CN107021999B

CN107021999B - Anti-candida polypeptide, application thereof and anti-candida drug

Info

Publication number: CN107021999B
Application number: CN201610074028.9A
Authority: CN
Inventors: 王灏; 吴子斌
Original assignee: Shenzhen Research Institute of CUHK
Current assignee: Shenzhen Research Institute of CUHK
Priority date: 2016-02-02
Filing date: 2016-02-02
Publication date: 2021-03-09
Anticipated expiration: 2036-02-02
Also published as: CN107021999A

Abstract

The invention provides an anti-candida polypeptide, application thereof and an anti-candida medicament, wherein the amino acid sequence of the polypeptide comprises any one of amino acid sequences shown by SEQ ID NO. 1-SEQ ID NO. 12. Experiments show that the polypeptide designed by the invention has a simple structure, has the effect of inhibiting candida and has the advantage of low hemolysis rate of human erythrocytes.

Description

Anti-candida polypeptide, application thereof and anti-candida drug

Technical Field

The invention relates to the field of antifungal medicines, in particular to an anti-candida polypeptide, application thereof and an anti-candida medicine.

Background

Candida species (Candida spp) are conditionally pathogenic bacteria that are present in the oral cavity, gastrointestinal tract and vaginal mucosa of normal humans. In healthy humans, candida does not cause any infection, but in immunocompromised persons candida causes severe infections. In recent years, the population with low immune function is increasing due to the increase of cancer patients and aids patients receiving chemotherapy, and the cases of candida infection are increasing year by year due to the wide application of invasive minimally invasive diagnosis and treatment and the increase of organ transplantation operations. Candida also has become one of the most common nosocomial infectious pathogens with high morbidity and mortality.

The results of the candida drug susceptibility survey showed that 100.0% candida glabrata, 4.9% candida tropicalis, 2.9% candida glabrata and 0.8% candida albicans were resistant to fluconazole; 9.8% of Candida tropicalis, 7.6% of Candida parapsilosis, 5.0% of Candida krusei and 0.6% of Candida albicans were resistant to voriconazole. Today, all echinocandins are effective against most candida species, and it has been reported that only 38.1% of candida glabrata are found to be sensitive to caspofungin.

The investigation result of people hospital in Shaoxing city in Zhejiang shows that 1003 pathogenic fungi are separated from inpatients in 2011, wherein the content of Candida albicans is 58.9%; the second is candida tropicalis, accounting for 23.4%. The susceptibility of candida tropicalis to voriconazole is only 0.9%, and the candida tropicalis has drug resistance to fluconazole, 5-flucytosine and itraconazole in different degrees; the sensitivity of candida glabrata to fluconazole and itraconazole is 79.8 percent; in 396 hospital candida infected specimens detected in subsidiary hospitals of Sichuan northern medical college and Sichuan Dahuaxi hospital, candida albicans (68.1%/70.2% of Sichuan northern medical college/Huaxi hospital), candida tropicalis (14.9%/9.0%), candida glabrata (5.7%/12.2%), candida krusei (3.4%/2.4%), and candida parapsilosis (2.1%/1.6%). These fungi are most sensitive to amphotericin B and have varying degrees of resistance to fluconazole. A survey in the affiliated Hospital of Guilin medical school, Guangxi, indicated that the 498 Candida strains tested were 59.84% Candida albicans and 18.27% Candida glabrata. The candida species have high drug resistance rates to fluconazole and itraconazole, which are respectively 22.8% and 56.8%, while the amphotericin B and 5-flucytosine have low drug resistance rates, which are respectively 7.9% and 7.3%.

The emergence of resistant strains is a problem to be faced worldwide. Because there is no effective antifungal vaccine available at present, the treatment of fungal infections has relied primarily on various antibiotics. When no new medicine is put on the market, most doctors use the medicine dosage and mixed medicine. Unfortunately, all antifungal drugs are cytotoxic, and the higher the concentration, the more obvious, the mixed drug is being discussed vigorously. In recent years, antibacterial peptides have a special mechanism of action, so that drug resistance is not easy to generate, and chemical synthesis processes are improved, so that the antibacterial peptides are attracted by researchers in various countries. These include defensins, cathelicidins, histatins and plant loop proteins. Therefore, the development of new drugs against candida is an effective solution to drug-resistant strains.

Disclosure of Invention

The invention provides an anti-candida polypeptide, application thereof and an anti-candida drug.

The technical problem of the invention is solved by the following technical scheme:

an anti-candida polypeptide, the amino acid sequence of which comprises any one of the amino acid sequences selected from the group consisting of:

SEQ ID NO:1：Lys Trp Lys Leu Arg Trp Cys Lys Gly Gly Phe Val Cys Lys Trp Arg；

SEQ ID NO:2：Lys Trp Lys Leu Arg Trp Arg Cys Lys Gly Gly Phe Val Cys Lys Trp Arg；

SEQ ID NO:3：Lys Trp Lys Leu Arg Trp Cys Lys Pro Pro Phe Val Cys Lys Trp Arg；

SEQ ID NO:4：Lys Trp Lys Leu Arg Trp Arg Cys Arg Ser Ser Gly Val Cys Lys Trp Arg；

SEQ ID NO:5：Lys Trp Lys Leu Arg Trp Arg Cys Arg Arg Ser Ser Gly Val Cys Lys Trp Arg；

SEQ ID NO:6：Lys Gly Lys Trp Arg Gly Arg Cys Arg Arg Ser Ser Gly Val Cys Lys Trp Arg

SEQ ID NO:7：Lys Trp Lys Leu Arg Trp Arg Cys Arg Arg Ser Ser Gly Val Cys Lys Pro Arg

SEQ ID NO:8：Lys Leu Arg Trp Arg Cys Arg Arg Ser Ser Gly Val Cys Lys Trp Arg Trp Lys

SEQ ID NO:9：Lys Trp Lys Trp Arg Trp Arg Cys Arg Arg Ser Ser Gly Val Cys Lys Trp Arg；

SEQ ID NO:10：Lys Trp Lys Leu Arg Trp Arg Arg Arg Ser Ser Gly Val Cys Lys Trp Arg；

SEQ ID NO:11：Lys Leu Arg Phe Arg Cys Arg Arg Ser Ser Gly Val Lys Trp Arg Phe Lys；

SEQ ID NO:12：Lys Leu Arg Trp Arg Arg Arg Ser Ser Gly Val Cys Lys Trp Arg。

preferably, the candida is at least one of candida albicans, candida krusei and candida glabrata.

The application of the polypeptide in preparing a medicament for treating candida infection.

An anti-candida medicament, wherein the active ingredient in the medicament is the polypeptide.

The beneficial effects of the invention include: experiments show that the polypeptide designed by the invention has a simple structure, has the effect of inhibiting candida and has the advantage of low hemolysis rate of human erythrocytes.

Drawings

FIGS. 1-3 are graphs showing the results of hemolysis test of human erythrocytes with the polypeptide in the preferred embodiment of the present invention.

Detailed Description

The invention will be further described with reference to the accompanying drawings and preferred embodiments.

The invention provides an anti-candida polypeptide, and in a specific embodiment, the amino acid sequence of the polypeptide comprises any one of the amino acid sequences shown as SEQ ID NO. 1-SEQ ID NO. 12. In a preferred embodiment, the amino acid sequence of the polypeptide may be any one of the amino acid sequences shown in SEQ ID NO. 1 to SEQ ID NO. 12. In other preferred embodiments, the amino acid sequence of the polypeptide can also be based on any one of the consecutive amino acid sequences shown in SEQ ID NO. 1-SEQ ID NO. 12, and at least one amino acid residue which does not affect the anti-candida activity is connected to the N-terminal and/or the C-terminal.

Hereinafter, preferred examples in which the amino acid sequence of the polypeptide is selected from any one of the amino acid sequences in the following table are described.

Table one: amino acid sequences of preferred embodiments of the invention

Meanwhile, in the embodiment, a group of comparative examples is also provided, and the polypeptide in the comparative example is selected from any one of the amino acid sequences in the following table two.

Table two: amino acid sequences in comparative examples

The 20 polypeptides are respectively synthesized by adopting a solid-phase polypeptide synthesis technology, specifically, DCM (dichloromethane) is used for swelling and activating 2-chlorotrityl chloride resin, amino acid containing Fmoc (fluorenylmethyloxycarbonyl acyl) protecting group is connected to the resin by an amide bond forming method, then the Fmoc protecting group is removed, the steps are repeated for a plurality of times, specific amino acid is connected to the resin according to each amino acid sequence, finally the polypeptide is cut off from the resin, and the polypeptide is separated and purified by HPLC (high performance liquid chromatography), and then the molecular weight is analyzed by mass spectrometry.

The above 20 polypeptides were subjected to the following antifungal activity test: referring to the study protocol of the national Committee for standardization of clinical laboratories (NCCLS) M27-A, the polypeptide to be tested was first dissolved in 100. mu.l of PBS buffer, and then diluted twice in a 96-well plate, and then 100ml of a suspension of 2000 cells/ml of the bacteria was added, and cultured at 37 ℃ for 48 hours, and the minimum dilution concentration for aseptic growth was used as the MIC (minimum inhibitory concentration) of the polypeptide. The antibacterial activity of the above 20 polypeptides was measured as shown in table three below.

Table three: antibacterial Activity (MIC) of polypeptide (Unit: μ M)

Note: sample is insoluble in RPMI solution; the strain is a clinical fluconazole-resistant strain;

-no test was performed;

candida albicans is Candida albicans; candida krusei is Candida krusei; candida glabrata is Candida glabrata. Wherein MYA-2876, 90028, 6258 and 90030 are ATCC numbers of the strain; 11B67479-5 is the number of strain archives in the Hospital of Weiersi parent King of hong Kong.

The polypeptide of the preferred embodiment of the present invention (i.e., the above amino acid sequences SEQ ID NO: 1-SEQ ID NO:12) was subjected to a hemolysis test on human erythrocytes as follows: first, 50. mu.l of each polypeptide was serially diluted two-fold with PBS in a 96-well plate, and 50. mu.l of human red blood cells diluted with PBS to a volume fraction of 1.5% of human red blood cells was added to each well. The 96-well plate was placed at 37 ℃ with a volume fraction of 5% CO₂For 24 hours. After the end of the incubation, the 96-well plate was centrifuged, and the supernatant was pipetted into another 96-well plate to measure the absorbance at 414 nm. Calculating the hemolysis rate of human red blood cell of each polypeptide under different concentrations. FIGS. 1-3 are polypeptide vs. human erythrocyteHemolysis test result of cells, wherein the test result is mean value ± standard deviation, and the test is repeated three times. In the bar graph of FIG. 1, the columns corresponding to each abscissa value from left to right, namely, C1, C2, C3 and C4, represent the test results of the polypeptides represented by SEQ ID NO 1 to SEQ ID NO 4, respectively; in the bar graph of FIG. 2, the columns corresponding to each abscissa value from left to right, namely, C7, C8, C9 and C10, represent the test results of the polypeptides represented by SEQ ID NO. 7 to SEQ ID NO. 10, respectively; in the histogram of FIG. 3, the columns corresponding to each abscissa value from left to right, namely, C5, C6, C11 and C12, represent the test results of the polypeptides shown in SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 11 and SEQ ID NO 12, respectively.

The polypeptide in the embodiment of the invention has about 20 amino acids, is short in polypeptide chain, easy to synthesize, simple in secondary structure, capable of inhibiting candida and low in human erythrocyte hemolysis rate, and the polypeptide has high cytotoxicity because the conventional partial antibacterial peptide, such as cathelicidin, has antibacterial property and high hemolysis rate.

The invention also provides the use of any one of the polypeptides of the above embodiments in the preparation of a medicament for the treatment of candida infection.

In addition, the invention also provides an anti-candida medicament, and the active ingredient of the anti-candida medicament is any polypeptide in the above specific embodiment.

The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several equivalent substitutions or obvious modifications can be made without departing from the spirit of the invention, and all the properties or uses are considered to be within the scope of the invention.

Claims

1. An anti-candida polypeptide, which is characterized in that: the amino acid sequence of the polypeptide is as follows:

SEQ ID NO:10：Lys Trp Lys Leu Arg Trp Arg Arg Arg Ser Ser Gly Val Cys Lys Trp Arg。

2. use of a polypeptide according to claim 1 in the manufacture of a medicament for the treatment of candida infection.

3. Use according to claim 2, characterized in that: the candida is at least one of candida albicans, candida krusei and candida glabrata.

4. An anti-candida drug, which is characterized in that: the polypeptide of claim 1 as an active ingredient in the medicament.