阅读说明：本技术 利巴韦林在制备抑制细菌群体感应的药物中的应用 (Application of ribavirin in preparation of medicine for inhibiting bacterial quorum sensing ) 是由 赵克雷 褚以文 王欣荣 林家富 刘超兰 宋涛 黄挺 张新宜 翟龙飞 于 2021-02-25 设计创作，主要内容包括：本发明公开了一种利巴韦林在制备抑制细菌群体感应的药物中的应用。可高效抑制革兰氏阴性病原菌铜绿假单胞菌(Pseudomonas aeruginosa)群体感应系统。解决传统抗生素治疗多重耐药病原菌相关感染有效药物选择少、周期长、病情易反复的问题。本发明包括如下特点：可同时抑制铜绿假单胞菌群体感应系统三个主控基因lasR、rhlR、pqsR及其下游功能基因的表达,可显著抑制铜绿假单胞菌群体感应系统控制的细菌毒力因子的合成,减轻感染症状。本发明可用于以铜绿假单胞菌为代表的多重耐药革兰氏阴性菌感染这类需要长期治疗的疾病,不容易产生耐药。(The invention discloses an application of ribavirin in preparation of a medicament for inhibiting bacterial quorum sensing. Can effectively inhibit gram-negative pathogenic bacteria pseudomonas aeruginosa Pseudomonas aeruginosa ) A quorum sensing system. Solves the problems of few effective drug choices, long period and easy relapse of the traditional antibiotic treatment for the infection related to multi-drug resistant pathogenic bacteria. The invention comprises the following characteristics: three main control genes capable of simultaneously inhibiting pseudomonas aeruginosa quorum sensing system lasR 、 rhlR 、 pqsR And the expression of downstream functional genes thereof can obviously inhibit the synthesis of bacterial virulence factors controlled by a pseudomonas aeruginosa quorum sensing system and relieve infection symptoms. The invention can be used for treating diseases needing long-term treatment, such as multiple drug-resistant gram-negative bacteria infection represented by pseudomonas aeruginosa, and the drug resistance is not easy to generate.)

1. Application of ribavirin in preparation of medicines for inhibiting bacterial quorum sensing.

2. The use of ribavirin for the manufacture of a medicament for the inhibition of bacterial quorum sensing according to claim 1 wherein the effective concentration of ribavirin is from 50 to 200 micromoles/liter.

3. The use of ribavirin for the manufacture of a medicament for inhibiting bacterial quorum sensing according to claim 1 wherein the ribavirin is in a pharmaceutically acceptable formulation comprising tablets, granules, capsules, injections, sprays or other forms that are absorbed by the human body.

4. The use of ribavirin for the manufacture of a medicament for inhibiting bacterial quorum sensing according to claim 1 wherein said bacteria are gram-negative bacteria.

5. The use of ribavirin for the manufacture of a medicament for inhibiting quorum sensing in a bacterium according to claim 4 wherein said bacterium is Pseudomonas aeruginosa.

Technical Field

The invention relates to a high-efficiency quorum sensing small molecule inhibitor and application thereof. In particular to a novel application of broad-spectrum antiviral drug Ribavirin (Ribavirin) which can inhibit the toxicity of multi-drug-resistant gram-negative bacteria through efficiently inhibiting a quorum sensing system. The compound of the invention can be used for treating diseases needing long-term treatment, such as multiple drug-resistant gram-negative bacterial infection represented by pseudomonas aeruginosa, and is not easy to generate drug resistance.

Background

Ribavirin is also called ribavirin or ribavirin, the chemical name is 1-D-ribofuranosyl-1H-1, 2, 4-triazole-3-carboxamide, the molecular formula is C₈H₁₂N₄O, a broad-spectrum antiviral drug, can inhibit non-retroviruses such as HCV, HIVl, RSV and the like, and has a chemical structure with pseudomonas aeruginosaPseudomonas aeruginosa) The parent nucleus structures of the quorum sensing signal molecule homoserine lactone of gram-negative bacteria have certain similarity.

The structural formula of ribavirin is shown in figure 1.

The bacterial quorum sensing is used for describing that after bacteria grow to a certain population density, individual bacteria can continuously receive homoserine lactone signal molecules secreted to the outside by other individuals in a unit environment, activate a bacterial intracellular complex regulation and control network, and further regulate and control release and metabolic pathways of virulence factors (such as extracellular protease, toxin substances and the like) in a large range, so that the survival fitness of the bacterial population is obviously improved. In view of the critical role of bacterial quorum sensing systems mediated virulence systems in infectious diseases, achieving a reduction in bacterial virulence through inhibition of bacterial quorum sensing systems, which is then cleared by the host immune system, is an antibiotic replacement drug development strategy. The strategy is different from the traditional antibiotic action mechanism, can not directly inhibit the growth and the reproduction of bacteria, but inhibit the exertion of bacterial toxicity and reduce the damage of bacterial toxin to host tissues and organs. The targeted small molecule compound developed based on the signal molecule of the bacterial quorum sensing system directly acts on the quorum sensing central control protein, and if the central control protein influences the drug effect of the targeted small molecule through mutation, namely drug resistance is generated, the change of the protein conformation of the central control protein can also influence the binding of the central control protein to the natural signal molecule, so that the quorum sensing system is actively inhibited. Therefore, whether the bacteria generate drug resistance through mutation or not, the application result of the targeted small molecule compound is that the bacterial quorum sensing system is inhibited, and the targeted small molecule compound is an anti-toxicity strategy which is stable in evolution and not easy to generate drug resistance.

Disclosure of Invention

The invention aims to provide application of ribavirin in preparation of a medicament for inhibiting quorum sensing of multiple drug-resistant gram-negative bacteria pseudomonas aeruginosa.

The invention is realized by the following steps:

application of ribavirin in preparation of medicine for inhibiting bacterial quorum sensing

The acting concentration of ribavirin is 50-200 micromole/L.

Ribavirin is presented in a pharmaceutically acceptable formulation comprising tablets, granules, capsules, injections, sprays or other forms that can be absorbed by the human body.

The bacteria are gram-negative bacteria.

The bacteria are pseudomonas aeruginosa.

Ribavirin and common gram-negative bacterium quorum sensing signal molecules have similar furan structure mother nuclei, can be combined with a plurality of central control proteins of a quorum sensing system in a targeted mode, inhibits the generation of extracellular protease, and significantly inhibits the expression of a plurality of central control genes and downstream functional genes of the quorum sensing system.

The bacteria are gram-negative bacteria represented by pseudomonas aeruginosa model strain PAO 1.

The ribavirin of the invention comprises the following characteristics: can act on a plurality of central control proteins of a quorum sensing system at the same time, is effective under a lower using dosage and can effectively inhibit the quorum sensing system. The ribavirin of the invention can be used for diseases needing long-term treatment, such as multi-drug resistant gram-negative bacterial infection represented by pseudomonas aeruginosa, and drug resistance is not easy to generate.

The ribavirin can simultaneously inhibit the expression of a plurality of central control genes and downstream functional genes thereof of a bacterial quorum sensing system, and inhibit the production of bacterial extracellular protease.

Ribavirin these effects were evaluated in vitro using the P.aeruginosa model strain PAO1 and the results are given in the examples.

In vitro, the small molecule compound ribavirin has no bacteriostatic activity on pseudomonas aeruginosa cultured under a nutrient-rich condition (such as LB culture medium), but can obviously inhibit the proliferation of the pseudomonas aeruginosa in an environment (such as M9-adenosin culture medium) which needs to activate a quorum sensing system for growth. Ribavirin can obviously inhibit central control gene of pseudomonas aeruginosa quorum sensing systemlasRThe functions of the recombinant strain and the synthesis of extracellular protease, and simultaneously inhibits the expression of three central control genes and downstream key virulence related functional genes of the currently known pseudomonas aeruginosa quorum sensing system, and can effectively protect nematodes from being infected by pseudomonas aeruginosa.

The application of ribavirin in preparing the medicine for inhibiting bacterial quorum sensing has the following advantages: completely different from the traditional antibiotic action mechanism, can avoid the direct action of multiple antibiotic resistance on a plurality of central control proteins of a quorum sensing system, and further effectively inhibit the quorum sensing system-mediated toxicity from exerting. The ribavirin of the invention can be used for diseases needing long-term treatment, such as multi-drug resistant gram-negative bacterial infection represented by pseudomonas aeruginosa, and is not easy to generate drug resistance.

Drawings

FIG. 1 is the structural formula of ribavirin.

FIG. 2 is a graph of the growth effect of ribavirin on Pseudomonas aeruginosa. A is LB culture medium; m9-adenosine culture medium.

FIG. 3 shows the effect of ribavirin on the induction regulation of gene expression in Pseudomonas aeruginosa populations.

FIG. 4 is a graph showing the protective effect of ribavirin on P.aeruginosa infected nematodes. Control, E.coli OP50 infected group. PAO1, pseudomonas aeruginosa infection panel. Ribavirin, a group of pseudomonas aeruginosa infections with Ribavirin intervention.

Detailed Description

The following examples are given to further illustrate the embodiments of the present invention. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. The invention covers all possible alternatives, modifications and equivalents within the scope of the claims. The specific techniques or conditions not mentioned in the following examples are all the conventional techniques or conditions, or the techniques or conditions described in the literature in the field, or the product specification.

Example 1

Ribavirin formulations inhibit pseudomonas aeruginosaP. aeruginosa PAO 1).

The ribavirin preparation is prepared by dissolving granules in sterile deionized water.

And (3) analyzing the bacteriostatic activity of ribavirin:

pseudomonas aeruginosaP. aeruginosa PAO1) （1.0×10⁶CFU) were inoculated into a common LB broth medium (Haibo Biotechnology Co., Ltd.) and M9-adenosin (0.1%, w/v) medium (Sigma-Aldrich), and 50, 100, 200. mu. mol/L ribavirin preparation was added to each medium for treatment, and an equal amount of sterile deionized water was added to control, and after incubation at 37 ℃ for 16 hours, OD was measured at optical density₆₀₀The density of the bacterial liquid was measured.

The result of the ribavirin bacteriostatic activity analysis is shown in figure 2. In the broth LB medium, the density of the inoculum after treatment of pseudomonas aeruginosa with ribavirin at each concentration did not change significantly compared to the control group (fig. 2A). In contrast, in the M9-adenosine medium, ribavirin can significantly inhibit the proliferation of Pseudomonas aeruginosa compared with the control group (FIG. 2A). In view of the Adenosine of Pseudomonas aeruginosaMetabolism needs to utilize quorum sensing to control geneslasRThe regulated intracellular hydrolase Nuh, the growth of the pseudomonas aeruginosa is weakened under the condition that the adenosine is the only carbon source in the presence of ribavirin, which indicates that the quorum sensing system is inhibited and the proliferation is influenced.

Ribavirin inhibition quorum sensing activity assay:

the preparation method comprises mixing 5 microliters of Pseudomonas aeruginosa (5.0X 10)⁶CFU) were inoculated into M9-skim mil (0.5%, w/v) (Sigma-Aldrich) and M9-adenosine (0.1%, w/v) agar plates (Sigma-Aldrich) containing varying concentration gradients of ribavirin (0, 50, 100, 200 micromole/L), respectively, the diameter of the proteolytic loop on the M9-skim mil plate was measured after 20 hours of incubation at 37 deg.C, and the growth of Pseudomonas aeruginosa on the M9-adenosine plate was examined.

Ribavirin inhibition quorum sensing activity assays are shown in table 1. On the M9-ski mil plates, ribavirin significantly inhibited the extracellular protease production of P.aeruginosa colonies, and inhibited P.aeruginosa growth on M9-adenosine plates. Whereas Pseudomonas aeruginosa requires the use of extracellular proteases regulated by quorum sensing for the metabolism of ski mil, the use of quorum sensing for the metabolism of adenosinelasRThe proteolysis cycle of the pseudomonas aeruginosa is reduced under the condition that the Skim milk is the only carbon source in the presence of the regulated intracellular hydrolase Nuh and ribavirin, and the growth of the pseudomonas aeruginosa is weakened under the condition that the adenosine is the only carbon source, which jointly indicates that the quorum sensing system is inhibited.

Ribavirin (micromole/L)	M9-skim milk (cm)	M9-adenosine1
			0	1.72 ± 0.148	+
50	1.43 ± 0.05****	-
			100	1.38 ± 0.076****	-
200	1.18 ± 0.083****	-

TABLE 1 Effect of ribavirin on the quorum sensing System of Pseudomonas aeruginosa

1: '+' represents normal growth; '-' represents no or weak growth

Analysis of the influence of ribavirin on quorum sensing regulatory gene expression:

pseudomonas aeruginosa (1.0X 10)⁶CFU) is inoculated in a liquid culture medium M9-skim mil (0.5%, w/v), 200 micromoles/L ribavirin preparation is added to a treatment group, equivalent sterile deionized water is added to a control group, thalli are collected and total RNA is extracted after 16-hour culture at 37 ℃, and Real-time PCR is utilized to detect three quorum sensing central control genes of pseudomonas aeruginosalasR, rhlR, pqsRAnd its downstream functional genelasB, rhlA, pqsA, pqsD, phzA, hcnAThe expression level of (3) was 16S rRNA as an internal reference gene.

The analysis result of the effect of ribavirin on quorum sensing regulatory gene expression is shown in figure 3. After the culture of the M9-ski milk culture solution, ribavirin can simultaneously and obviously inhibit three groups of pseudomonas aeruginosaBody-sensing central control genelasR, rhlR, pqsRAnd a functional gene downstream thereoflasB, rhlA, pqsA, pqsD, phzA, hcnAThe expression of (a) was also significantly inhibited. The results are combined to show that the broad-spectrum antiviral drug ribavirin can effectively inhibit the pseudomonas aeruginosa quorum sensing system.

Example 2

Protecting against Pseudomonas aeruginosa infection nematodes.

Ribavirin, which is prepared into tablets, is ground into dry powder and dissolved in sterile deionized water, and the dry powder is centrifuged to obtain supernatant serving as test solution.

Analysis of the protective effect of ribavirin on pseudomonas aeruginosa infected nematodes:

50 microliters of Pseudomonas aeruginosa (5.0X 10)⁷CFU) is inoculated on a PGS (peptone-glucose-sorbent) culture plate (Sigma-Aldrich), 200 micromoles/L of ribavirin test solution is added into the corresponding culture plate in a treatment group, an equal amount of sterile deionized water is added into a control group, the control group is placed at room temperature for cooling after being cultured for 24 hours at 37 ℃, then 10 newly cultured nematode adults (stage L4) are inoculated on each plate, and the culture is continued at 25 ℃ and the survival condition of the nematodes is observed. The negative control group was initially inoculated with E.coli OP50, and the other culture conditions were identical to those of the P.aeruginosa infection group.

The analysis result of the protective effect of ribavirin on pseudomonas aeruginosa infected nematodes is shown in fig. 4. The lethal effect of pseudomonas aeruginosa on nematodes in the control group is gradually enhanced along with the extension of infection time, while the survival condition of nematodes in the ribavirin treatment group is obviously improved and has very significant statistical difference with the untreated group (the lethal effect of pseudomonas aeruginosa on nematodes in the ribavirin treatment group is reduced by the control group, and the lethal effect of pseudomonas aeruginosa on nematodes in the ribavirin treatment group is reduced by the control groupP <0.0001), indicating that ribavirin can better protect nematodes from being infected by pseudomonas aeruginosa.

Application of ribavirin in preparation of medicine for inhibiting bacterial quorum sensing

The LB broth and agar medium referred to in the examples were purchased from Haibo Biotechnology Ltd. The main components of M9-adenosine and M9-skim mil Medium, adenosine and skim mil, and PGS (peptone-glucose-sorbent) Medium, PGS, were purchased from Sigma-Aldrich.