A compound targeting staphylococcus aureus pgl or gnd enzyme and use thereof

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a compound targeting Staphylococcus aureus PGL or GND enzymes and its applications. Based on the high-resolution three-dimensional structures of Staphylococcus aureus GND and PGL proteins and the identification of their enzyme active centers, this invention uses molecular docking to virtually screen for novel small molecule compounds that may target these two proteins. These compounds are the first successfully screened novel small molecule compounds with GND or PGL inhibitory activity. Their application against Staphylococcus aureus is reported for the first time, and it is also the first time that these compounds exert their antibacterial activity by targeting Staphylococcus aureus PPP pathway metabolic enzymes. This discovery provides a new candidate entity with a clear target for the treatment of drug-resistant Staphylococcus aureus infections.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a compound that targets Staphylococcus aureus PGL or GND enzymes and its applications. Background Technology

[0002] staphylococcus( Staphylococcus Staphylococcus spp. is a Gram-positive, non-motile, coagulase-producing spherical bacterium belonging to the phylum Firmicutes. To date, the genus *Staphylococcus* has been identified, comprising 52 species and 28 subspecies, among which *Staphylococcus aureus* (*S. aureus*) is the most abundant. Staphylococcus aureus Due to its high pathogenicity, resistance to multiple antibiotics, and the presence of various drug resistance genes, it has become one of the most clinically significant conditional pathogens.

[0003] Staphylococcus aureus, due to its strong adaptability, can colonize the human nasal cavity and skin mucosa in a symbiotic manner. When the body's immunity is low or the skin and mucosal barriers are damaged due to chronic skin diseases, wounds, or surgical interventions, this bacterium can enter underlying tissues or the bloodstream, causing various diseases and even systemic infections, such as skin and soft tissue infections, sepsis, osteomyelitis, peritonitis, pneumonia, and endocarditis. From onset to resolution or potential host death, infection is inherent and dynamic, and this versatility manifests as physiological adaptation to a constantly changing environment. In addition to its own secreted toxin proteins that can directly affect host health, Staphylococcus aureus can also disrupt the microbial composition of host tissues, mucosa, or body surfaces by expressing and secreting various enzymes, ultimately causing host mucosal immune dysfunction and increasing the host's risk of infection with other pathogens. The mechanisms involved may include surface colonization, competitive acquisition of nutrients, synthesis and secretion of antimicrobial substances, and activation of specific host defense mechanisms. The toxins secreted by Staphylococcus aureus can induce the upregulation of antimicrobial peptide expression in the body, further killing other bacteria and causing dysbiosis of the host's normal flora, thus becoming the dominant strain and posing further harm to the body. The bacterial cell wall forms the interface between Staphylococcus aureus and the human host; this component is not only crucial for bacterial survival but also remains dynamic during growth and interacts with the host's immune system. Furthermore, Staphylococcus aureus can form biofilms, making patients with invasive medical devices (such as peripheral and central venous catheters and urinary catheters) more susceptible to Staphylococcus aureus infection, significantly increasing the difficulty of treating infections or diseases caused by this bacterium in clinical practice. According to literature and clinical reports, the incidence of diseases caused by Staphylococcus aureus varies depending on the size and demographics of the study population, sampling quality, and the culture techniques used, but the number of infections and deaths caused by this coccus continues to rise annually.

[0004] In recent years, the resistance rate of Staphylococcus aureus to mupirocin has increased significantly. While the resistance rate has remained around 10% in many countries, it has reached 30% or even higher in some regions of the United States and Asia. Therefore, there is an urgent need to develop novel antibacterial strategies or agents to kill or eliminate overgrowth of Staphylococcus aureus in the body. However, new drug development is a lengthy process with high time and human resource costs, making this strategy equally challenging. Currently, developing novel antibacterial inhibitors to reverse existing resistance in Staphylococcus aureus has become a novel and important antibacterial strategy. Although some inhibitors cannot completely eradicate Staphylococcus aureus, they can cause disruption or damage to the normal physiological functions of the bacteria. Combining these inhibitors with existing or resistant antibiotics can also achieve the goal of killing the bacteria—a process known as "drug repurposing."

[0005] Currently, the Newman strain of Staphylococcus aureus (NEWMAN) was first isolated from human infection samples in 1952. Due to its clear genome, ease of genetic manipulation, and stable phenotypic characteristics, it has become the laboratory model strain. This strain carries typical virulence factors (such as α-hemolysin, protein A, and coagulase). Methicillin-sensitive Staphylococcus aureus (MSSA) is widely used for establishing animal models of staphylococcal diseases because it lacks virulence islands compared to clinically prevalent strains. Methicillin-resistant Staphylococcus aureus (MRSA)... Staphylococcus aureus Methicillin (MRSA) was first discovered in the UK in 1961. At that time, it was widely used in the treatment of clinical patients with infections. However, due to its high toxicity, it has been largely replaced by penicillin-type drugs of the same type and more stable, and is no longer used for patient treatment, such as oxacillin, flucloxacillin, and dicloxacillin.

[0006] Furthermore, vancomycin holds a significant position as the first-line drug for treating Staphylococcus aureus infections, especially those caused by MRSA. Since its clinical introduction in the 1950s, it has significantly reduced the morbidity and mortality of MRSA-related sepsis, endocarditis, and osteomyelitis. However, with the increasing prevalence of vancomycin heterogeneous resistance (hVISA) and intermediate resistance (VISA) strains, its efficacy faces considerable challenges. Intravenous administration of vancomycin, daptomycin, or linezolid is commonly used to treat MRSA. Clindamycin, trimethoprim-sulfamethoxazole, and doxycycline are alternative drugs for treating mild to moderate Staphylococcus aureus infections, depending on antibiotic susceptibility testing. For uncomplicated skin abscesses, the use of clindamycin or trimethoprim-sulfamethoxazole combined with incision drainage has been shown to improve clinical cure rates in emergency departments and other outpatient settings. Oral therapy should generally be avoided in the initial treatment phase of severe infections. Current clinical treatment recommendations for severe MRSA infection include intravenous vancomycin or daptomycin for bacteremia and intravenous vancomycin or linezolid for hospital-acquired pneumonia. For severe infections, oral linezolid should not be the initial treatment; it is recommended to switch to oral linezolid only if the patient's condition is stable and tolerates the oral route. Because Staphylococcus aureus can form biofilms, patients with medical devices (such as central venous catheters, urethral catheters, etc.) and associated infections often need to have these devices removed, if possible, to achieve successful treatment.

[0007] It is evident that despite the availability of a wide variety of antibiotics in clinical practice, in the decade following its initial discovery, MRSA continued to cause outbreaks and deaths in hospitals worldwide, and the term "methicillin-resistant Staphylococcus aureus" remained in use. While infections caused by this type of Staphylococcus aureus initially occurred primarily in hospitals or communities, researchers later discovered a shift in the prevalence of MRSA. The bacteria could also be detected in populations without prior healthcare exposure, particularly in Australian Aboriginal populations in the 1980s and in healthy adults and children in the United States in the 1990s. Since the mid-2000s, infections and diseases caused by this bacterium in various livestock have been frequently reported. Besides methicillin resistance, MRSA often exhibits resistance to other types of antibiotics. In fact, Staphylococcus aureus can acquire resistance genes through plasmid transfer or transposons, potentially leading to resistance to any antibiotic. This presents significant challenges and difficulties in selecting treatments for Staphylococcus aureus, both now and in the future.

[0008] In recent years, there have been numerous reports on the development of antibacterial or bacteriostatic agents targeting the virulence factors or key enzyme molecules of Staphylococcus aureus. Researchers have found that the important sorting enzyme A (SrtA) and the important toxin protein α-hemolysin, which are involved in the adhesion and invasion processes of Staphylococcus aureus, play a crucial role in bacterial infection. Therefore, the flavonoid compound isopyranin was synthesized targeting SrtA. This compound can effectively inhibit the activity of Staphylococcus aureus SrtA and further affect the normal physiological processes of the bacteria, ultimately reducing the infection efficiency. The results also indicate that although this compound cannot completely eliminate the bacteria in the body, it can effectively reduce the morbidity rate of the host after infection with this bacterium. In addition, Staphylococcus aureus shikimate dehydrogenase (SaSDH) plays a vital role in the growth of Staphylococcus aureus, and this enzyme is absent in mammals; therefore, SaSDH is an important target for the development of antibacterial drugs. Researchers have found that gallate and rhodiola rosea are inhibitors of this bacterium, and in vitro and in vivo studies show that both inhibitors have good antibacterial activity. Furthermore, SaeR, together with the histidine kinase SeaS, forms a two-component system that is a response regulator of Staphylococcus aureus. Multiple studies have shown that the absence of this regulator leads to a significant reduction in the infection efficiency of Staphylococcus aureus. This also indicates that SaeR has potential application value as a target for antibacterial agents. Researchers designed corresponding inhibitor molecules and verified their antibacterial activity in vitro and in vivo. The results showed that using inhibitors targeting SaeR can effectively reduce the infection efficiency of Staphylococcus aureus.

[0009] Therefore, the field looks forward to developing more compounds with anti-Staphylococcus aureus properties, which can effectively alleviate the limitations of drug resistance. Summary of the Invention

[0010] The first objective of this invention is to provide a compound that targets Staphylococcus aureus PGL or GND enzymes, said compound can target and inhibit the expression of Staphylococcus aureus PGL or GND enzymes, thereby inhibiting Staphylococcus aureus by inhibiting the pentose phosphate pathway; A second object of the present invention is to provide a pharmaceutical composition for treating Staphylococcus aureus.

[0011] To address the aforementioned technical problems, the present invention provides a compound targeting Staphylococcus aureus PGL enzyme, the compound comprising at least one of the following structural compounds: , .

[0012] (PGL_8)(PGL_9) The present invention also provides the use of the compound in the preparation of a Staphylococcus aureus PGL enzyme inhibitor.

[0013] The present invention also provides a compound that targets Staphylococcus aureus GND enzyme, said compound comprising at least one of the following structural compounds: , , .

[0014] (GND_4) (GND_6) (GND_7) The present invention also provides the use of the compound in the preparation of an inhibitor targeting Staphylococcus aureus GND enzyme.

[0015] The present invention also provides a pharmaceutical composition comprising the compound targeting Staphylococcus aureus PGL enzyme and / or the compound targeting Staphylococcus aureus GND enzyme.

[0016] Specifically, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0017] Specifically, the dosage forms of the pharmaceutical composition include non-gastrointestinal dosage forms and / or gastrointestinal dosage forms.

[0018] Specifically, in the pharmaceutical composition, the non-gastrointestinal dosage form includes at least one of the following: injection dosage form, cavity dosage form, mucosal dosage form, and skin dosage form; Specifically, the drug composition includes at least one of the following dosage forms for gastrointestinal administration: tablets, granules, capsules, solutions, powders, sustained-release preparations, emulsions, suspensions, syrups, and drops.

[0019] The present invention also provides the use of the compound that targets Staphylococcus aureus PGL enzyme or the compound that targets Staphylococcus aureus GND enzyme or the pharmaceutical composition for the preparation of an anti-Staphylococcus aureus drug.

[0020] This invention also provides the use of a Staphylococcus aureus PGL enzyme inhibitor or a Staphylococcus aureus GND enzyme inhibitor for the preparation of an anti-Staphylococcus aureus drug; wherein, The Staphylococcus aureus PGL enzyme inhibitor includes the compound that targets Staphylococcus aureus PGL enzyme; The Staphylococcus aureus GND enzyme inhibitor includes the compound that targets Staphylococcus aureus GND enzyme.

[0021] This invention is based on research into the pentose phosphate pathway of Staphylococcus aureus, targeting key enzymes in this pathway—GND and PGL enzymes—to screen for safe and effective unknown small-molecule antibacterial compounds. The main functions of GND and PGL enzymes are to provide substances and energy for bacterial proliferation and host infection. Targeting these two metabolic enzymes can reduce their enzyme activity, further disrupting downstream physiological processes and directly or indirectly inducing bacterial death. This invention, targeting GND and PGL enzymes of Staphylococcus aureus, screens for safe and effective unknown small-molecule antibacterial compounds, which can mitigate the risk of drug-resistant strains spreading drug-resistant genes.

[0022] This invention is based on Ag + This invention aims to exert antibacterial activity by targeting and binding to key enzymes (GND and PGL) in the metabolic pathway of Staphylococcus aureus. The crystal structures of these enzymes are resolved at high resolution, and their active sites are identified. Subsequently, molecular docking methods are used for high-throughput prediction of small molecule compounds with potential antibacterial activity. This invention utilizes virtual screening methods such as molecular docking, ranking compounds according to their docking scores, and combining molecular structural characteristics, physicochemical properties, and drug-likeness to screen for potential antibacterial small molecules. Further screening is then conducted on 12 small molecule compounds (5 targeting GND and 7 targeting PGL) that have not yet been reported for anti-Staphylococcus aureus activity, performing antibacterial experiments. For unknown small molecule compounds with good antibacterial effects, further enzyme activity experiments, safety, and efficacy evaluations are performed.

[0023] This invention predicts and preliminarily verifies 12 previously unreported small molecule inhibitors against Staphylococcus aureus (5 targeting GND and 7 targeting PGL). Antibacterial activity tests were then performed, further screening to identify 5 compounds with antibacterial activity (3 targeting GND and 2 targeting PGL). Enzyme inhibition experiments and safety evaluations were then conducted on these compounds, ultimately validating a safe and effective antibacterial inhibitor targeting both GND and PGL against Staphylococcus aureus. This discovery provides a novel candidate with a clearly defined target for combating drug-resistant bacterial infections and expands the antibiotic development pipeline.

[0024] This invention uses key enzymes required for the growth of Staphylococcus aureus as targets for antibacterial agents, specifically GND enzymes and / or PGL enzymes, to screen for drugs. The screening yields compounds that inhibit GND and / or PGL enzymes. These inhibitors exhibit significant inhibitory activity against Staphylococcus aureus, effectively addressing the problems of low in vitro and in vivo activity, poor stability, and easy induction of bacterial resistance in existing inhibitors designed targeting molecules such as SrtA, SaSDH, and SaeR. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of this disclosure, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This refers to the pentose phosphate pathway (PPP) metabolic process and the three-dimensional conformation and amino acid sequence of the protein; where A: pentose phosphate pathway (PPP) metabolic process; B: three-dimensional conformation of GND protein; C: amino acid sequence of GND protein; D: three-dimensional conformation of PGL protein; E: amino acid sequence of PGL protein; Figure 2 (including) Figure 2-1 , Figure 2-2 , Figure 2-3 , Figure 2-4 , Figure 2-5 , Figure 2-6 The results show the antibacterial activity of different small molecule compounds against Staphylococcus aureus (Newman strain). Figure 3 The results show the enzyme inhibitory activities of different small molecule compounds on GND / PGL; Figure 4 Data on the efficacy of combined medication. Detailed Implementation

[0027] In the following embodiments of the present invention, antibacterial drugs were screened and validated based on the key proteins GND enzyme and PGL enzyme of the pentose phosphate pathway. Firstly, Ag was identified using liquid chromatography-gel electrophoresis-inductively coupled plasma mass spectrometry (LC-GE-ICP-MS). + The target proteins (GND and PGL) were identified, and the crystal structures of the two enzymes were resolved at high resolution and their active sites were identified. Subsequently, molecular docking was used to screen for compounds that could potentially bind to GND or PGL, i.e. Staphylococcus aureus inhibitors. The screened inhibitors were then subjected to in vitro antibacterial activity assays, enzyme activity experiments, and in vitro safety evaluations to assess the potential of small molecule compounds as anti-Staphylococcus aureus inhibitors.

[0028] Previous research on this invention has revealed that the pentose phosphate pathway (PPP) is a form of glucose oxidative degradation that occurs in the cytoplasm. Because this pathway begins with glucose-6-phosphate (G-6-P), it is also known as the hexose phosphate bypass. This process involves the metabolism of one molecule of glucose-6-phosphate to produce NADPH and ribose-5-phosphate, which function by degrading carbohydrates. PPP comprises two phases: oxidative and non-oxidative. In the oxidative phase, glucose-6-phosphate is converted to ribulose-5-phosphate and CO2, generating two molecules of NADPH. In the non-oxidative phase, ribulose-5-phosphate is isomerized to ribose-5-phosphate or converted to two intermediate metabolites in glycolysis, fructose-6-phosphate and glyceraldehyde-3-phosphate, which can then re-enter the glycolytic pathway for metabolism and energy supply. The two dehydrogenation steps in the oxidation phase of the pentose phosphate pathway are irreversible under physiological conditions and are the rate-limiting reactions of the entire pathway. G6PDH and 6PGDH (GND), which catalyze these two steps, are both rate-limiting enzymes in this pathway. Besides being regulated by G6PDH and GND, the pentose phosphate pathway is also regulated by intracellular NADPH. When [NADPH] / [NADP]... + An excessively high ratio of NADP to 6-phosphate gluconate will inhibit the activity of both enzymes. GND catalyzes the oxidative decarboxylation of 6-phosphate gluconate to ribulose 5-phosphate (R5P) and CO2, while simultaneously releasing NADP. + Reduction to NADPH further affects cellular oxidative stress homeostasis. In the PPP pathway, PGL is primarily responsible for catalyzing the hydrolysis of 6-phosphogluconeolactone to produce 6-phosphogluconic acid. The pentose phosphate pathway reaction process is as follows... Figure 1 As shown in Figure A.

[0029] Based on the preliminary research of this scheme, Ag + It has the ability to inhibit S. aureus Activity, the main antibacterial mechanism is Ag + It can target and inhibit the activity of the two enzymes GND and PGL, thereby affecting the energy required for normal bacterial physiological processes and the synthesis of substances such as nucleic acids. Therefore, it is speculated that the development of small molecule inhibitors targeting GND and PGL may be beneficial for the treatment of diseases caused by GND and PGL. S. aureus The resulting infection has significant clinical implications.

[0030] This invention resolves the protein structures of GND and PGL from Staphylococcus aureus Newman strain. The crystal structure of GND has been uploaded to the PDB database (7CB0), while the protein structure of PGL has not yet been publicly disclosed. The GND protein contains 468 amino acids, has a molecular weight of approximately 51 kDa, and does not contain a signal peptide or transmembrane region. Its structure is primarily composed of α-helices and random coils. Its structure and amino acid sequence are as follows: Figure 1 As shown in Figures B and C. The PGL protein contains 342 amino acids, has a molecular weight of approximately 38 kDa, and its structure consists of seven consecutive β-plates arranged symmetrically along the pseudo-C2 crystal axis, forming a β-propeller structure. Its structure and amino acid sequence are shown in Figures B and C, respectively. Figure 1 As shown in D and E in the diagram.

[0031] The detailed technical solutions of the present invention are shown in the following embodiments.

[0032] Example 1 This embodiment screens antibacterial agents targeting Staphylococcus aureus GND and PGL enzymes.

[0033] In this embodiment, a virtual screening method was used to screen for potential antibacterial agents targeting Staphylococcus aureus GND and PGL proteins, and AutoDock Vina was used for screening. A total of 100,000 small molecules were collected from public drug databases and small molecule databases. Using the Chemoinformatics Python library RDKit in Python 2.7, a universal force field (UFF) was used to generate the three-dimensional conformations of these compounds. The Python script "prepare_ligand4.py" developed using AutoDockTools was used to convert the small molecule structures into the PDBQT format required for the AutoDock Vina virtual screening process. Using Staphylococcus aureus GND and PGL proteins as docking acceptors, MGLTool was used to convert the acceptor PDB structures into PDBQT format. AutoDock Vina was then run to predict the potential activity of the compounds based on their docking scores with GND or PGL.

[0034] In this embodiment, 26 compounds were selected based on their top ranking. Among them, 14 compounds targeted GND and were named GND_1-GND_14, and 12 compounds targeted PGL and were named PGL_1-PGL_12. The specific compound structure information is shown in Table 1 below.

[0035] Table 1 Information on screened compounds

[0036] Of the compounds mentioned above, GND_1, GND_2, GND_3, GND_5, GND_8, GND_9, GND_10, GND_11, GND_14, PGL_1, PGL_2, PGL_3, PGL_4, and PGL_5 are known drugs; the remaining 12 (5 targeting GND and 7 targeting PGL) are small molecule compounds that have not been previously reported to be effective against Staphylococcus aureus.

[0037] In this embodiment, potential antimicrobial compounds targeting Staphylococcus aureus GND and PGL proteins were screened using a virtual screening method. Based on the docking scores of the compounds with GND or PGL, combined with structural characteristics, physicochemical properties and drug-likeness analysis, the docking scores of the 12 small molecule compounds (5 targeting GND and 7 targeting PGL) that had not been previously reported to be applicable to the fight against Staphylococcus aureus were shown in Table 2 below.

[0038] Table 2. Docking scores of candidate small molecule compounds for antibacterial activity assay

[0039] It is evident that, except for PGL_7, PGL_10, PGL_11, and PGL_12, which are analogs of products (6PG) catalyzed by PGL enzymes, the docking fractions of the remaining compounds with their corresponding enzymes are ≤ -7.0 kcal / mol, indicating that the compounds bind strongly to their corresponding enzymes. In particular, the docking fractions of compounds GND_4, GND_7, and GND_13 are < -9.0 kcal / mol, indicating that the compounds have extremely strong binding ability to their corresponding enzymes.

[0040] Example 2 This embodiment tests the antibacterial activity of 12 previously unreported small molecule compound inhibitors that were screened and can be used against Staphylococcus aureus.

[0041] Staphylococcus aureus (Newman strain) frozen at -80 ℃ was streaked onto BHI agar medium and incubated at 37 ℃ for 16-18 h. Single colonies were picked and inoculated into BHI broth medium and cultured to the logarithmic growth phase (37 ℃, 200 rpm). The bacterial cells were then recovered by centrifugation (3000 g, 2 min) and resuspended in BHI broth medium. The bacterial turbidity was adjusted to 5 × 10⁻⁶. 5CFU / mL was used for subsequent antibacterial experiments. Various compound molecules were dissolved in their respective solvents and diluted to different concentrations using BHI broth. 50 µL of the compound solution and an equal volume of bacterial culture were added to sterile 96-well cell culture plates. Negative controls (bacterial group) and blank controls (culture medium group) were set up. The 96-well plates were then incubated at 37 ℃ for 16-20 h. OD was measured. 600 Values ​​determine the minimum inhibitory concentration (MIC) and half-inhibitory concentration (IC50) of a compound against bacteria. 50 ).

[0042] In this embodiment, different small molecule compound inhibitors resist... S. aureus The activity test results and the antibacterial fitting curve of Staphylococcus aureus treatment are shown in Figure 2.

[0043] In this embodiment, vancomycin was selected as a positive control in the small molecule compound antibacterial activity experiment. Its IC50 against Staphylococcus aureus Newman strain... 50 The value was 1.12 μg / mL. In previous antibacterial activity experiments targeting 5 compounds from GND and 7 compounds from PGL, GND_4, GND_6, GND_7, PGL_8, and PGL_9 exhibited antibacterial activity, with an IC50 value of 1.12 μg / mL. 50 The values ​​were 8.8 μg / mL, 14.1 μg / mL, 15.8 μg / mL, 1.1 μg / mL, and 3.1 μg / mL, respectively. The IC50 values ​​for the remaining compounds were... 50 Values ​​>40 μg / mL indicate poor antibacterial activity.

[0044] Example 3 In this embodiment, based on previous antibacterial activity experiments, five compounds with antibacterial activity (GND_4, GND_6, GND_7, PGL_8, and PGL_9) were further screened. To verify the targeting, enzyme activity inhibition experiments were conducted on these five compounds.

[0045] Freshly prepared GND / PGL enzymes were incubated with different compound solutions at room temperature for 1 hour in Tris-HNO3 buffer (35 mM Tris-HNO3, 100 mM NaNO3, pH = 7.4). The activity of compound GND was determined by measuring the forward reaction rates of 6PG oxidation and decarboxylation, and the absorbance of the product NADPH was monitored at 340 nm. A typical reaction mixture contained 35 mM Tris-HNO3 buffer (pH = 7.4), 10 mM MgCl2, and 0.5 mM NADP. +And 2 mM 6PG. All enzyme experiments were performed at 25 °C to a final volume of 100 µL. The reaction was initiated by adding enzyme to a final concentration of 10 nM. The absorbance at 340 nm was continuously monitored for 20 minutes. PGL activity was measured by measuring the hydrolysis of 6-phosphogluconate-δ-lactone to 6-phosphogluconate, which is accompanied by the reduction of NADP to NADPH in the presence of 6-phosphogluconate dehydrogenase (6PGL), with the absorbance of the product NADPH monitored at 340 nm. A typical reaction mixture contained 35 mM Tris-HNO3 buffer (pH = 7.4), 10 mM MgCl2, and 0.5 mM NADP. + 0.15–0.2 units / mL GND and 0.2 mM 6PGL were used. All enzyme experiments were performed at 25 °C to a final volume of 100 µL. Reactions were initiated by adding enzyme to a final concentration of 80 nM. The absorbance at 340 nm was continuously monitored for 20 minutes.

[0046] In this embodiment, the enzyme activity inhibition fitting curves for the five compounds (GND_4, GND_6, GND_7, PGL_8, and PGL_9) screened out with antibacterial activity are shown in the figure below. Figure 3 As shown.

[0047] The results showed that, except for GND_6, GND_4 and GND_7 exhibited significant concentration-dependent inhibitory effects on the enzyme activity of GND, with the half-maximal inhibitory concentration (IC50) being less than 100%. 50 The values ​​were 16.2 μM and 1.2 μM, respectively; PGL_8 and PGL_9 also showed significant concentration-dependent inhibitory effects on the enzyme activity of PGL, with half-maximum inhibitory concentrations (IC50) of 16.2 μM and 1.2 μM, respectively. 50 The values ​​were 1.8 μM and 2.5 μM, respectively.

[0048] Example 4 In this embodiment, based on previous antibacterial activity experiments, five compounds with antibacterial activity (GND_4, GND_6, GND_7, PGL_8 and PGL_9) were further screened and their inhibitor safety was evaluated.

[0049] In this embodiment, to evaluate the safety of the screened compounds with antibacterial activity, the toxicity of the compounds with antibacterial activity to human liver cancer cells (HepG2) was determined using the CCK8 method.

[0050] In this embodiment, the cytotoxicity of the compound was detected using the CCK8 assay kit. The compound was administered at a dose of 1 × 10⁻⁶ ppm one day prior to the experiment. 5Human hepatocellular carcinoma cells (HepG2) were seeded into 96-well plates at a ratio of cells / mL. The cell supernatant was then discarded, and different concentrations of inhibitor solutions, pre-diluted with cell culture medium, were added to the wells for co-culture. After 24 h, the medium was discarded, and 100 µL of fresh cell culture medium containing 10% CCK8 was added. The plates were incubated at 37 °C for 1 h, and the absorbance at 450 nm was measured. Cell viability and the half-maximal toxicity concentration (CMC) of the corresponding compounds were calculated and analyzed. 50 The selection index (SI) was calculated in conjunction with antibacterial activity experiments.

[0051] In this embodiment, the CC of different compounds 50 The SI values ​​are shown in Table 3 below.

[0052] Table 3 Safety evaluation indicators for small molecule compounds

[0053] The results showed that GND_4 had a half-maximal toxicity concentration (CMC) of 5% to HepG2. 50 >1280 µg / mL, Selectivity Index (SI) >145; PGL_9 toxicity median concentration (CC) for HepG2 toxicity 50 The concentration of the compound was 330.8 µg / mL, and the Selective Index (SI) was 108. Both SI values ​​were >100, indicating that these two compounds have good safety.

[0054] Example 5 In this embodiment, PGL_8 and PGL_9 inhibitors are further used as examples to conduct combined antibacterial experiments with different inhibitors.

[0055] Staphylococcus aureus Newman strain was cultured to the logarithmic growth phase, and the bacteria were collected by centrifugation and diluted to 5 × 10⁻⁶. 5 CFU / mL. 25 µL of different concentrations of PGL_8 or PGL_9 inhibitor, 25 µL of antibiotic solution, and 50 µL of bacterial culture were added to a sterile 96-well plate using a checkerboard method. The plate was then incubated at 37 °C for 16–20 h, after which the OD was measured. 600 The values ​​and analysis of the antibacterial effects of combined drug therapy were performed. The fractional inhibitory concentration index (FICI) = MIC com1 / MIC1+ MIC com2 / MIC2, where MIC1 and MIC2 are the MIC values ​​when the antimicrobial peptide or antibiotic is used alone. com1 and MIC com2is the MIC value after the combined use of inhibitors. FICI ≤ 0.5 indicates a synergistic effect between the two drugs; 0.5 FICI ≤ 1 indicates an additive effect between the two drugs; when 1<FICI ≤ 2, it indicates no interaction between the two drugs; when FICI>2, it indicates an antagonistic effect between the two drugs.

[0056] In this example, the checkerboard method is used to detect the combined antibacterial activity of PGL_8 and linezolid, as well as PGL_9 and ciprofloxacin. The results are shown in Figure 4 as shown.

[0057] It can be seen that in the anti-Newman strain experiment, the FICI value of the combination of PGL_8 and linezolid is 0.63, and the FICI value of the combination of PGL_9 and ciprofloxacin is 0.63, indicating that these two combinations have an additive antibacterial effect.

[0058] It can be seen that PGL_8 and PGL_9 screened by the scheme of the present invention can be used in combination with commonly used therapeutic S. aureus anti-infective antibiotics to reduce the administration concentration.

[0059] Example 6 This example discloses a pharmaceutical preparation using the compound targeting Staphylococcus aureus PGL enzyme and / or the compound targeting Staphylococcus aureus GND enzyme as active ingredients or as the sole active ingredient. The pharmaceutical preparation has an anti-Staphylococcus aureus effect. It is understandable that the compound, as the active ingredient of the drug, can exert a good bacteriostatic effect.

[0060] In this example, the pharmaceutical preparation may select the compound targeting Staphylococcus aureus PGL enzyme and / or the compound targeting Staphylococcus aureus GND enzyme as the sole active ingredient of the drug; it may also be compatible with other bacteriostatic components well known in the art.

[0061] In this example, the pharmaceutical preparation comprises a bacteriostatically effective amount of the compound targeting Staphylococcus aureus PGL enzyme and / or the compound targeting Staphylococcus aureus GND enzyme. It should be noted that the term "bacteriostatically effective amount" refers to the dosage of the drug required to produce an effective effect. This "bacteriostatically effective amount" can be adjusted and changed according to the actual situation, and is ultimately determined by medical personnel. The factors considered include the route of administration and the nature of the preparation, the general conditions such as the weight and age of the recipient, as well as the nature and severity of the disease to be treated.

[0062] In this embodiment, the pharmaceutical formulation further includes other pharmaceutically acceptable carriers and / or excipients compatible with the compound. It should be noted that "other pharmaceuticals" here refers to drugs that are compatible with the compound, do not cause inactivation, reduced activity, or hydrolysis, and whose synergistic effect with the compound enhances its therapeutic efficacy. The aforementioned carriers can be commonly used drug carriers in the art, such as chitosan, liposomes, alginate, agar, fibrin, collagen, and synthetic polymer carriers. The aforementioned excipients are physiologically inactive, do not affect the efficacy, content determination, or stability of the active pharmaceutical ingredient, and are primarily intended to facilitate formulation preparation and clinical application. For example, they can be commonly used starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, light magnesium oxide, calcium carbonate, dry starch, sodium hydroxymethyl starch, low-substituted hydroxypropyl cellulose, effervescent disintegrants, crospovidone, etc.

[0063] In this embodiment, the pharmaceutical preparation is a pharmaceutically acceptable oral dosage form. Examples include powders, granules, pills, capsules, tablets, ointments, liquid preparations, gels, etc. Accordingly, depending on the actual dosage form, the administration method can be selected from inhalation, inhalation, nasal administration, sublingual administration, parenteral administration, etc.

[0064] In summary, this invention utilizes the high-resolution three-dimensional structures of Staphylococcus aureus GND and PGL proteins and identifies their enzyme active centers. Through molecular docking, 12 novel small molecule compounds were virtually screened to potentially target these two proteins. Subsequently, their antibacterial activity was tested, further identifying 5 compounds with antibacterial activity. Next, enzyme inhibition experiments and safety evaluations were conducted on these antibacterial compounds, ultimately identifying inhibitors targeting Staphylococcus aureus GND and PGL with good antibacterial activity. These compounds represent the first successful screening of novel small molecule compounds with both GND and PGL inhibitory activities. Their application against Staphylococcus aureus is reported for the first time, and it is also the first time that these compounds exert their antibacterial activity by targeting Staphylococcus aureus PPP pathway metabolic enzymes. This discovery provides new candidate entities with clear targets for the treatment of drug-resistant Staphylococcus aureus infections and expands the antibiotic research and development pipeline.

[0065] The above description is merely an embodiment of this disclosure and is not intended to limit this disclosure. Any modifications, equivalent substitutions, improvements, etc., made within the principles of this disclosure should be included within the protection scope of this disclosure.

Claims

1. A compound targeting Staphylococcus aureus PGL enzyme, characterized in that, The compound includes at least one of the following structural compounds: 、 (PGL_8)(PGL_9).

2. Use of the compound of claim 1 for the preparation of a PGL enzyme inhibitor targeting Staphylococcus aureus.

3. A compound targeting Staphylococcus aureus GND enzyme, characterized in that, The compound includes at least one of the following structural compounds: 、 、 (GND_4) (GND_6) (GND_7).

4. Use of the compound of claim 3 for the preparation of an inhibitor targeting Staphylococcus aureus GND enzyme.

5. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the compound of claim 1 that targets Staphylococcus aureus PGL enzyme and / or the compound of claim 3 that targets Staphylococcus aureus GND enzyme.

6. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutical composition also includes pharmaceutically acceptable excipients.

7. The pharmaceutical composition according to claim 5 or 6, characterized in that, The dosage forms of the pharmaceutical composition include non-gastrointestinal and / or gastrointestinal dosage forms.

8. The pharmaceutical composition according to claim 7, characterized in that: The non-gastrointestinal drug delivery dosage forms include at least one of injection dosage forms, cavity dosage forms, mucosal dosage forms, and skin dosage forms; The gastrointestinal dosage forms include at least one of tablets, granules, capsules, solutions, powders, sustained-release preparations, emulsions, and suspensions.

9. Use of a compound targeting Staphylococcus aureus PGL enzyme as described in claim 1, or a compound targeting Staphylococcus aureus GND enzyme as described in claim 3, or a pharmaceutical composition as described in any one of claims 5-8, for the preparation of an anti-Staphylococcus aureus drug.

10. The use of a Staphylococcus aureus PGL enzyme inhibitor or a Staphylococcus aureus GND enzyme inhibitor in the preparation of an anti-Staphylococcus aureus drug, characterized in that: The targeted Staphylococcus aureus PGL enzyme inhibitor includes the compound of claim 1; The targeted Staphylococcus aureus GND enzyme inhibitor includes the compound of claim 3.

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