Use of norzodronaldehyde to inhibit nmd-1 enzyme activity

Abstract

This invention provides the application of norzelamin in the preparation of NDM-1 enzyme inhibitors. Through cefotaxime hydrolysis assays, checkerboard minimum inhibitory concentration (MIC) tests, growth curves, bactericidal curves, hemolysis tests, and *Gnaphalium affine* infection experiments, it was demonstrated that norzelamin can inhibit NDM-1 enzyme activity and exhibits good synergistic antibacterial activity with the carbapenem antibiotic meropenem, enhancing the protective effect of meropenem against *Gnaphalium affine* larval infection models. Therefore, norzelamin, as a potential adjuvant for β-lactam antibiotics, can enhance the antibacterial effect of β-lactam antibiotics by inhibiting NDM-1 enzyme activity, which is of great significance for the clinical development of safe and effective β-lactam antibiotic adjuvants.

Description

Technical Field

[0001] This invention discloses a novel use of the natural compound norzelaminaldehyde, specifically its application in inhibiting carbapenemase NDM-1, belonging to the field of pharmaceutical technology. Background Technology

[0002] The emergence and spread of drug-resistant bacteria have led to a continuous increase in the number of morbidities and deaths caused by bacterial infections worldwide. In 2016, the World Health Organization (WHO) established a priority list of drug-resistant bacteria, excluding Mycobacterium tuberculosis. Among the more important clinical priority bacteria are carbapenem-resistant Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae resistant to carbapenems and third-generation cephalosporins. New Delhi metallo-β-lactamase (NDM-1), which emerged in 2008, is an important carbapenemase that can hydrolyze most β-lactam antibiotics, including carbapenems, making infections caused by drug-resistant bacteria difficult to treat. Researching and developing effective NDM-1 enzyme inhibitors in combination with β-lactam antibiotics is one effective approach to treating NDM-1-positive bacterial infections. Although several β-lactamase inhibitors, such as clavulanic acid and sulbactam, are currently in clinical use, there is still no enzyme inhibitor specifically targeting NDM-1. Therefore, developing novel, safe, and effective NDM-1 enzyme inhibitors is of great significance.

[0003] Norzelaminaldehyde is a triterpenoid compound extracted from Tripterygium wilfordii, and it is found in high concentrations in the plant. It possesses anti-inflammatory, immunosuppressive, and antitumor activities. Norzelaminaldehyde can significantly reduce atherosclerosis and can also block typical and atypical TGF-β signaling pathways, inhibiting the invasion of triple-negative breast cancer cells. However, there are no reports of norzelaminaldehyde as an antibiotic adjuvant; therefore, this study investigated its use as an adjuvant for meropenem in inhibiting NDM-1 activity. Summary of the Invention

[0004] The CAS number of the nor-Zera aldehyde described in this invention is 107316-88-1, and its molecular formula is C. 29 H 36 O6 has a molar mass of 480.59 g / mol.

[0005] This study screened a natural compound library for an NDM-1 enzyme inhibitor, norzelamin, using a cefotaxime hydrolysis assay. Norzelamin effectively inhibited NDM-1 activity. A checkerboard minimum inhibitory concentration (MIC) assay demonstrated a good synergistic antibacterial effect between norzelamin and the carbapenem antibiotic meropenem. Growth and bactericidal curve assays further confirmed the synergistic bactericidal effect of the two compounds. Furthermore, a large wax moth infection model validated that norzelamin effectively enhanced the in vivo therapeutic effect of meropenem.

[0006] The positive effects of this invention are as follows:

[0007] The application of norzelamin in the development of NDM-1 enzyme inhibitors is provided, and it is disclosed that norzelamin can inhibit the enzyme activity of NDM-1 and effectively enhance the antibacterial activity of meropenem. Attached Figure Description

[0008] Figure 1 The inhibitory effect of norzelaminaraldehyde on NDM-1 enzyme activity;

[0009] Figure 2 The results of the growth curve experiment of norzelamin combined with meropenem on the NDM-1 clinical isolate E. coli ZJ487;

[0010] Figure 3 The results of the bactericidal curve test of norzelamin combined with meropenem against the NDM-1 clinical isolate E. coli ZJ487;

[0011] Figure 4 The results of a test on the protection rate of zearalenone combined with meropenem against E. coli ZJ487-infected larvae of the large wax moth; Detailed Implementation

[0012] The present invention is further illustrated by the following embodiments, which are not intended to limit the invention in any way. Any modifications or alterations made to the present invention that are easily implemented by those skilled in the art without departing from the technical solutions of the present invention shall fall within the scope of the claims of the present invention.

[0013] 1. Cefuroxime hydrolysis test

[0014] Based on the principle of color change produced by the hydrolysis of cefotaxime by NDM-1, an appropriate amount of NDM-1 protein was premixed with PBS in a 175 μL PBS buffer system. Different concentrations of norzelaminaraldehyde (0, 4, 8, 16, 32, 64 μg / mL) were added simultaneously, and the mixture was incubated at 37℃ for 20 min. Then, 25 μL of cefotaxime was added, and after shaking to mix, the mixture was incubated at 37℃ for 30 min. The absorbance at 492 nm was measured using a microplate reader. The experimental results are shown below. Figure 1 .

[0015] Conclusion: Norzelamin can effectively inhibit NDM-1 enzyme activity, IC50 50 =13.46μg / mL.

[0016] 2. Minimum inhibitory concentration test using the checkerboard method

[0017] According to the standards published by the American Society for Clinical and Laboratory Standards, meropenem and norzelamin were serially diluted using LB, with 100 μL each added to a 96-well plate. Simultaneously, 1 μL of bacterial culture (1×10⁻⁶) was added to each well. 8 (CFUs / mL). After incubation at 37℃ for 16-24 hours, observe the experimental results. The minimum concentration at which no bacteria grow is the minimum inhibitory concentration (MIC). Partial inhibitory concentration index (FICI) calculation method: FICI = MIC (meropenem combined with other drugs) / MIC (meropenem alone) + MIC (norzelamin combined with other drugs) / MIC (norzelamin combined with other drugs).

[0018] Table 1. MIC and FIC values ​​of norzelamin combined with meropenem against NDM-1 positive bacteria.

[0019]

[0020] Conclusion: Norzemarin combined with meropenem has a good synergistic antibacterial effect against NDM-1 positive engineered bacteria E. coli (pET28a-SP-ndm-1) and clinical isolate E. coli ZJ487.

[0021] 3. Growth curve experiment

[0022] Overnight cultured *E. coli* ZJ487 was transferred to fresh LB medium and allowed to oxidize. 600nm The concentration was adjusted to 0.1–0.3, and aliquoted into 50 mL centrifuge tubes. Different concentrations of norzelaminaraldehyde were added to achieve final concentrations of 0, 8, 32, and 128 μg / mL. The tubes were continuously incubated at 37°C and 180 rpm / min, with OD values ​​measured every 1 hour. 600nm The absorbance values ​​were recorded and plotted as curves.

[0023] Conclusion: Norzemarin at concentrations below 128 μg / mL had almost no effect on the growth of Escherichia coli ZJ487.

[0024] 4. Sterilization curve test

[0025] For Escherichia coli ZJ487, the following groups were set up: a positive control, meropenem alone (4 μg / mL), norzelamin alone (64 μg / mL), and a combined treatment group; the bacterial count in each group was 5 × 10⁶.5 CFUs were incubated statically at 37°C. Samples were taken at 0, 4, 8, 12, and 24 hours, diluted to different ratios, and plated. The number of colonies was counted and recorded after 24 hours.

[0026] Conclusion: The combined use of norzelamin and meropenem can completely kill the NDM-1 positive clinical isolate E. coli ZJ487 within 24 hours.

[0027] 5. Protection rate test of large wax moth larvae

[0028] Large wax moth larvae were purchased from Tianjin Huiyude Biotechnology Co., Ltd., stored at 18℃, and used within two weeks. Overnight cultured *E. coli* ZJ487 bacterial suspension was diluted 1:100 and amplified into fresh LB granules until the bacterial suspension reached OD500. 600nm When the bacterial count is between 0.6 and 0.8, collect the bacteria and wash three times with PBS to adjust the bacterial count to 7 × 10⁸. 7 CFUs / mL, 10 μL of bacterial suspension was administered to the right side of the last pair of abdominal legs of each large wax moth. Infected large wax moths were randomly divided into four groups: infection group, 25 mg / kg norzelamin group, 1 mg / kg meropenem group, and combined treatment group. The right side of the second-to-last pair of abdominal legs was treated with different drugs, and the moths were incubated at 37°C for observation and mortality.

[0029] Conclusion: The combination of 25 mg / kg norzelamin and 1 mg / kg meropenem can increase the protective effect of meropenem from 50% to 70%, which is significantly higher than that of the control group or norzelamin alone.

Claims

1. The application of norzelaminaraldehyde in the preparation of NDM-1 enzyme inhibitors, characterized in that, The inhibitory effect of norzelamin on NDM-1 enzyme activity, the application of which is the use of norzelamin in the preparation of β-lactam antibiotic adjuvants.

Images