Application of L-histidine in preparation of NDM enzyme inhibitor, medicine for treating bacterial infectious diseases or antibiotic synergist

Abstract

The invention discloses application of L-histidine in preparation of an NDM enzyme inhibitor, a medicine for treating bacterial infectious diseases or an antibiotic synergist. The invention discloses that L-histidine can inhibit the hydrolytic activity of NDM enzyme for the first time, has a relatively strong effect of resisting drug-resistant pathogenic bacteria in vivo and in vitro when being combined with the antibiotic meropenem for use, and can effectively improve the curative effect of meropenem; when the L-histidine and meropenem combined medicine is used for treating gram-negative bacteria infectious diseases, the survival rate of mice can be remarkably increased and the inflammation level of organisms can be reduced by the combined medicine of the L-histidine and meropenem. The invention provides a new thought for the control of pathogenic bacteria of drug-resistant bacteria, and also provides a new strategy for the prevention and treatment of increasingly serious gram-negative bacterial infection.

Description

TECHNICAL FIELD

[0001] The application relates to application of L-histidine in preparation of an NDM enzyme inhibitor, a drug for treating a bacterial infectious disease or an antibiotic sensitizer, and belongs to the technical field of medicines. BACKGROUND

[0002] The discovery and wide application of antibiotics not only saved countless lives, but also played a crucial role in the prevention and control of bacterial diseases in livestock and poultry breeding, ensuring the stable supply of animal-derived food. However, the emergence of antibiotic-resistant strains has seriously weakened the effectiveness of this traditional anti-infective treatment strategy. The problem of antibiotic resistance has become one of the biggest challenges facing global public health. Meropenem (MEM), as a representative of carbapenem antibiotics, is widely considered as the last line of defense for the treatment of multidrug-resistant gram-negative bacterial infections. Its bactericidal mechanism lies in the interaction with the penicillin-binding proteins (PBPs) of the outer membrane of gram-negative bacteria, inhibiting cell wall synthesis, leading to the leakage of cell contents, and thus causing bacterial death. However, bacteria have evolved multiple resistance mechanisms: producing inactivating enzymes (such as metallo-beta-lactamases) to destroy drug structures; activating efflux pump systems to expel drugs out of the cell; changing membrane permeability to prevent drug entry; modifying drug targets (PBPs) to reduce drug affinity. Among them, the plasmid-mediated bla NDM New Delhi metallo-beta-lactamase (NDM) encoded by the gene can hydrolyze the beta-lactam ring of meropenem, making it inactive, which is a key mechanism for the spread of drug resistance. Since its first discovery in 2008, bla NDM It has spread widely in more than 70 countries around the world and has evolved into multiple variants. Its high transferability allows it to spread among multiple human pathogenic bacteria such as Enterobacteriaceae, Pseudomonas and Acinetobacter, and often coexists with other drug-resistant genes, leading to multiple drug resistance of host bacteria to almost all commonly used antibiotics.

[0003] Although the research and development of new antibacterial drugs continues to advance, since the 21st century, only more than 20 new antibiotics have entered clinical application worldwide. Although new drugs can temporarily curb the spread of drug-resistant bacteria, bacteria quickly develop resistance, leading to the emergence of multiple drug-resistant bacteria with even wider resistance spectrum or "superbugs". Therefore, simply relying on new drug development has been difficult to cope with the drug resistance crisis, and the sensitization strategy to enhance the efficacy of existing antibiotics has become increasingly important.

[0004] L-histidine (L-His) as an essential amino acid, plays multiple physiological functions in proton buffering, metal ion chelation, free radical scavenging and immune regulation. Recent studies have found that changes in bacterial metabolic state significantly affect their growth and survival, and metabolic overload can even directly lead to bacterial death. This suggests that there is great potential for enhancing the bactericidal efficiency of antibiotics through metabolic reprogramming. Summary of the Invention

[0005] Objective of the Invention: The first objective of this invention is to provide the application of L-histidine in the synergistic treatment of bacterial infectious diseases by inhibiting NDM enzymes and antibiotics. The second objective of this invention is to provide an NDM enzyme inhibitor containing L-histidine and a pharmaceutical composition.

[0006] Technical solution: The application of L-histidine described in this invention in the preparation of NDM enzyme inhibitors, drugs for treating bacterial infectious diseases, or antibiotic potentiators.

[0007] Furthermore, the molecular formula of the L-histidine is C2H9N3O2.

[0008] Furthermore, the bacteria are Gram-negative.

[0009] Furthermore, the Gram-negative bacteria are bla NDM Drug-resistant bacteria.

[0010] Furthermore, the aforementioned bla NDM Drug-resistant bacteria are those that carry... bla NDM-1 or bla NDM-5 Drug-resistant bacteria.

[0011] Furthermore, the aforementioned bla NDM Drug-resistant bacteria selected E. coli C3 ( bla NDM-1 ) , E. coli G6 ( bla NDM-5 ) 、 P. cibarius HNCF44W( bla NDM-1 ), E. coli MG1655 (PUC19- bla NDM-1 ).

[0012] Furthermore, the antibiotic is a carbapenem antibacterial drug.

[0013] Furthermore, the carbapenem antibacterial drug is meropenem.

[0014] Furthermore, the final concentration of the L-histidine is 2.5~20 mM. Furthermore, the final concentration of meropenem is 16~192 μg / mL.

[0015] The NDM enzyme inhibitor described in this invention has L-histidine as its active ingredient.

[0016] The pharmaceutical composition of the present invention comprises L-histidine and meropenem.

[0017] Furthermore, the mass ratio of L-histidine to meropenem is 2 to 194:1.

[0018] Beneficial Effects: Compared with the prior art, the present invention has the following significant advantages: The present invention discloses for the first time that L-histidine has the effect of inhibiting the hydrolytic activity of NDM enzyme, and when used in combination with the antibiotic meropenem, it has a strong anti-Gram-negative pathogen effect both in vivo and in vitro, specifically manifested in its ability to effectively inhibit bacterial growth and reduce bacterial load; used for the treatment of bla NDM In diseases caused by Gram-positive bacteria, combination therapy can significantly improve the survival rate of mice and reduce the level of inflammation in the body. Current research has found that the use of L-histidine in combination with meropenem can improve the sensitivity of drug-resistant bacteria to antibiotics. This discovery provides a new perspective for the development of novel drug resistance control strategies and also offers a new method for the control of increasingly serious Gram-negative bacterial infections. Attached Figure Description

[0019] Figure 1 L-histidine enhances the effectiveness of antibiotics in various applications. bla NDM Evaluation of antibacterial activity in positive bacteria, where Both refers to the combination therapy group.

[0020] Figure 2 To evaluate the effect of L-histidine on inhibiting the hydrolytic activity of NDM-5.

[0021] Figure 3 To evaluate the efficacy of L-histidine and meropenem in the treatment of peritoneal resistant bacterial infection in mice. Detailed Implementation

[0022] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0023] The LB broth medium used in the examples was an aqueous solution containing 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl; the MHB broth medium was an aqueous solution containing 2 g / L beef meal, 1.5 g / L soluble starch, and 17.5 g / L acid-hydrolyzed casein; and the M9 medium was composed of 6.8 g / L Na2HPO4, 3.0 g / L KH2PO4, 1.0 g / L NH4Cl, 0.5 g / L NaCl, 4 g / L glucose, 1 mM MgSO4, and 1 mM CaCl2.

[0024] The specific information on the strains and sources used in the examples is shown in Table 1.

[0025] Table 1

[0026]

[0027] Note: Document 1 is Shi, J. et al. Amphipathic peptide antibiotics with potentactivity against multidrug-resistant pathogens. Pharmaceutics 13, 438 (2021). Document 2 is Fang, D. et al. Methionine-driven methylation modificationovercomes plasmid-mediated high-level tigecycline resistance. Nat Commun 16,417 (2025). Reference 3 is Li, P. et al. Targeted elimination of bla NDM-5 gene in Escherichia coli by conjugative CRISPR-Cas9 system. Infect Drug Resist 15,1707–1716 (2022). Example 1: L-histidine enhances antibiotics in multiple... bla NDM Evaluation of antibacterial activity in positive bacteria Multiple amino acids combined with the antibiotic meropenem were replenished. bla NDM Positive-positive drug-resistant bacteria were screened for substances that could effectively enhance antibiotic efficacy. Bacterial survival rates under different treatments were determined using a dropper plate counting method. Single colonies were selected. E. coli G6 ( bla NDM-5The culture was incubated overnight at 37 °C in a shaker at 200 r / min, then diluted 1:100 to fresh MH broth and cultured for 4 h to reach the logarithmic growth phase. The culture was then washed and resuspended in M9 basal medium. A blank control group was set up, consisting only of bacterial suspension resuspended in M9. Experimental groups included meropenem (MEM, 32 μg / mL) as a single-drug group; and meropenem (MEM, 32 μg / mL) combined with different metabolites L-Pro (L-proline, 30 mM), L-His (L-histidine, 30 mM), L-Asp (aspartic acid, 30 mM), L-Trp (L-tryptophan, 30 mM), L-Cys (L-cysteine, 30 mM), or L-Hse (L-homose, 10 mM) as a combination therapy group, with an action time of 4 h. Subsequently, 50 μL of bacterial suspension from either the single-drug group or the combination-drug group was serially diluted tenfold, and 50 μL of the bacterial suspension was spotted onto LB agar plates and incubated overnight at 37 ℃. After 18 h, the colony count was determined, and the bacterial load for each group was calculated. Three biological replicates were set up. The results showed that meropenem combined with L-histidine had the most significant bactericidal effect. Figure 1 A).

[0028] To further evaluate the applicability of the synergistic activity between L-histidine and meropenem, we selected standard strains. E. coli ATCC 25922, Positive engineered bacteria E. coli MG1655 (PUC19- bla NDM-1 ) and clinically derived positive bacteria E. coli C3 ( bla NDM-1 ), E. coli G6 ( bla NDM-5 )and P. cibarius HNCF44W ( bla NDM-1 A checkerboard experiment was performed. L-histidine (240 mM) and meropenem (4-fold MIC) were serially diluted twice in 96-well plates pre-filled with MHB broth, and the corresponding bacterial cultures (single colonies incubated overnight and then diluted with MHB to 1.0 × 10⁻⁶) were added respectively. 7 Add 100 μL of CFU / mL per well and incubate overnight at 37°C. After 18 h, measure the absorbance of the bacterial culture at 600 nm using a microplate reader. Each combination was performed in triplicate, and the partial inhibitory concentration index (FIC) was calculated using the average value according to the following formula: FIC = MIC a,comb / MIC a,alone + MIC b,coma / MIC b,alone(MIC a,comb and MIC b,coma These represent the MIC values ​​of drugs a and b against the test strain under combined conditions. a,alone and MIC b,alone (These represent the MIC values ​​when drugs a and b are used alone, respectively). The results showed that L-histidine and meropenem had a synergistic effect against all the aforementioned positive bacteria, significantly reducing their FIC values, but had no such effect against negative bacteria. Figure 1 B). Regarding E. coli C3 ( bla NDM-1 ), E. coli G6( bla NDM-5 )and P. cibarius HNCF44W( bla NDM-1 The time-killing curve was determined. The pretreatment method was the same as that used for the survival rate determination. Groups were set up as follows: blank control group, meropenem alone (1 / 2 MIC), L-histidine alone (10 mM), meropenem (1 / 2 MIC) + L-histidine (10 mM), and meropenem (1 / 2 MIC) + L-histidine (10 mM) + Zn. 2+ (100 μM) group. Droplet counts were performed at 4 h, 8 h, 24 h, and 48 h after drug administration, with three biological replicates. Results showed that the combined use of L-histidine and meropenem resulted in a sustained decrease in bacterial count. Figure 1 CE). E. coli G6 ( bla NDM-5 The effect of L-histidine and meropenem dosages on the synergistic effect was further tested in this study. Meropenem (MEM, 32 μg / mL) and L-histidine (L-His, 5, 10, 20 mM) were added simultaneously as the combination therapy group. Figure 1 F), L-histidine (L-His, 5 mM) and meropenem (MEM, 32, 64, 192 μg / mL) were added simultaneously as the combination therapy group. Figure 1 G). The results showed that the synergistic effect of L-histidine and meropenem was dose-dependent. To determine the effect of L-histidine on the evolution of meropenem resistance, continuous passages were performed in media containing only meropenem (1 / 4 MIC) or in combination with L-histidine (10 mM). E. coli G6 ( bla NDM-5 )12 generations, with MIC detected every 2 generations. The results show ( Figure 1In the presence of meropenem (H), the MIC value increased 8-fold. In contrast, the meropenem MIC was only increased 2-fold in the meropenem and L-histidine combination group, indicating that the addition of L-histidine effectively prevented the evolution of meropenem resistance.

[0029] Example 2: Evaluation of the effect of L-histidine on inhibiting the hydrolytic activity of NDM-5 Since the combined effects of meropenem and L-histidine differ in positive and negative bacteria, this suggests that their synergistic effect is likely attributed to the inhibition of NDM by L-histidine. We explored the potential binding modes of L-histidine and NDM-5 using molecular docking via Autodock. Figure 2 (AB). The results showed that L-His could be located in the catalytic pocket of NDM-5 and bind to two zinc ions in the active pocket of NDM-5 via hydrogen bonds. Next, we performed an NDM-5 enzyme activity inhibition experiment. 0.2 μM NDM enzyme solution was added to a 96-well plate, and L-histidine was added to the first well to make a concentration of 40 mM, which was then serially diluted. The plate was incubated at 37 °C for 20 min, and then 100 μL of 0.2 mM cefuroxime was added and incubated for 15 min. The absorbance of each well was measured at 492 nm using a microplate reader. Three biological replicates were set up. The positive control was performed in an enzyme-containing system without inhibitors, and the negative control was performed in an enzyme-free system. Residual activity was calculated using the formula (A-A0) / (A 100 The result is calculated as (-A0)×100%, where A represents the absorbance of the inhibitor group at 492 nm, and A0 and A... 100 The values ​​represent 0% and 100% activity values ​​obtained through negative and positive controls, respectively. The results indicate that L-histidine can significantly reduce the hydrolytic activity of NDM-5. Figure 2 C). To demonstrate that this inhibition is achieved by affecting zinc ions in NDM, we further determined the hydrolytic activity of L-histidine in the presence of zinc ions, as well as the synergistic effect and time-killing curve of L-histidine and meropenem. Zinc ions at a concentration of 250 μM were serially diluted in the enzyme solution, and L-histidine (10 mM) was added to each well. The mixture was incubated at 37 °C for 20 minutes, followed by the addition of 100 μL of 0.2 mM cefnitrothiophene. The absorbance of each well at 492 nm was continuously measured for 60 minutes using a microplate reader. The synergistic effect was determined using a checkerboard assay, with 100 μM zinc ions pre-added to MHB broth before subsequent operations. The results showed that the addition of zinc ions restored the hydrolytic activity of NDM-5, and the higher the zinc ion concentration, the faster the recovery. Figure 2 D), the checkerboard experiment results showed that zinc ions could weaken the synergistic effect of L-histidine and meropenem ( Figure 2E). The time-killing curve also showed that the combination of L-histidine and meropenem had no effect on bacterial count changes after the addition of zinc ions, restoring the bacterial count to the same level as the control group. Figure 1 To demonstrate that L-histidine inhibits the hydrolysis of antibiotics by NDM, we further determined the extracellular antibiotic content using liquid chromatography-mass spectrometry (LC-MS). Different concentrations (16, 32, and 64 µg / mL) of meropenem were added to bacterial cultures containing or without L-histidine (20 mM) and incubated for 1.5 h. The supernatant was then collected by centrifugation at 10,000 rpm for 20 min, filtered through a 0.2 µm filter, and used for subsequent analysis. LC separation conditions: mobile phase A (10 mM potassium dihydrogen phosphate aqueous solution) and mobile phase B (acetonitrile solution); column: C18 column (4.6 mm × 250 mm). The flow rate was set to 0.5 mL / min, and the gradient elution program was as follows: 0–0.5 min, 99% A; 0.5–1.5 min, 99%–70% A; 1.5–2.0 min, 70% A; 2.0–3.0 min, 70%–10% A; 3.0–3.5 min, 10% A; 3.5–3.6 min, 10%–99% A; 3.6–5.0 min, 99% A. The injection volume was 5 µL. Results showed that extracellular meropenem levels were below the detection limit without the addition of L-histidine (even with the addition of 64 µg / mL), while significant accumulation of meropenem was detectable with the addition of 20 mM L-histidine. Figure 2 F).

[0030] Example 3: Evaluation of the efficacy of combined L-histidine and meropenem therapy in an animal infection model. Mouse model of intraperitoneal infection: Female BALB / c mice (Comparative Medicine Center, Yangzhou University, 6-8 weeks old) were randomly divided into 4 groups of 10 mice each. 1 × 10⁻⁶ mg / L of the solution was injected into the right side of the peritoneum of each mouse. 9 CFU E. coli G6 (100 μL per mouse). Four hours post-infection, infected mice were treated with a blank control (PBS, 200 μL), MEM (20 mg / kg), or L-His (150 mg / kg) as single or combined therapy. Mice survival was monitored for 7 days. To determine the bacterial load in mouse organs, mice were treated in the same manner, and all mice were euthanized 12 hours post-infection. Liver, spleen, and kidneys were aseptically removed for CFU estimation. The liver was divided into two parts for simultaneous HE staining, and blood was collected for ELISA detection.

[0031] ELISA was used to determine serum inflammatory cytokine levels. An inflammatory cytokine ELISA kit was used, including pro-inflammatory cytokines IL-1β and IL-6, and anti-inflammatory cytokines IL-4 and IL-10 (Enzyme-Linked Biotechnology Co., Ltd., ml098416, ml098430, ml064310, ml037873). First, standards and serum samples were prepared, with each sample tested in triplicate. 50 μL of sample was added to each well. 50 μL of pre-labeled biotinylated antibody (HRP-IL-1β, HRP-IL-6, HRP-IL-4, HRP-IL-10) was added to each well containing the sample. The plate was gently shaken to ensure thorough mixing, and then incubated at 37 °C for 1 h. After incubation, the liquid in the wells was completely poured out, and each well was washed thoroughly with sufficient washing buffer. After each wash, the plate was gently shaken for 30 seconds to remove unbound material, and then the bottom of the well was gently patted with absorbent paper to ensure cleanliness. This washing process needs to be repeated three times to ensure that all non-specific bindings are removed as much as possible. After the washing step, 80 μL of avidin-labeled HRP (horseradish peroxidase) is added to each well, gently vortexed again, and incubated again at 37 °C for 30 min. After incubation, the previous washing steps are repeated to ensure the removal of all unreacted enzyme labels. Next, 50 μL of substrate A and substrate B are added to each well simultaneously in the dark. These substrates react with the enzyme-labeled antibody to produce a color change. After incubation at 37 °C in the dark for 10 min, the reaction is stopped by adding 50 μL of stop solution. Finally, the absorbance of each well is measured at 450 nm using a microplate reader. The concentration of specific inflammatory factors in the sample is calculated by comparing the absorbance of the sample with a pre-prepared standard curve.

[0032] The experimental results are shown in Figure 3 A mouse model of intraperitoneal infection showed that mice treated with meropenem or L-histidine alone had a survival rate of 20% or 0% within 7 days post-infection. In contrast, combination therapy achieved a 70% survival rate, significantly higher than monotherapy. Figure 3 A). Combined treatment can also reduce the pathogen load in the liver, spleen, and kidneys of mice ( Figure 3 (BD) reduces inflammation levels. Simultaneously, compared to the single-drug treatment group, the combination therapy group showed decreased levels of pro-inflammatory cytokines IL-1β and IL-6, and upregulated levels of anti-inflammatory cytokines IL-4 and IL-10, suggesting that the immune system played a positive role in the early stages of infection. Figure 3 Consistent with this, hematoxylin and eosin (HE) staining was performed simultaneously to assess liver damage. In the liver, the control group and the single-treatment group showed more severe pathological lesions, such as inflammatory cell infiltration, while the lesions in the meropenem and L-histidine combined treatment group were milder.

[0033] In summary, the combined use of meropenem and L-histidine can target bla NDM Positive bacteria can exert potential in vivo and in vitro antibacterial effects, and this effect is due to L-histidine inhibiting the hydrolytic activity of NDM against antibiotics and further promoting antibiotic accumulation.

Claims

1. Application of L-histidine in the preparation of NDM enzyme inhibitors, drugs for treating bacterial infectious diseases, or antibiotic potentiators.

2. The application according to claim 1, characterized in that, The bacteria in question are Gram-negative.

3. The application according to claim 2, characterized in that, The Gram-negative bacteria are bla NDM Drug-resistant bacteria.

4. The application according to claim 2, characterized in that, The bla NDM Drug-resistant bacteria are those that carry... bla NDM-1 or bla NDM-5 Drug-resistant bacteria.

5. The application according to claim 2, characterized in that, The bla NDM Drug-resistant bacteria selected E. coli C3( bla NDM-1 ) E. coli G6 ( bla NDM-5 ) P. cibarius HNCF44W ( bla NDM-1 ), E. coli MG1655(PUC19- bla NDM-1 ).

6. The application according to claim 1, characterized in that, The antibiotic is a carbapenem antibacterial drug.

7. The application according to claim 6, characterized in that, The carbapenem antibacterial drug is meropenem.

8. An NDM enzyme inhibitor, characterized in that, The active ingredient in the inhibitor is L-histidine.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises L-histidine and meropenem.

10. The pharmaceutical composition according to claim 9, characterized in that, The mass ratio of L-histidine to meropenem is 2 to 194:1.

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