CN118812657A - A computer-aided designed antifungal peptide CDAFP5 and its application in treating tinea barbae - Google Patents

Abstract

The present invention relates to the technical field of antimicrobial peptides, and in particular to a computer-aided designed antifungal peptide CDAFP5 with anti-Candida and Trichophyton mentagrophytes activity and its application in anti-tinea barbae. The antifungal peptide CDAFP5 provided by the present invention has anti-Candida and Trichophyton mentagrophytes activity, with a minimum inhibitory concentration of 1.9 μg/mL for tropical Candida albicans and 15.6 μg/mL for Candida parapsilosis, and a minimum inhibitory concentration of 64 μg/mL for Trichophyton mentagrophytes, and has strong antibacterial activity. Increasing the concentration of CDAFP5 to 2×MIC kills planktonic Candida albicans and fluconazole-resistant stubborn Candida albicans. The antifungal peptide CDAFP5 provided by the present invention does not produce drug resistance to Candida and Trichophyton mentagrophytes, has a therapeutic effect on animal models of tinea barbae, and is expected to become a new candidate drug for treating Trichophyton mentagrophytes and Candida epidermis and mucosal infections, and has good application prospects.

Description

A computer-aided designed antifungal peptide CDAFP5 and its application in the treatment of tinea barbae

Technical Field

The present invention relates to the technical field of antimicrobial peptides, in particular to a computer-aided designed antifungal peptide CDAFP5 with anti-Candida and Trichophyton mentagrophytes activities and its application in anti-tinea barbae.

Background Art

Trichophyton mentagrophytes is a type of infectious fungus that can be transmitted between humans and animals. It is one of the common pathogens of fungal skin diseases and is closely related to the occurrence of skin fungal infections such as tinea capitis, tinea corporis, tinea cruris, and tinea barbae. Among the pathogens that cause superficial skin fungal diseases, Trichophyton is the most common dermatophyte. Among the Trichophyton species, Trichophyton mentagrophytes has become the second most common dermatophyte after Trichophyton rubrum. Among the total number of dermatophyte cases, Trichophyton mentagrophytes accounts for approximately 10%. Trichophyton mentagrophytes is a keratinophilic fungus that usually only exists in the stratum corneum of the skin. It secretes keratinase to decompose keratin in humans and animals, causing serious skin infections. Ringworm resistance is a challenge in the treatment of psoriasis.

Candida is also one of the common pathogens of superficial infections in humans and animals. Candida is widely present in nature and parasitizes on normal human skin, oral cavity, gastrointestinal tract, anus and vaginal mucosa without causing disease. It is a typical conditional pathogen. Among them, Candida albicans accounts for the highest proportion, about 70%. When the human immune function is impaired, these pathogens will transform into pathogenic bacteria, leading to skin and mucosal infections, forming facial acne, stubborn vaginitis, oral ulcers and other skin and mucosal inflammations. Candida resistance is one of the difficulties in the treatment of skin and mucosal inflammations.

Due to the single mechanism of action, most antibiotics are easily resistant to pathogenic fungi when used externally. Unlike traditional antibiotics, which mainly work by inhibiting a certain biosynthetic pathway (such as cell wall, protein), most antimicrobial peptides inhibit or kill pathogens through multi-pathway and multi-target mechanisms. The antimicrobial peptide CDAFP5 inhibits spore germination, destroys the integrity of pathogen cell membranes, increases the level of intracellular reactive oxygen species, and destroys cell membrane potential to exert antibacterial effects. Its unique mechanism of action enables CDAFP5 to kill pathogens, and pathogens are not easy to develop resistance to it. Therefore, CDAFP5 has great potential in the clinical treatment of skin and mucosal infections caused by Candida albicans and tinea barbae caused by Trichophyton mentagrophytes.

Summary of the invention

In order to solve the problems of skin and mucosal infections caused by Candida and tinea barbae caused by Trichophyton mentagrophytes, the present invention provides a computer-aided designed antifungal peptide CDAFP5 with anti-Candida and Trichophyton mentagrophytes activities and its application.

To achieve the above object, the present invention provides the following technical solutions:

The present invention provides a computer-aided designed antimicrobial peptide CDAFP5, the amino acid sequence of which is NH ₂ -FLPKLGKAIKRLL-CONH ₂ .

Preferably, the carboxyl terminus of the antimicrobial peptide CDAFP5 is amidated.

The present invention also provides an anti-infection drug, wherein the active ingredient of the anti-infection drug comprises the antimicrobial peptide CDAFP5 according to claim 1 or 2.

Preferably, the pathogen is a fungus.

Preferably, the fungus is Candida albicans (C.albicans), Candida tropicalis (C.tropicalis), Candida parapsilosis (C.parapsilosis) and Trichophyton mentagrophytes (T.mentagrophytes).

Preferably, CDAFP5 can kill planktonic Candida albicans and fluconazole-resistant recalcitrant Candida albicans.

Preferably, the Candida albicans (C.albicans) is not likely to develop drug resistance to CDAFP5.

Preferably, the Trichophyton mentagrophytes (T. mentagrophytes) is not likely to develop resistance to CDAFP5.

Preferably, the minimum inhibitory concentration of the antimicrobial peptide CDAFP5 against Candida tropicalis is 1.9 μg/mL, the minimum inhibitory concentration against Candida albicans and Candida parapsilosis is 15.6 μg/mL, and the minimum inhibitory concentration against Trichophyton mentagrophytes is 64 μg/mL.

Beneficial effects:

The present invention provides an antifungal peptide CDAFP5 with anti-Candida and Trichophyton mentagrophytes activity, wherein the amino acid sequence is _NH2- FLPKLGKAIKRLL- _CONH2 . The antifungal peptide CDAFP5 provided by the present invention comprises 13 amino acids, and the amino acids contained are all L-type natural amino acids, and the synthesis cost is low. It is expected to become a new antifungal candidate drug, has good application prospects in the treatment of body surface and mucosal Candida infections and tinea barbae, and has great potential in solving the drug resistance of pathogenic fungi.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the prior art solutions, the drawings required to be used in the embodiments are briefly introduced below.

FIG1 is a curve showing the killing of Candida albicans by the antifungal peptide CDAFP5.

FIG. 2 shows the results of the antimicrobial peptide CDAFP5 killing fluconazole-resistant and stubborn Candida albicans.

FIG. 3 shows the results of the antifungal peptide CDAFP5 anti-Candida albicans drug resistance experiment.

FIG. 4 shows the results of the antifungal peptide CDAFP5 anti-Trichophyton mentagrophytes drug resistance experiment.

FIG5 is the scoring result of antifungal peptide CDAFP5 against tinea barbae lesions;

DETAILED DESCRIPTION

The present invention provides an antifungal peptide CDAFP5 having anti-Candida and anti-Trichophyton mentagrophytes activities, wherein the amino acid sequence is NH ₂ -FLPKLGKAIKRLL-CONH ₂ .

The antifungal peptide CDAFP5 provided by the present invention comprises 13 amino acids, and the amino acids contained are all L-type natural amino acids. In vitro antibacterial experiments show that the antifungal peptide CDAFP5 exhibits a high antibacterial effect on Candida albicans (C.albicans), Candida tropicalis (C.tropicalis), Candida parapsilosis (C.parapsilosis) and Trichophyton mentagrophytes (T.mentagrophytes), and the drug resistance tendency of Candida albicans and Trichophyton mentagrophytes is lower than that of fluconazole and terbinafine. Antibacterial mechanism experiments show that CDAFP5 increases cell membrane permeability, dissipates membrane potential, increases the level of active oxygen, and inhibits hyphae formation and spore germination.

In the present invention, the preparation method of the antifungal peptide CDAFP5 preferably includes a polypeptide solid phase synthesis method. The present invention has no special requirements for the polypeptide solid phase synthesis method, and a method well known to those skilled in the art can be used.

In the present invention, the carbon terminus of the antifungal peptide CDAFP5 is preferably amidated. The present invention has no special requirements for the modification method, and methods well known to those skilled in the art can be used.

The present invention provides the use of the antifungal peptide CDAFP5 described in the above technical solution in the preparation of drugs for resisting pathogenic bacteria infection.

In the present invention, the pathogenic bacteria are preferably fungi; the fungi include one or more of Trichophyton mentagrophytes, Candida albicans, Candida tropicalis, and Candida parapsilosis.

The present invention provides an anti-infection drug, wherein the effective ingredient of the anti-infection drug comprises the antifungal peptide CDAFP5 described in the above technical solution.

In the present invention, the minimum inhibitory concentration of the antifungal peptide CDAFP5 is 1.9-64 μg/mL.

In order to further illustrate the present invention, the antifungal peptide CDAFP5 and its application provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the protection scope of the present invention.

Example 1 Antimicrobial peptide CDAFP5 sequence and its preparation

The sequence of antimicrobial peptide CDAFP5 and its preparation are as follows:

The target peptide was synthesized by solid phase Fmoc method from C-terminus to N-terminus.

(1) Resin activation. Weigh 0.5 g of Rink Amide-AM resin using an analytical balance and add it to a solid-phase peptide synthesis tube. Add 5 mL of dichloromethane (DCM) to the tube, seal the tube, shake it up and down, and release the air three times. Place the solid-phase peptide synthesis tube on a rotary shaker for 8 hours to fully activate and swell the Rink Amide-AM resin, and then remove the solvent and soluble reagents under vacuum with a vacuum pump.

(2) Removal of Fmoc protecting group and Kaiser test detection: Add 5mL of 20% methylpiperidine/N,N-dimethylformamide (DMF) deprotection solution to the solid phase peptide synthesis tube containing activated resin Resin-NH-Fmoc, and react for 30 minutes to remove Fmoc protection. Filter under vacuum to remove the deprotection solution. Cross-clean the resin with N,N-dimethylformamide (DMF) and dichloromethane (DCM) solution, and filter under vacuum until no solution flows out, three times each. The last time, wait for colorless liquid to flow out, and continue to filter for 1 minute to ensure that the solution is fully drained. Take a small amount of resin and add it to a glass tube (10mm×100mm), add 30μL of Kaiser test, and heat it in a 110℃ heated metal bath for 1 minute. A positive Kaiser test (dark blue) indicates the presence of free amino groups, and the Fmoc protecting group is successfully removed to obtain Resin-NH2.

(3) Synthesis of Resin-CDAFP5: 1-Hydroxybenzotriazole (HOBT) (2.33 mmol, 3 eq) and Fmoc-L-leucine (Fmoc-Leu-OH) (2.33 mmol, 3 eq) were dissolved in 4 mL N,N-dimethylformamide (DMF), vortexed to dissolve thoroughly, and then all were added to a solid phase peptide synthesis tube containing 0.5 g Resin-NH2, followed by 200 μL N,N'-diisopropylcarbodiimide (DIC) (2.33 mmol, 3 eq). The synthesis tube was sealed, shaken up and down, and vented 3 times. The solid phase peptide synthesis tube was placed on a rotary shaker for 3 hours, and Kaiser test was performed. When the Kaiser test was negative (colorless), it indicated that Fmoc-Leu-OH had been successfully coupled to obtain Resin-NH-Leu-Fmoc. The coupling agent, uncoupled Fmoc-Leu-OH and other solvents were removed in vacuo. Cross-clean the resin with N,N-dimethylformamide (DMF) and dichloromethane (DCM) solutions, elute the residual substances, and filter under vacuum decompression until no solution flows out, three times each, and wait for the colorless liquid to flow out for the last time, continue to filter for 1 minute to ensure that the solution is fully drained. Add 5mL of 20% methylpiperidine/N,N-dimethylformamide (DMF) deprotection solution to the peptide synthesis tube, react for 30 minutes to remove Fmoc protection. Filter under vacuum decompression to remove the deprotection solution. Cross-clean the resin with N,N-dimethylformamide (DMF) and dichloromethane (DCM) solutions, and filter under vacuum decompression until no solution flows out, three times each, and wait for the colorless liquid to flow out for the last time, continue to filter for 1 minute to ensure that the solution is fully drained. Take a small amount of resin and add it to a glass tube (10mm×10～100mm), add 30μL of Kaiser test, and heat it in a 110℃ heating metal bath for 1 minute. A positive Kaiser test (dark blue) indicated the presence of free amino groups, and the Fmoc protecting group was successfully removed to obtain Resin-NH-Leu-NH2.

Repeat the above steps to sequentially couple Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Phe-OH and other amino acids. Finally, the Fmoc group at the end of the last amino acid was removed with 20% piperidine to obtain Resin-NH-Leu-Leu-Arg(Pbf)-Lys(Boc)-Ile-Ala-Lys(Boc)-Gly-Leu-Lys(Boc)-Pro-Leu-Phe-NH2.

(4) Antimicrobial peptide cleavage. Add 4 mL of cleavage solution (trifluoroacetic acid/dimethylresorcinol/triisopropylsilane/thioanisole/water/phenol/=35:1:1:2:2:3 V/V) to the solid phase peptide synthesis tube containing Resin-NH-Leu-Leu-Arg(Pbf)-Lys(Boc)-Ile-Ala-Lys(Boc)-Gly-Leu-Lys(Boc)-Pro-Leu-Phe-NH2, seal the synthesis tube, shake up and down, release air 3 times, and place the solid phase peptide synthesis tube on a heated shaker for reaction at 37°C and 250 rpm for 2 h. Then collect the cleavage solution in a 50 mL centrifuge tube, then wash the resin twice with 2 mL of trifluoroacetic acid, and collect the washing solution in a centrifuge tube. Add 3 mL of cleavage solution to the solid phase peptide synthesis tube again. Repeat the above operation. Add ice ether to all the filtrates to precipitate the crude antimicrobial peptide. The volume ratio of the filtrate to ice ether is (1:5). Shake well to make the antimicrobial peptide precipitate flocs. Place the centrifuge tube at -20℃ and let it stand for 10 minutes to allow the crude antimicrobial peptide to precipitate. Then centrifuge at 3500rpm for 5 minutes. Remove the supernatant, add ice ether, vortex and shake to wash thoroughly, repeat the above steps, and wash twice. Use a rotary evaporator to reduce pressure and concentrate to obtain a white crude CDAFP5 antimicrobial peptide. The crude product was purified by HPLC.

Example 2 Antimicrobial spectrum of antimicrobial peptide CDAFP5

The antimicrobial spectrum of antimicrobial peptide CDAFP5 was determined by the following steps:

(1) Weigh and dissolve the protein. Prepare the CDAFP5 phosphate solution. Set the initial concentration of the first column to 500 μg/mL and the final concentration to 0.975 μg/mL. Then prepare a 2 mg/mL CDAFP5 stock solution. Dissolve CDAFP5 in 20 mmol/L pH 6.0 sodium phosphate buffer and filter the solution with a 0.22 μm sterile filter membrane to sterilize.

(2) Using a pipette, add 100 μL of 20 mmol/L pH 6.0 sodium phosphate buffer to each well of a 96-well plate to dilute CDAFP5.

(3) Use a pipette to pipette 100 μL of CDAFP5 stock solution into each well in the first column of a 96-well plate.

(4) Repeatedly pipette the solution in the first column of the plate 6 to 8 times to mix well without splashing.

(5) Pipette 100 μL from the first column and add it to the second column. Repeat pipetting and mixing 6 to 8 times, and then pipette 100 μL to the third column. Repeat this step to the tenth column.

(6) Discard the 100 μL aspirated from the 10th column and do not add it to the 11th column, which is the negative control well.

(7) Add 100 μL of bacterial suspension to columns 1 to 11 of the 96-well plate in order, and pipette and aspirate 6 to 8 times to mix. Do not add the bacterial suspension to column 12. Column 12 is the blank control well.

(8) Incubate the 96-well plate at rest. If it is fungi, it needs to be cultured at 30°C for 48 hours; if it is bacteria, it needs to be cultured at 37°C for 16 hours.

(9) Add 20 μL of 5 mg/mL MTT solution to each well and continue incubation for 4 h. Aspirate the supernatant and add 100 μL of dimethyl sulfoxide (DMSO). Incubate at 37°C for 20 min with occasional shaking. After the crystals are completely dissolved, measure the OD value at 490 nm using a microplate reader.

Inhibition rate (%) = [(OD ₅₇₀ (sample)−OD ₅₇₀ (blank)]/[OD ₅₇₀ (negative)−OD ₅₇₀ (blank)]×100.

MIC ₁₀₀ was defined as the lowest concentration of CDAFP5 that completely inhibited the growth of the test bacteria.

(10) Each experiment was repeated three times.

The results are shown in Table 1. The results show that CDAFP5 has the strongest activity against Candida tropicalis, with a 100% inhibition concentration of 1.9 μg/mL; it is second to Candida albicans and Candida parapsilosis, with a 100% inhibition concentration of 15.6 μg/mL. However, in terms of activity against filamentous fungi, CDAFP5 is more active, with a 100% inhibition concentration of 64 μg/mL against Trichophyton mentagrophytes. Although lower than naphthol (0.9 μg/mL), it is obviously higher than fluconazole (128 μg/mL). CDAFP5 is also active against the bacterium Staphylococcus aureus, with a 100% inhibition concentration of 62.5 μg/mL.

Table 1 Minimum inhibitory concentration of antimicrobial peptide CDAFP5 against test pathogens (μg/mL)

Example 3 Antimicrobial peptide CDAFP5 killing activity of planktonic Candida albicans

Specific steps: The concentration of the Candida albicans suspension in the logarithmic phase was adjusted to 1×10 ⁶ CFU/mL, 100 μL of the bacterial suspension was mixed with 100 μL of the antimicrobial peptide CDAFP5 in a new 96-well plate to a final concentration of 1, 2, 4, and 8×MIC, and incubated for 9 hours. 10 μL of the sample was applied to the SDA plate every hour, and the colonies were counted after culturing at 37°C for 24 hours, and the bactericidal kinetic curve was drawn. PBS was used as a blank control, and each group of experiments was repeated three times. The results showed that the antimicrobial peptide CDAFP5 killed bacteria in a concentration-dependent manner, and the higher the concentration, the shorter the killing time. At a concentration of 2×MIC, all cells can be completely killed in 6 hours; at a concentration of 4×MIC, all cells can be completely killed within 30 minutes (Figure 1 of the specification).

Example 4 Antimicrobial peptide CDAFP5 killing activity of fluconazole-resistant recalcitrant Candida albicans

Specific steps: Pick a single colony of Candida albicans in SD liquid culture medium and culture it in a constant temperature culture shaker at 28°C and 180rpm to the logarithmic phase. Dilute with SD culture medium to 1×10 ⁸ CFU/mL. Take 100μL of bacterial suspension and transfer it to a 96-well plate and culture it at 37°C for 24h. Add 100μL of fluconazole (1×MIC) to the bacterial suspension and culture it at 37°C for 24h. After washing with PBS three times, remove the attached adherents and sonicate for 5min to obtain fluconazole-resistant stubborn Candida albicans. The bactericidal activity of the antimicrobial peptide CDAFP5 against stubborn Candida albicans was determined according to the method for killing planktonic Candida albicans described in Example 3. The results are shown in Figure 2 of the specification. The results show that the concentration of the antimicrobial peptide CDAFP5 killing stubborn Candida albicans is 31.2μg/mL.

Example 5 Antimicrobial peptide CDAFP5 against Candida albicans drug resistance

The tendency of the antimicrobial peptide CDAFP5 to produce drug resistance against Candida albicans was evaluated by continuous subculture. A single colony was picked and cultured in 2 mL SD medium at 28°C and 180 rpm for 24 h, and then 20 μL of bacterial solution was taken and cultured in 2 mL of fresh SD medium with a final concentration of 0.5×MIC of CDAFP5 or fluconazole at 28°C and 180 rpm for 24 h. The drug concentration was doubled every ten generations, and antibacterial experiments were performed on the induced strains, control strains, and original strains every five generations, and resistance was evaluated by MIC values. The results showed that the MIC of the fluconazole-treated group did not change in the first 15 generations, and the MIC changed by 2 to 8 times from 20 to 50 generations, while the MIC of the CDAFP5-treated group did not change within 50 generations (Table 3 and Figure 3 of the specification). The results showed that Candida albicans had a low tendency to resist CDAFP5.

Table 3 Drug resistance of antimicrobial peptide CDAFP5 against Candida albicans (×MIC)

Example 6 Antimicrobial Peptide CDAFP5 Against Drug Resistance of Trichophyton mentagrophytes

The tendency of the antimicrobial peptide CDAFP5 to develop drug resistance against Trichophyton mentagrophytes was evaluated by continuous subculture. First, spores of Trichophyton mentagrophytes (1×10 ⁵ CFU/mL) were cultured in SD medium containing 0.5×MIC of CDAFP5 for 24 h, and then the hyphae were collected by centrifugation and transferred to SD medium containing 0.5×MIC of CDAFP5 for culture. The drug resistance tendency of Trichophyton mentagrophytes was evaluated by continuous subculture in the presence of subinhibitory concentration (0.5×MIC) of CDAFP5. The clinical antifungal drug terbinafine was used as a control. The results showed that the MIC of the terbinafine-treated group increased by 4 times in the second generation and 8 times in the fourth generation, while the MIC value of CDAFP5 remained unchanged in the fifth generation (Table 4 and Figure 4 of the specification). This shows that Trichophyton mentagrophytes has a low tendency to develop drug resistance to CDAFP5.

Table 4 Drug resistance of antimicrobial peptide CDAFP5 against Trichophyton mentagrophytes (×MIC)

Example 6 Results of animal experiments on antifungal peptide CDAFP5 against tinea barbae

The specific steps of the CDAFP5 anti-tinea barbae animal experiment are as follows:

(1) Establishment of animal model of tinea mentagrophytes and animal experimental treatment plan. Twenty-five female guinea pigs aged 1 to 2 months were randomly selected, and an immunocompromised guinea pig model was established by intraperitoneal injection of cyclophosphamide (100 mg/kg) into each guinea pig for three consecutive days. When the guinea pigs were inoculated with skin, they were anesthetized with sodium pentobarbital (30 mg/100 g) by intraperitoneal injection, and then the hair on the back of the guinea pigs was gently shaved and cleaned with 75% alcohol. Then, the back skin was gently polished with sterile sandpaper to form a 2 cm × 2 cm scratch, and 200 μL of Trichophyton mentagrophytes spore suspension (1 × 10 ⁸ CFU/mL) was suspended in physiological saline and applied to the back skin of guinea pigs (n = 20). The medication started on the third day. Each guinea pig in the treatment group was topically applied with 200 μL of CDAFP5 solution to the infected area twice a day with a sterile pipette tip. The uninfected guinea pigs in the control group did not receive any form of treatment. Since the concentration of many drugs for treating psoriasis on the market is 1% (w/v), CDAFP5 was prepared into 0.1% and 1% PBS solutions in this embodiment. The experimental animals were respectively set as a control group (5 animals), a 0.1% CDAFP5 treatment group (5 animals), a 1% CDAFP5 treatment group (5 animals), and a 1% terbinafine treatment group (5 animals).

(2) Lesion scoring. Lesions were scored during the treatment. The specific scoring criteria are as follows: 0, no signs or normal; 1, a small amount of mild erythema on the skin; 2, moderate erythema spread over the entire site, with inflammatory reactions or epidermal abrasions in some parts; 3, large areas of obvious redness, crusting, scaling, bald spots and ulcers; 4, severe erythema, accompanied by large areas of bleeding. The average score of each group was calculated every day to determine the average score of the lesions. The larger the score, the more severe the lesions. The results of lesions on the 0th, 4th, 7th and 10th days of treatment for guinea pigs receiving different treatments are shown in Figure 5 of the specification. As shown in Figure 5A, on the 0th day of treatment, there was no significant difference between the four infection groups, with skin peeling and mild redness. On the 4th day of treatment, the negative control group and the 0.1% CDAFP5 treatment group developed large areas of scaly skin patches, hair loss and obvious redness, while the 1% terbinafine and 1% CDAFP5 treatment groups had very little scaling and hair loss, indicating that an early therapeutic effect had occurred. On the 7th day of treatment, the negative control group and the 0.1% CDAFP5 treatment group showed large areas of desquamation, curry-like hair loss and bald spots, while the guinea pigs in the 1% terbinafine treatment group were basically cured, with smooth and vibrant hair. In contrast, the guinea pigs in the 1% CDAFP5 treatment group had basically normal hair, but not as ideal as the hair in the terbinafine treatment group. On the 10th day of treatment, the negative control group showed symptoms such as large areas of bleeding, skin ulceration, and redness and swelling. Although the group treated with 0.1% CDAFP5 was less severe than the negative control group, there was no improvement compared with the 7th day of treatment, and there were still large areas of scabs and scales. At this time, the 1% CDAFP5 and 1% terbinafine treatment groups had basically recovered, similar to the positive control group. Figure 5B shows the skin lesion scores. On the 0th day of treatment, the skin lesion scores of the four infection groups were all 1, but on the 2nd day of treatment, the skin lesion scores of the 1% CDAFP5 and 1% terbinafine treatment groups were different from those of the 0.1% CDAFP5 and untreated groups, indicating that the efficacy of the drug began to emerge. The 1% CDAFP5 treatment group was basically cured on the 9th day of treatment. On the 10th day of treatment, the treatment effects of 1% terbinafine and 1% CDAFP5 were similar, with an average skin lesion score of less than 1 point, while the skin lesion scores of the 0.1% CDAFP5 and untreated groups showed an upward trend, showing no efficacy.

In summary, the antimicrobial peptide CDAFP5 provided by the present invention has a broad-spectrum antibacterial activity, has candidal activity, has activity to kill recalcitrant candida resistant to fluconazole, has a low tendency of Candida albicans and Trichophyton mentagrophytes to resist it, has a therapeutic effect on tinea barbae, is expected to become a candidate drug for infections of anti-epidermal and mucosal fungi (including Candida and Trichophyton mentagrophytes), and has good application prospects in skin and mucosal fungal infections.

Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims (9)

1. A computer-aided designed antifungal peptide CDAFP with anti-candida and trichophyton mentagrophytes activity, characterized by the following amino acid sequence: NH ₂-FLPKLGKAIKRLL-CONH₂.

2. The antifungal peptide CDAFP of claim 1 wherein the carboxy terminus of the antifungal peptide CDAFP5 is amidated modified.

3. An anti-infective drug characterized by comprising the antifungal peptide CDAFP according to claim 1 or 2 as an active ingredient.

4. The use according to claim 3, wherein the infection is a fungal infection.

5. Use according to claim 4, characterized in that the fungi comprise, but are not limited to, infections caused by: candida albicans (c.albicans), candida tropicalis (c. tropicalis), candida parapsilosis (c.parapsilosis), and trichophyton mentagrophytes (t. mentagrophytes).

6. The antifungal peptide CDAFP of claim 1 or claim 2, wherein it kills planktonic candida albicans and fluconazole-resistant candida albicans.

7. The antifungal peptide CDAFP according to claim 1 or claim 2, wherein candida albicans is less susceptible to developing resistance thereto.

8. The antifungal peptide CDAFP of claim 1 or claim 2, wherein trichophyton mentagrophytes are less susceptible to developing resistance thereto.

9. An anti-infective drug according to claim 3, characterized in that the antimicrobial peptide CDAFP has a minimum inhibitory concentration of 1.9 μg/mL for candida tropicalis, 15.6 μg/mL for candida albicans and candida parapsilosis, and 64 μg/mL for trichophyton mentagrophytes.