CN104817619B - Antimicrobial compound and its application - Google Patents

Abstract

The invention discloses an antibacterial compound and its application. The structural formula of the compound is shown in formula (I). The present invention utilizes Bacillus subtilis (Bacillus subtilis) ZJU007 CGMCC NO.4140 to obtain the lead compound Chlorotetaine, and takes the drug delivery process as a theoretical basis to carry out homology modeling to construct the virtual structure of the small molecule peptide transporter, and through the small molecule peptide transporter The virtual structure of the lead compound Chlorotetaine was optimized, and a new tripeptide compound with antifungal ability was obtained. The minimum inhibitory concentration (MIC) of Candida fungi such as Trichoptera is 0.81 μg/mL; it can also resist Aspergillus such as Aspergillus niger, and the minimum inhibitory concentration is 1.63 μg/mL.

Description

Antibacterial compounds and their applications

technical field

The invention relates to the technical field of medicinal chemistry, in particular to an antibacterial compound and its application.

Background technique

The invention and use of antibiotics is one of the greatest inventions in the 20th century. Its wide application in the field of antibacterial has relieved a lot of human suffering and brought great blessings. However, in recent decades, whether in the field of agriculture, breeding or human medicine, the abuse of antibiotics has brought huge challenges to the ecosystem and global medical and health issues. At the same time, with the continuous outbreak of drug-resistant strains and the continuous improvement of the public's requirements for public health, people's requirements for new antibiotics, especially new antifungal drugs, are becoming more and more urgent.

Antimicrobial peptides (AMPs) are a class of positively charged amphiphilic (having both hydrophobic and hydrophilic parts) small molecule peptides. The main difference between antimicrobial peptides lies in the amino acid composition and the length of the peptide chain (6 ~100 amino acids). Antimicrobial peptides are an integral part of the innate immune system of most organisms, including humans. From the perspective of biosynthesis, antimicrobial peptides are mainly divided into two categories: antimicrobial peptides synthesized by non-ribosomal pathways and antimicrobial peptides synthesized by ribosomal pathways. From the perspective of biochemical properties and structural characteristics, antimicrobial peptides are usually divided into cationic antimicrobial peptides, anionic antimicrobial peptides, aromatic antimicrobial peptides, and peptides derived from oxygen-binding proteins.

Compared with synthetic and semi-synthetic antibiotics, antimicrobial peptides have better bactericidal activity, which makes antimicrobial peptides attract a lot of scientific interest. For example, antimicrobial peptides have a very broad antibacterial spectrum, such as β-defensins, indocidin, cecropin A and magainins, which can effectively kill bacteria, fungi, parasites and even viruses. Antimicrobial peptides also exhibit rapid bactericidal or fungicidal activity, limited immunogenicity, and some antimicrobial peptides are not even easily degraded by enzymes, making them stable antimicrobial coating materials. More importantly, antimicrobial peptides showed strong inhibitory activity against drug-resistant superbugs, such as methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, and quinolone-resistant Enterobacteriaceae, indicating that antimicrobial peptides may be through The mechanism is different from the existing antibiotics to achieve the effect of killing pathogenic bacteria. Since antimicrobial peptides are less likely to cause resistance in pathogenic bacteria, their use in medical implant coatings appears attractive.

Compared with traditional antifungal drugs, antimicrobial peptides have obvious advantages: 1) Antimicrobial peptides have selective toxicity to microbial targets, which is a necessary condition for any antibacterial drug. Due to the differences in membrane structure composition, transmembrane potential, polarization, and other structural properties between microbial cells and mammalian cells, AMPs are almost non-toxic to mammals. 2) Broad-spectrum biological activity. A large number of studies have shown that AMPs can not only act as antibacterial and antiviral drugs, but also be an important effector and regulator of the innate immune system. In addition, antimicrobial peptides can also inhibit the formation of biofilms, cause the dissolution of formed biofilms, attract phagocytes chemotactically and regulate non-opsonized phagocytosis. 3) AMPs can neutralize endotoxin, but are not affected by conventional antibiotic inhibition mechanisms. 4) So far, the most promising potential application of antimicrobial peptides lies in their synergistic therapeutic effect, which improves the activity of existing drugs by promoting the entry of antibacterial drugs into biological cells. Different from conventional antibiotics, AMPs are easy to cause changes in the structure of microbial cell membranes, and it is not easy for microorganisms to develop resistance to them.

Therefore, it is necessary to provide more antimicrobial peptide drugs to solve the problems caused by the use of traditional antifungal drugs.

Contents of the invention

The invention provides an antibacterial compound and application thereof. The antibacterial compound has high antifungal activity and can be used to prepare antifungal drugs.

A compound whose structural formula is shown in formula (I):

(I)

The above-mentioned compound is a tripeptide compound, which is obtained by connecting lysine to the terminal amino group of the lead compound through a peptide bond. The lead compound is Chlorotetaine, and its chemical structure is shown in (II):

(II)

The present invention first inoculates Bacillus subtilis ( Bacillus subtilis ) ZJU007 CGMCC NO.4140 into the fermentation medium and cultivates it. After separation and purification, the lead compound Chlorotetaine is obtained; then, the virtual structure of the small molecule peptide transporter is constructed by using homology modeling , and confirm the rationality of the virtual structure of the small molecule peptide transporter according to the information of the Lagren diagram; then use the software to simulate the connection of 20 common amino acids to the terminal amino group of the lead compound Chlorotetaine to form a new compound; Modeling and construction of the virtual structure of the small molecule peptide transporter was carried out for simulated docking, and the number of hydrogen bonds and docking free energy were used as reference standards to screen the degree of binding, and 5 compounds with high binding degrees were obtained; finally, through the antifungal activity Detecting, it is known that the compound with the best antifungal effect is: the tripeptide compound Chlorotetaine-Lys.

In the present invention, the tripeptide compound Chlorotetaine-Lys is subjected to various fungal resistance experiments, which proves that the tripeptide compound Chlorotetaine-Lys has antifungal property and good antifungal effect.

Therefore, the present invention also provides the application of the compound in antifungal, especially in the preparation of antifungal drugs. The compound can be mixed with conventional adjuvants to make injections, ointments, sprays and the like.

The fungus is a yeast fungus or a fungus of the genus Aspergillus, and preferably, the yeast fungus is Candida albicans, Candida parapsilosis, Candida krusei or Candida pseudopolymorpha.

The fungus of the genus Aspergillus is Aspergillus niger.

The present invention uses Bacillus subtilis ( Bacillus subtilis ) ZJU007 CGMCC NO.4140 to obtain the lead compound Chlorotetaine, and takes the drug delivery process as the theoretical basis to carry out homology modeling to construct the virtual structure of the small molecule peptide transporter, through the small molecule peptide transporter The virtual structure of the lead compound Chlorotetaine was analyzed, and a new tripeptide compound with antifungal ability was obtained by designing and synthesizing a new type of antifungal compound. This compound can not only inhibit Candida albicans, Candida parapsilosis, Gram Candida fungi such as Candida ruckeri and Candida pseudopolymorpha have a minimum inhibitory concentration (MIC) of 0.81 µg/mL; they can also inhibit Aspergillus fungi such as Aspergillus niger with a minimum inhibitory concentration (MIC) of up to 1.63 µg/mL.

Description of drawings

Figure 1 is a schematic diagram of the antibacterial mechanism of the lead compound Chlorotetaine.

Figure 2 is a graph showing the changes in reducing sugar, dry cell weight, total sugar and antibiotic titer during the fermentation of Bacillus subtilis ZJU007.

Fig. 3 is the mass spectrogram and chemical structural formula of lead compound Chlorotetaine;

A: The positive ion mass spectrum of Chlorotetaine; B: The negative ion mass spectrum of Chlorotetaine C: The chemical structure of the lead compound Chlorotetaine.

Figure 4 is a schematic diagram of the homology modeling structure and structural identification results of small molecule peptide transporters;

A: Schematic diagram of the structure of small molecule peptide transporters after homology modeling; B: Lag diagram of molecular peptide transporters identified by PROCHECK software; C: WHATCHECK identification results; D: WHATCHECK identification results.

Figure 5 is a schematic diagram of the connection between the lead compound Chlorotetaine and an amino acid (R is an amino acid).

Figure 6 is a schematic diagram of the interaction between five new compounds obtained through virtual screening and transporter docking; among them, the amino acids connected to the lead compound Chlorotetaine are: 1: Lys; 2: Gln; 3: Asn; 4: Thr; 5 : Glu.

Fig. 7 is the antifungal experiment of the novel compound of the synthetic lead compound Chlorotetaine-Lys;

Wherein, 1 is Chlorotetaine-Gln; 2 is Chlorotetaine-Asn; 3 is Chlorotetaine-Lys; 4 is Chlorotetaine; 5 is Chlorotetaine-Thr; 6 is Chlorotetaine-Glu.

Figure 8 is the chemical structural formula of the new compound Chlorotetaine-Lys.

Fig. 9 is a mass spectrum data diagram of the new compound Chlorotetaine-Lys.

Detailed ways

The present invention will be further explained and illustrated below in conjunction with the accompanying drawings and specific embodiments.

The strains involved in the following examples are: Bacillus subtilis (Bacillus subtilis ) ZJU007 CGMCC NO.4140 used for fermentation, isolation and purification of the lead compound Chlorotetaine and five purchased common fungal pathogens, specifically as follows:

(1) Bacillus subtilis ( Bacillus subtilis ) ZJU007 CGMCC NO.4140, preserved in the China General Microorganism Culture Collection Management Center (CGMCC), located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing It is CGMCC No.4140, and the preservation time was September 03, 2010; the strain has also been disclosed in the application number 201010521030.9, entitled "A strain of Bacillus subtilis and its application".

(2) Candida albicans, preserved in the China General Microorganism Culture Collection Management Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, the Institute of Microbiology, Chinese Academy of Sciences, with the preservation number ATCC10231-3147.

(3) Aspergillus niger, kept in the laboratory.

(4) Candida parapsilosis, preserved in the China General Microorganism Culture Collection Center (CGMCC), located at No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Beijing, with the preservation number JCM1785.

(5) Candida cruzii, preserved in the China General Microorganism Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Beijing, with the preservation number CBS573.

(6) Candida pseudopolymorpha, preserved at the China General Microorganism Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences, with the preservation number ATCC 26012.

Example 1

1. Fermentation of lead compound

Bacillus subtilis ( Bacillus subtilis ) ZJU007 CGMCC NO.4140 was fermented, separated and purified to obtain the lead compound Chlorotetaine, as follows:

i medium

(1) Incline medium: LB medium.

(2) Seed medium (g·L ^-1 ): glucose 15, yeast powder 10, K ₂ HPO ₄ 1, MgSO ₄ 0.5, FeSO ₄ 0.01, NaCl 10, adjust pH to 7.0 and add CaCO ₃ 0.4.

(3) Fermentation medium (g·L ^-1 ): yeast powder 2.72, soluble starch 26.67, (NH ₄ ) ₂ SO ₄ 3.95, K ₂ HPO ₄ 2.4, NaCl 10, MgSO ₄ 0.5, CaCO ₃ 0.72, CuSO ₄ 0.02, FeSO ₄ 0.02, MnSO ₄ 0.02, pH 6.5.

(4) Candida albicans culture medium (g·L ^-1 ): glucose 20, peptone 10, agar 20.

ii method

Strain culture conditions Take 1 loop of activated strains and put them into a seed bottle, fill 500 mL Erlenmeyer flask with 100 mL medium, the initial pH value is 7.0, and culture at 200 r/min at 30 °C for 24 h. Then, the seed liquid was introduced into a 5 L fermenter (60% of the liquid content) with an inoculum volume of 10% (v/v), the ventilation volume of the fermenter was 400 L·h ^-1 , and the rotation speed was 200 r·min ^-1 , fermented at 28 ° C for 50 h, and took samples every 4 h.

iii Determination of biological potency of antibiotics

Using the tube-and-disk method, wash the slant of Candida albicans cultured at 37°C for 2 days with 30 mL of sterile saline to make a bacterial suspension, and put 2 mL to 100 mL in a solid medium at about 50°C After mixing, draw 20 mL into a petri dish with a diameter of 9 cm. After cooling, place an Oxford cup on the surface of the culture medium. Take 150 µL of the solution to be tested and add it to the Oxford cup. Incubate at 37 °C for 18 h, and measure the diameter of the transparent zone of inhibition , converted into the corresponding activity:

Nystatin standard curve: y = 1.2297x -1.58778, R ² =0.9998

(where U—Nystatin potency (U/mL), r—Inhibition zone radius (cm), Nystatin (potency 6593 U/mL).

The corresponding potency of antibiotics can be calculated according to the above relational formula. )

It can be seen from Figure 2 that the pH value has been decreasing from 0 to 28 h, which is mainly because the bacteria are in the growth period, and the bacteria use the nutrients in the fermentation broth, respiration releases CO ₂ and decomposes to produce organic acids . The pH tends to be stable in the 28-48 h stage, and at this stage, the bacteria are also in a stable stage. After the bacteria were introduced into the fermenter, the total sugar concentration in the fermentation broth gradually decreased with the growth of the bacteria, and the total sugar consumption was faster in the 8-28 h stage, and the bacteria were also in the logarithmic growth phase at this time. The bacterial concentration reached the maximum at 28 h, and maintained at a relatively stable level, and did not begin to decrease until 44 h after fermentation, which may be related to the autolysis of the bacterial cells during this period. When the bacteria grew rapidly, the titer of the fermentation broth also increased rapidly, indicating that the antibiotic production process of this strain was coupled with the growth of the bacteria. During the fermentation process, the titer of the fermentation broth reached the highest at the 28th hour, and the fermentation period of the strain was determined to be 24 hours. Compared with antibiotic-producing filamentous bacteria, this bacterial strain produces antibiotics with a shorter cycle.

2. Separation and purification of lead compounds

The fermented liquid obtained by fermentation is firstly filtered through a ceramic membrane filter to remove bacteria and macromolecular foreign proteins produced by partial fermentation. Then, the filtered fermented liquid was concentrated 10 times under low pressure using a rotary evaporator. According to the concentration of 1:4 (v/v, fermentation broth: ethanol), the concentrated solution was subjected to alcohol precipitation, centrifuged at 500rpm/min to remove the precipitate, and concentrated under low pressure to recover the supernatant. The recovered supernatant was passed through XAD1600 macroporous resin (400 mm x Φ 55 mm), deionized water was used as mobile phase, and the flow rate was 5mL/min for macroporous adsorption chromatography to remove most of the pigment and fat-soluble impurities. The eluate was concentrated and recovered under low pressure, and then reversed-phase gradient chromatography was performed using MCI reversed-phase chromatography resin (400 mm x Φ 100 mm). First, use 0.1% trifluoroacetic acid solution as the mobile phase, 22 mL/min, and elute for 2.5 column volumes. Then use 0.1% trifluoroacetic acid solution + 10% methanol solution as the mobile phase, 22 mL/min, and elute for 3 column volumes. Collect the methanol eluate, concentrate and recover. The methanol eluate was subjected to reverse-phase C18 chromatography (400 mm x Φ 40 mm) at a flow rate of 12 mL/min, and an automatic collector was used to collect one tube every 50 s, and then antibacterial experiments were performed on each tube to collect samples with antibacterial activity. eluent. Chlorotetaine with a purity of >95% can be obtained after the second antibacterial peak is subjected to reversed-phase C18 chromatography for the second time.

2. Structural identification of lead compound Chlorotetaine

The structure analysis of the lead compound Chlorotetaine was carried out by mass spectrometry and nuclear magnetic resonance techniques.

The result is as follows:

As can be seen from the mass spectrograms shown in Figure 3 (A, B), the relative molecular mass of the compound in Figure 3 A is M+1=289,

The relative molecular mass of the compound in Figure 3 B is 2M-1=575, and the calculated relative molecular mass of the lead compound Chlorotetaine is M=288, and the chemical structural formula in Figure 3 C can be obtained by synthesizing the NMR data of the lead compound Chlorotetaine in Table 1 , namely: the lead compound Chlorotetaine.

Table 1 Chlorotetaine NMR data. Chlorotetaine ¹³ C (600MHz) and ¹ H in D ₂ O

(600 MHz) NMR data. P.: position; qua. : quaternary carbon.

3. Homology modeling of small molecule peptide transporter (PTR22 (Accession: XP_712738))

Figure 1 shows the schematic diagram of the antibacterial mechanism of the lead compound Chlorotetaine. It can be seen from Figure 1 that after the lead compound Chlorotetaine is produced by Bacillus subtilis ( Bacillus subtilis ) ZJU007 CGMCC NO.4140, under the action of a specific transporter, it is transported from the cell to Transport from inside to outside the cell; when the lead compound Chlorotetaine comes into contact with pathogenic microorganisms, under the action of the transporter in the pathogenic microorganisms, the lead compound Chlorotetaine is transported from outside the cell to the inside of the pathogenic microorganism; when the lead compound Chlorotetaine enters the cell , under the action of peptidase, the lead compound Chlorotetaine of the dipeptide is decomposed into alanine and an uncommon amino acid (as shown in Figure 1). This uncommon amino acid is a structural analogue of glutamine, which can competitively inhibit the activity of glucosamine-6-phosphate synthase, thereby hindering the synthesis of corresponding macromolecular substances such as dextran and peptidoglycan, thereby destroying The structure of the cell membrane and cell wall of pathogenic bacteria achieves the purpose of sterilization.

It can be seen that the lead compound Chlorotetaine must be transported into the cell through the small molecule peptide transporter to exert its bactericidal effect, and the transport efficiency determines the bactericidal effect of the bactericidal compound. Therefore, the present invention optimizes the structure of the lead compound Chlorotetaine and improves its binding to the small molecule peptide transporter by performing homology modeling on the small molecule peptide transporter (Gene ID: 3645634) in Candida albicans (ATCC 10731) Spend.

The present invention adopts the membrane protein homology modeling module in the Discovery Studio software, and uses three transporter structures GKPOT (Accession: Q5KYD1), Pep Tst (Accession: Q5M4H8), Pep Tso (Accession: Q8EKT7) with the highest sequence similarity As a template, perform homology modeling.

The obtained membrane protein structure is shown in Figure 4 A. In the figure, the ball-and-stick model represents its active center. In order to better reflect the function of the transmembrane protein and clarify the solvation properties of the protein in the implicit solvent model, the present invention is The model adds simulated cell membrane structures. The spherical region represents the binding region of the small molecule peptide transporter. Then, by using the PROCHECK software to obtain the Laplace diagram (as shown in Figure 4 B), it is found that 95% of the amino acid residues are located in the reasonable range, and only 0.2% of the amino acid residues are located in the unreasonable range, which shows that the model has a good Stereochemical stability; and the identification results obtained by WHATCHECK software show that the Z-scores are all within a reasonable range (as shown in Figure 4D), which illustrates the rationality of the local environment of amino acids in the simulated structure of small molecule peptide transporters.

4. Design, screening and chemical synthesis of new antifungal drugs

By connecting 20 common different amino acids to the lead compound Chlorotetaine at the R site in the chemical structural formula in Figure 5, a new compound is formed, and then, using the model constructed in step (3) as the target enzyme, the above-mentioned new compound is used as a small molecule substrate for virtual screening.

Through the CDOCKER module in Discovery Studio, the new compound is molecularly docked with the constructed small molecule peptide transporter to achieve the purpose of virtual screening. CDOCKER is an accurate molecular docking technology, which places the ligand molecule at the active site of the receptor molecule, and then evaluates the interaction between the ligand and the receptor in real time according to the principles of geometric complementarity, energy complementarity and chemical environment complementarity. bad, and find the optimal binding mode between the two molecules. The results are shown in Table 2.

Since H bonds play an important role in immobilizing compounds during the transmembrane transport of peptides, virtual screening was performed using the number of hydrogen bonds and docking free energy as reference standards. By referring to the free energy of docking and comparing the optimal conformation generated by docking, the reasonable interaction relationship is determined. As can be seen from the results in Table 2, the lead compound Chlorotetaine connected to the new compound of lysine, glutamine, aspartic acid, threonine and glutamic acid is more suitable. Figure 6 shows the interaction relationship between the above five new compounds and the small molecule peptide transporter. It can be seen from Figure 6 that these five compounds can fully fill the active region of the transporter, and can interact with nearby key amino acid residues to form a stable complex structure. Therefore, the above new compounds were entrusted to China Peptide Biochemical Co., Ltd. for chemical synthesis.

Table 2 Results of virtual screening using molecular docking.

5. Activity detection and structure identification of new antifungal compounds.

Candida albicans (ATCC 10731) preserved in the laboratory was used as the test strain, and the lead compound Chlorotetaine was used as the positive control for activity detection. As shown in Figure 7 (No. 4 is the positive control, No. 1, 2, 3, 5, and 6 are new synthetic antifungal drugs), only two new compounds have antifungal activity, namely: Chlorotetaine-Lys (No. 3 ) and Chlorotetaine-Gln (No. 1), and only one new compound (Chlorotetaine-Lys) had significantly improved antifungal effect.

The structure of Chlorotetaine-Lys was determined by Fourier transform infrared transform mass spectrometry, and the relative molecular weight of Chlorotetaine-Lys was determined to be 416.2118. The structural formula is shown in formula (I) and Figure 8.

Example 2

Utilize the novel antibacterial compound Chlorotetaine-Lys that embodiment 1 obtains to carry out in vitro antifungal experiment, concrete steps are as follows:

(1) Five strains of common fungal pathogens purchased from the China Microbial Culture Collection Center were activated and cultured in vitro; the above five strains of common fungal pathogens were Candida albicans (ATCC10231), Aspergillus niger (preserved in the laboratory), Candida parapsilosis (JCM1785), Candida krusei (CBS573), Candida pseudopolymorpha (ATCC 26012)

The formula of the fermentation medium of Candida and Aspergillus niger is: 4% glucose and 1.0% peptone. Place the above-mentioned five common fungal pathogenic bacteria in the above-mentioned culture medium at 28°C, and cultivate them for 18-24h.

(2) Using a 96-well plate, adopt the double dilution method to prepare the new antibacterial compounds to a final concentration between 100 µg/mL and 0.2 µg/mL;

(3) Inoculate 5 µL of the test strain (~2.03×10 ⁵ cells/mL) into a 96-well plate, culture it at 28°C for 24 hours, and detect the minimum inhibitory concentration of the compound against different fungi by a microplate reader, to determine Fluconazole with better water solubility was used as a positive control, and the results are shown in Table 3.

It can be seen from Table 3 that Chlorotetaine-Lys has a good antibacterial effect on the tested strains, especially on Candida krusei (CBS573), its antibacterial effect even exceeds the level of fluconazole, and has a good application prospect.

Table 3 The minimum inhibitory concentration results of new antifungal compounds against different fungi.

Claims (1)

1. application of a kind of compound in preparing antifungal drug, shown in the structural formula such as formula (I) of the compound：

The fungi is Candida class fungi and aspergillus fungi；

The Candida class fungi is Candida albicans, Candida parapsilosis, Candida krusei or quasi- multiform vacation silk ferment It is female；The aspergillus fungi is aspergillus niger.