC was removed using a Superdex 200 10 / 300GL column pre-equilibrated with 10 mM Tris-HCl (pH 8.0). Labeled and unlabeled PBP were then separated using a 5 mL HiTrap Q Sepharose FF column, followed by elution with a 200 mL 0-250 mM NaCl gradient at a flow rate of 1 mL / min. -1After collecting MDCC-PBP, its concentration was calculated by correcting the absorbance of MDCC to A280. The calculation formula is referenced in (Brune, M., Hunter, JL, Corrie, JET & Webb, MR Direct, real-time measurement of rapid organic 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-ecPBP was concentrated to 3.7 mg / mL. -1 It was flash-frozen with liquid nitrogen and stored at -80 °C.

[0069] (6) Standard: Sodium dihydrogen phosphate (NaH2PO4, 25 μM aqueous solution, used to standardize the response of MDCC-ecPBP to inorganic phosphoric acid).

[0070] (7) Solvent control: Dimethyl sulfoxide (DMSO).

[0071] 1.2 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; a black-bottomed, transparent 96-well half-plate (Corning 3880); a multi-channel pipette and corresponding tips.

[0072] 2. Experimental Methods Add an appropriate amount of assay buffer to a 96-well half-well plate, then add purified spPlsY to 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 ratio of product formation to fluorescence value change using 1 μM NaH2PO4. Add 5 μL of 20 mM G3P to start the reaction, with a final reaction volume of 45 μL. Read every 30 seconds for a total of 40 min.

[0073] 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.

[0074] 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).

[0075] Example 3: Half-maximal inhibitory concentration (IC5) of compound T5 50 ) Measurement To characterize the enzymatic inhibitory effect of compound T5, the IC50 of this compound was measured. 50 The value was measured.

[0076] 1.1 Experimental Materials (1) Reagents: Concentration gradient solutions for the test compounds: 50 mM T5 (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). All other reagents were identical to those listed in Part 1 of Example 2 (including spPlsY protein, Acyl-P, G3P, MDCC-ecPBP, and a DMSO-containing solvent control, etc.).

[0077] The molecular formula of T5 is: C 28 H 16 Cl3NO4; The structural formula is: O=C1N(C(=C / C1=C\C=2OC(=CC=2)C=3 / C=C( / [Cl])C([Cl])=CC=3)C4=CC=CC=C4)C5=CC(C(=O)O)=C([Cl])C=C5; .

[0078] (2) Instruments and consumables: Completely consistent with the contents listed in section 1.2 of Example 2 (including multi-functional microplate reader, black 96-well half-well plate, multi-channel pipette, etc.).

[0079] 2. Experimental Methods In a 96-well half-well plate, add the following in sequence: Assay buffer, acyl-P (final concentration 2.5 μM), MDCC-ecPBP (final concentration 8 μM), the analyte compound (final concentrations of 0, 6.25, 12.5, 25, 50, and 100 μM), and spPlsY protein (final concentration 0.02 μg / mL). Perform replicates for each concentration. Add an equal volume of the corresponding DMSO solution (without the compound) to the solvent control wells. Set the microplate reader to 26℃ and read the fluorescence using bottom-reading (excitation / emission wavelengths 425 / 466 nm). To initiate the experiment, add G3P (final concentration 2 mM), with a final system volume of 50 μL. Read the fluorescence every 30 seconds for 40 minutes.

[0080] 3. Data Analysis Calculate the reaction rate at each compound concentration. Define the enzyme activity at 0 μM of the compound as 100%, and normalize the enzyme activity at other concentrations based on this (calculate the relative percentage of enzyme activity).

[0081] 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.

[0082] 4. Experimental Results The results showed that plotting and fitting the relative reaction rate against concentration revealed that the half-inhibition concentration (IC5) of compound T5 was... 50 30.3 μM ( Figure 5 It has value for further research and improvement.

[0083] Example 4: Determination of the minimum inhibitory concentration (MIC) of T5 compound - human pathogenic bacteria To investigate the antibacterial effect of compound T5, the growth capacity of five human pathogens was tested in the presence of different concentrations of compound T5. Because pathogens were involved, all the following procedures were performed in a P2 laboratory.

[0084] 1. Experimental Materials 1.1 Strains and Reagents: (1) Glycerol-preserved strains: methicillin-sensitive Staphylococcus aureus RN4220, methicillin-resistant Staphylococcus aureus MRSA-USA300, Staphylococcus epidermidis ATCC1457, Streptococcus pneumoniae ATCC49619, and Streptococcus pyogenes ATCC12344. Source: Purchased from ATCC.

[0085] (2) CAMHB liquid culture medium.

[0086] (3) Test compound T5: 26.84 mg / mL stock solution prepared with DMSO.

[0087] (4) MTT solution (5 mg / mL, dissolved in PBS or deionized water, diluted to the required concentration before use).

[0088] (5) Negative control: CAMHB medium containing 0.238% DMSO.

[0089] (6) Sterile physiological saline or CAMHB medium (for dilution of bacterial solution).

[0090] 1.2 Instruments and Consumables: 37℃ constant temperature incubator; ELISA reader; sterile 96-well cell culture plate (full-well plate); biosafety cabinet; constant temperature shaker; pipettes and sterile pipette tips.

[0091] 2. Experimental Methods (1) Preparation of bacterial culture: Five types of glycerol bacteria stored at -80℃ were rapidly streaked onto CAMHB plates and incubated at 37℃ for about 16 hours. Single clones were picked and transferred to CAMHB, and cultured overnight at 37℃ with shaking. Then, they were transferred to the same medium at a 1:100 dilution and cultured with shaking until the OD reached the target value. 600 Approximately 0.6, then dilute the bacterial culture to OD using CAMHB medium. 600 = 0.002, reserved.

[0092] (2) Compound dilution and plate preparation: The 26.84 mg / mL DMSO stock solution of compound T5 was diluted to 64 μg / mL using CAMHB medium. This intermediate solution was mixed with CAMHB medium containing 0.238% DMSO at different ratios to prepare a series of dilutions with concentrations of 0, 1, 2, 4, 8, 16, and 32 μg / mL (the final volume of each concentration must meet the requirements for subsequent sample loading). 50 μL of the above-mentioned compound dilution at one concentration was added to each well of a sterile 96-well plate.

[0093] (3) Inoculation and culture: Add 50 μL OD to each well 600 =0.002% bacterial suspension to make the final volume of each well 100 μL, with final compound concentrations of 0, 0.5, 1, 2, 4, 8, 16, and 32 μg / mL. Wells containing no compound and only an equal volume of 0.238% DMSO and bacterial suspension were set up as growth controls. After sealing the plates with breathable sealing film, they were incubated at 37°C for approximately 16-18 hours.

[0094] (4) Activity detection and reading: After the culture was completed, 10 μL of MTT solution was added to each well (to make the final concentration approximately 0.5 mg / mL). After culturing for another 30 minutes, the absorbance (OD) of each well at 595 nm was measured using a microplate reader. 595 Record the OD values ​​of each compound concentration well and the control well. 595 Values. Using OriginPro software, the final concentration of the compound is plotted on the x-axis, corresponding to the OD value. 595 Use the values ​​as the ordinate to plot a scatter plot.

[0095] 3. Experimental Results The results showed that the MIC value of compound T5 against all five pathogenic bacteria was 4 μg / mL. Figure 6 ).

[0096] Example 5: Determination of the minimum inhibitory concentration of compound T5 - Tomato Ulcer Bacterium To further investigate the antibacterial effect of compound T5 on plant pathogens, the minimum inhibitory concentration (MIC) of Clavibactermichiganensis subsp. Michiganensis was determined.

[0097] 1. Experimental Materials 1.1 Strains and Reagents: (1) Test strain: Clavibacter michiganensis subsp. michiganensis (strain number JCM1370), which was cryopreserved with glycerol; the strain was purchased from Taisto Biotechnology Co., Ltd.

[0098] (2) TSA + 5% sheep blood plate (tryptone soybean agar + 5% defibrinated sheep blood); (3) TSB liquid culture medium.

[0099] (4) Test compound T5: 26.84 mg / mL stock solution prepared with DMSO.

[0100] (5) MTT solution (5 mg / mL, dissolved in PBS).

[0101] (6) Negative control: TSB medium containing 0.2% DMSO.

[0102] 1.2 Instruments and Consumables: 37℃ constant temperature incubator; ELISA reader; sterile 96-well cell culture plate (full-well plate); biosafety cabinet; constant temperature shaker; pipettes and sterile pipette tips.

[0103] 2. Experimental Methods (1) Preparation of bacterial culture: The glycerol bacillus of *Tomato Ulcer Bacterium* stored at -80℃ was rapidly streaked onto TSA+5% sheep blood agar plates and cultured overnight at 28℃. Single clones were picked and cultured overnight on TSB medium for about 20 h. The next day, they were transferred to fresh medium at a dilution of 1:100 and cultured until OD. 600 Approximately 0.4.

[0104] (2) Compound dilution and plate preparation: The 26.84 mg / mL DMSO stock solution of compound T5 was diluted to 53.7 μg / mL using TSB medium. 50 μL of this concentration of compound was added to a sterile 96-well plate.

[0105] (3) Inoculation and culture: Add 50 μL OD to each well 600 =0.002 JCM1370 bacterial suspension to make the final volume of each well 100 μL, at which point the T5 concentration is 26.8 ug / mL. A well containing only an equal volume of 0.2% DMSO and bacterial suspension served as a growth control. After sealing the plate with breathable sealing film, it was incubated at 37°C for approximately 20 hours.

[0106] (4) Activity detection and reading: After the culture was completed, 10 μL of MTT solution was added to each well (to make the final concentration approximately 0.5 mg / mL). After culturing for another 30 minutes, the absorbance (OD) of each well at 595 nm was measured using a microplate reader. 595 Record the OD values ​​of each compound concentration well and the control well. 595 value.

[0107] 3. Experimental Results The results showed that 26.8 ug / mL of compound T5 could completely inhibit the growth of *Bacillus cankerii* in tomato. Figure 7 ).

[0108] In summary, to avoid the drawbacks of cross-resistance caused by the homogeneity of existing antibacterial drug targets, this invention develops a novel antibacterial research and development scheme targeting a novel target. Using the key bacterial phospholipid synthesis enzyme PlsY as the target site, the screened compound T5 can potently inhibit PlsY bioactivity and significantly antagonize pathogenic strains such as Streptococcus pneumoniae and Staphylococcus aureus. Since the human body does not possess PlsY homologous proteins, the safety window for drug use is improved. The chemical structure of this molecule differs from known antibiotics and substrate analogs, facilitating structural optimization based on structure-activity relationships. Furthermore, it exhibits inhibitory activity against various crop pathogenic bacteria, demonstrating broad application value. Therefore, this invention pioneers a new path for PlsY-targeted antibacterial research and development, providing a differentiated leading framework for the prevention and control of drug-resistant bacterial infections, and possesses promising development prospects in both human anti-infective drugs and agricultural fungicides.

[0109] 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. The application of small molecule compound T5 in the preparation of PlsY inhibitors, characterized in that, The PlsY inhibitor is used to prepare a drug that inhibits pathogenic bacteria in humans. The structure of the small molecule compound T5 is shown below: 。 2. The application according to claim 1, characterized in that, The human pathogenic bacteria are at least one of methicillin-sensitive Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, or Streptococcus pyogenes.

3. The application according to claim 1, 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.

4. The application according to claim 3, characterized in that, The dosage form of the drug is an oral preparation, an injectable preparation, an inhaled preparation, or a transdermal preparation.

5. The application of small molecule compound T5 in the preparation of PlsY inhibitors, characterized in that, The PlsY inhibitor is used to prepare agricultural fungicides that inhibit plant pathogens. The structure of the small molecule compound T5 is shown below: 。 6. The application according to claim 5, characterized in that, The plant pathogen is *Tomato Ulcer Bacterium*.

7. The application according to claim 5, characterized in that, The agricultural fungicide also includes pesticide-acceptable excipients selected from at least one of water, dispersants, wetting agents, fillers, stabilizers, cosolvents, adhesives, and defoamers.

8. The application according to claim 7, characterized in that, The formulation of the agricultural fungicide is an aqueous solution, suspension, wettable powder, granule, or emulsifiable concentrate.

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