Introduction

In 1979, Japanese researchers (Takagi et al. 1979) discovered Kyotorphin (KTP), a dipeptide consisting of L-Tyrosine and L-Arginine, originally isolated from bovine brain (Takagi et al. 1979). Subsequently, this compound was identified in the brain synaptosomes of various mammals, including humans. When directly administered into the central nervous system (CNS) or used at high concentrations (200 mg/kg), KTP demonstrates opioid and analgesic effects (Fukui et al. 1983). Limited effects arise upon systemic administration due to KTP's limited ability to traverse the blood–brain barrier (BBB), as revealed in studies (Chen et al. 1998), thereby presenting a significant obstacle for its potential as a drug candidate.

Enzymatic degradation susceptibility poses another significant challenge for the potential use of KTP as a pharmaceutical agent. To address this concern, numerous research groups have undertaken diverse approaches involving the incorporation and alteration of the original peptide structure (Chen et al. 1998; Rybal’chenko et al. 1999; Wang et al. 2001; Lopes et al. 2006a, b; Ribeiro et al. 2011a, b; Dzimbova et al. 2014; Serrano et al. 2015). Within this context, two KTP analogues that are the focal point of the present study were synthesized: GABA-KTP-NH2, achieved by introducing aminobutyric acid (GABA) at the N-terminus, and Indol-KTP-NH2, which involves substituting the tyrosyl residue with an indole moiety (Serrano et al. 2015). These alterations have exhibited noteworthy influences on permeability and analgesic efficacy, suggesting their potential to traverse the blood–brain barrier through passive diffusion across lipid bilayers (Serrano et al. 2015).

Certain bioactive peptides are capable of eliciting multiple physiological responses within living systems, earning them the designation of multifunctional peptides (MPs). An instance of such MPs is found in lactoferricin hydrolysates, a 25-sequence pepsin hydrolysate of lactoferrin, which has demonstrated attributes encompassing anticancer, antitumor, antiparasitic, and antimicrobial properties (Eliassen et al. 2002; Korhonen and Pihlanto 2006). Similarly, the KTP derivatives are classified as MPs due to their capacity to serve as analgesics, neuromodulators, antimicrobial peptides, biomarkers in Alzheimer’s disease, and more (Perazzo et al. 2017; Ueda 2021).

In terms of its antimicrobial effects, KTP-NH2 prompted membrane blebbing, disruption, and lysis of Staphylococcus aureus, as observed through atomic force microscopy experimentation (Ribeiro et al. 2011b). Additionally, the IbKTP-NH2 variant demonstrated intriguing antifungal capabilities against Candida species, as highlighted in the research conducted by Martins de Andrade et al. (2020). The advancement of antimicrobial agents, particularly peptides known as AMPs, holds significant importance, given the substantial bacterial resistance to antibiotics. This avenue serves as an alternative approach to counteract such resistance. Antibiotics, commonly employed to combat bacterial infections, encounter challenges in penetrating the blood–brain barrier (BBB) and attaining therapeutic levels within the brain. The success of antibiotics in treating central nervous system (CNS) infections hinges on their capacity to access the infection site and unleash their antimicrobial effects. However, the BBB acts as a barrier that blocks the entry of numerous antibiotics into the brain, curtailing their effectiveness against CNS pathogens (Haddad et al. 2022). We hypothesized that derivatives such as GABA-KTP-NH2 and Indol-KTP-NH2, with their potential to translocate the BBB, might not only display analgesic properties but also present antimicrobial and antibiofilm activity.

The association of microorganisms within biofilms presents a protective mechanism for their growth, rendering them resilient to various environments, the host's immune system, and antimicrobial agents (Dunne 2018). Within the biofilm's matrix, the extracellular polymeric substances (EPS) are composed of polysaccharides, proteins, and nucleic acids (Stoodley et al. 2002). This matrix, characterized by porosity, contains water channels that facilitate the exchange of nutrients, oxygen, and excreted metabolites (Davey and O'toole 2000). EPS plays a pivotal role in diminishing antimicrobial penetration across all biofilm regions, acting as a physical hindrance to diffusion by sequestering most antimicrobial agents (Nichols et al. 1988). Microbial adhesion and the formation of biofilms are critical processes in pathogenesis, contributing to the development of persistent infections, as highlighted by the US agency "National Institutes of Health" (Costerton et al. 1999; Wilson et al. 2017).

The assessment of potential drug prototypes’ toxicity commonly initiates with in vitro studies employing diverse cell lines across varying sample concentrations. Subsequently, these prototypes are subjected to testing in different animal models, historically including mice and rats, before proceeding to clinical trials. As elucidated across multiple references, the utility of alternative animal models in intermediate toxicity assessment is underscored (Rizzo et al. 2013; D'amora and Giordani 2018). Moreover, driven by ethical considerations, financial constraints, evolving safety requisites, and alignment with the 3Rs principle (Refinement, Reduction, Replacement), the utilization of mammalian experimental animals is increasingly curtailed to meet essential needs. Consequently, the establishment of standardized alternative models becomes imperative (Russell and Burch 1959; Hartung 2010). Alternative methods or models are designed to complement or substitute traditional mammalian tests in both biomedical research and education. Within this framework, the significance of employing experimental models like Galleria mellonella and Danio rerio (zebrafish) for studying host–pathogen interactions and conducting preclinical evaluations of new drugs is expanding (Martins de Andrade et al. 2020; Skalska et al. 2020), which coincidentally were the models adopted in the present study.

In the last years, methodologies have emerged for in vivo infection investigations using invertebrates, including the G. mellonella moth larva. Research has demonstrated that despite the taxonomic distinctions between invertebrates and mammals, they share analogous attributes within their innate immune systems. This invertebrate model confers numerous advantages compared to other counterparts, encompassing economical breeding, minimal technical constraints, uncomplicated management, and notably, swift outcomes (Mylonakis et al. 2005). The larvae can be readily and precisely subjected to inoculation via injection, forced feeding, or overlay with a spore layer. Notably, this approach allows for the examination of diverse parameters based on the rapid responses to infection, encompassing mortality rates, melanization processes, immune modifications, microbial loads, gene expression alterations, and shifts in the proteome (Arvanitis et al. 2013; Kavanagh and Sheehan 2018). The evaluation of survival post-infection was carried out to determine suitable concentrations of the derivatives studied in zebrafish.

Zebrafish emerges as an optimal vertebrate model for toxicity screening due to its ability to emulate the intricate interactions within the human body during embryo development. Possessing a substantial genomic similarity (70%) and biological system resemblance to humans, zebrafish embryos allow the monitoring of phenotypic and genotypic abnormalities subsequent to exposure to various compounds. The assessment of alterations in organ dimensions and structures during development serves as a straightforward yet valuable gauge of toxicity. The cost-effective maintenance, swift developmental timeline, and ease of manipulation enable high-throughput and economically viable preclinical screenings (Yang et al. 2018; Batista-Filho et al. 2020).

Finally, in an attempt to find new activities associated with KTP derivatives, Indol-KTP-NH2 and GABA-KTP-NH2 and evaluate their toxicity, the present study evaluated the inhibition of biofilm form of selected microorganisms strains, followed by in vitro analysis of cell viability. Additionally, the toxicity and anti-infective action of the KTP derivatives were investigated in vivo in G. mellonella and D. rerio.

Materials and Methods

Synthesis of KTP Derivatives

Details on peptide synthesis of Indol-KTP-NH2 (331,2 g.mol-1) and GABA-KTP-NH2 (422,2 g.mol-1) are provided in Supplementary Material. Briefly, both derivatives of KTP, were synthesized by solution phase synthesis and analyzed for purity on high performance liquid chromatography (HPLC). Moreover, the two kyotorphin derivatives synthesized, as well as the intermediate compounds of the synthetic sequence of GABA-KTP-NH2, were completely characterized by nuclear magnetic resonance (NMR) and mass spectra under electrospray ionization (ESI–MS) (see Supplementary Material).

Determination of the Minimum Inhibitory Concentration (MIC)

The antimicrobial activity of kyotorphins was determined using a modified broth microdilution method according to the Clinical and Laboratory Standards Institute (Komatsuzawa et al. 2000; Ouhara et al. 2008) the following microorganisms: Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 15442) as gram-negative bacteria and Streptococcus pneumoniae (ATCC 25922) as gram-positive bacteria. It also tested three Candida’s species: C. krusei (ATCC 6258), C. parapsilosis (ATCC 63) and C. tropicalis (140-S). Briefly, a cell suspension of each pathogens species was adjusted to 103 CFU/ml using brain heart infusion (BHI) broth (Sigma) or Mueller Hinton Broth media (MH) for bacteria. Aliquots (100 mL) of the suspension were dispensed in polystyrene, flat-bottomed, 96-well microtiter plates. Peptide stock solutions were diluted in BHI broth, and 100 mL of the dilution was added to the wells at a final concentration ranging from 25 µM to 1000 µM. The plates were incubated at 37 C for 24 h. Absorbance was measured at an optical density of 590 nm using a microplate reader (Synergy H1 Hybrid Reader, BIOTEK, USA). The assays were repeated independently three times using different cell suspensions. All the tests were performed in triplicates. MIC50 was defined as the peptide concentration that inhibited 50% of the isolates from each group and MIC90 was defined as the peptide concentration that inhibited 90% of the isolates from each group. MIC50 and MIC90 were calculated as follows: MIC50 = nº. of isolates (n) × 0.5 and MIC90 = nº. of isolates (n) × 0.9.

Determination of Biofilm Inhibition

The inhibition of the biofilm formation was evaluated using the protocol to static biofilm culture (Kwasny and Opperman 2010). For this, it was chosen only the microorganisms that were inhibited in the MIC90 assay, E. coli, S. pneumoniae, and C. krusei. It was transferred 190 μL of the inoculum in the concentration of 106 and 10 μL of the peptide for the well of a sterile 96-well assay plate, also having a triplicate of each concentration. Amphotericin B and Chloramphenicol (Sigma, USA) were used as positive controls according to CLSI (Clinical and Laboratory Standards Institute, 2008; Clinical and Laboratory Standards Institute, 2023). After the 18 h incubation at 37 °C, it was measured the (OD) at λ = 600 nm to quantify overall growth. For evaluation of the biomass of biofilm, the medium was removed from each well and washed three times with NaCl 0.9% solution, ensuring the preservation of the biofilm structure at the bottom of each assay well. The washed plate was incubated at 60 °C for 1 h to fix the biofilm. For S. pneumoniae and C. krusei, it was transferred 200 µL of a 0.06% solution of crystal violet to each well to stain the biofilm for 5 min at 60 °C. For E. coli was used safranin (0.06%) to stain the biofilm. The dye was removed from each well and washed with NaCl 0.9%, followed by addition of 200 µL of acetic acid 30% in the wells and the samples were transferred to a new plate. For the evaluation of the viable cells on biofilms, colony forming unit (CFU) counting assay (Wilson et al. 2017) was conducted removing sample aliquots from the biofilm formation (without staining) to identify the number of viable cells. After incubation for 24 h at 37 °C, microorganism colonies were counted, and viable microorganisms (CFU/mL) were reported as percentage of the control.

Cytotoxicity Test

Cell viability was monitored using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (Riss et al. 2013; Formaggio et al. 2022) with mice embryonic fibroblasts (MEF) and murine melanoma tumor cells (B16-F10-Nex2). A total of 1 × 104 MEF and B16-F10-Nex2 per mL were sown in a 96-well microplate with Dulbecco’s Modified Eagle Medium (DMEM) culture medium. The cells were maintained for 24 h in DMEM in the incubator with 5% CO2 at 37 °C. After this period, the medium was removed, and the peptides were added in triplicate into microplate wells together with 240 µL of DMEM. Dimethyl sulfoxide (DMSO) was used as the positive control, and culture medium was used as the negative control. The plate was incubated for 24 h at 37 °C and 5% CO2. The culture medium was removed, and the cells were washed in phosphate-buffered saline (PBS). MTT (5 mg/mL, prepared in PBS) was added to each well, and the plates were incubated at 37 °C and 5% CO2 for 3 h. Then, MTT was removed, and DMSO was added to each well. Absorbance was measured in a spectrophotometer (Synergy H1 Hybrid Reader, BIOTEK, EUA) at 540 nm.

G. mellonella Toxicity and Infection Assay

G. mellonella larvae were kept in a metal container at 25 °C in the dark and fed a specific manipulated feed. For the assays, larvae without color changes and with adequate weight (150–200 mg) were selected and kept without food in petri dishes at 37 °C in the dark for 24 h prior to the tests. The toxicity activity of Indol-KTP-NH2 and GABA-KTP-NH2 was conducted according to the methodology by Allegra et al. (2018). Ten G. mellonela larvae were used in each of eight treatment groups (Indol-KTP-NH2 and GABA-KTP-NH2 in 200, 100 and 50 µM). A volume of 10 µL of each different peptide concentrations were inoculated in each group. One control group (inoculated with PBS) was included and served as a control for overall viability. The larvae were treated and counted for 7 consecutive days in three independent experiments and the toxicity was measured by the number of live larvae. For the infection assay, initially the lethal concentration of microorganisms for the larvae was standardized (Martins de Andrade et al. 2020). Ten G. mellonella larvae were infected with 10 µL of selected strains and kyotorphin derivatives by injection in the last pair of prolegs of each larva (Cotter et al. 2000). The negative control gets 10 µL of saline (NaCl 0.9%). For all experiments, larvae were incubated at 30 °C in standard petri dishes. The larvae were treated and counted for 7 consecutive days in three independent experiments and the peptides treatment against infection was measured by the number of live larvae.

Evaluation of KTP Derivatives Interference in Zebrafish Survival and Development

The in vivo toxicity activity of Indol-KTP-NH2 and GABA-KTP-NH2, was tested with the zebrafish model. Wild-type strains of zebrafish were conditioned under standard conditions: 28 °C and pH 7.0 with a light:dark photo cycle of 12:12. Adult animals are kept in individual aquariums (Alesco) suitable for breeding zebrafish. The experiments were carried out under the laws of the National Council for Animal Experiment Control (CONCEA) and approved by the UNIFESP Animal Use Ethics Commission (CEUA nº 8861040323). The experiment begins with the separation of fertilized and unfertilized embryos. The fertilized embryos (n = 20) visualized on the Leica EZ4W stereomicroscope (Leica Microsystems, Cambridge, United Kingdom) were individually transferred to 24-well polystyrene plates containing different concentrations of the peptides to be tested. The E2 0.5 × medium was used as a control. In order to ensure the reproducibility of the tests, the embryos were kept in ovens at a constant temperature of 28 °C and the tests were carried out in triplicate. The tests were performed according to the Fish Embryo Acute Toxicity (FET) Test, according to OECD #236. Embryos were observed daily with Leica M205C stereomicroscope. Four parameters were evaluated as indicators of acute lethality in fish (according to FET test protocol): (1) coagulation of fertilized eggs; (2) absence of somite formation; (3) lack of separation between the tail and the yolk sac and, finally, the absence of a heartbeat. Parameters indicative of teratogenicity were also observed according to data obtained from Lantz-McPeak et al. (2015) and Brannen et al. (2010).

Statistical Analysis

The values were presented as the mean ± standard deviation. All experiments were conducted independently in triplicate. Statistical analyses were performed for each assay as follows: MIC statistical analysis employed a Two-way ANOVA followed by Dunnett's test; Biofilm inhibition and Quantification of viable cells underwent statistical analysis with ANOVA followed by Tukey’s test; Quantification of cell viability in the MTT assay was subjected to a Two-way ANOVA followed by Dunnett's test; Toxicity test and Survival percentage in G. mellonella were analyzed using the Log-rank test and Gehan-Breslow-Wilcoxon test; Mortality percentage of zebrafish embryos was statistically assessed via Two-way ANOVA followed by Tukey's test. Data were analyzed utilizing GraphPad Prism 7.00 (GraphPad Software, Inc., California, CA, USA), with a significance threshold set at p < 0.005.

Results

The antimicrobial activities of KTP derivatives against the microorganism were determined using microdilution method to obtain MIC50 and MIC90 of planktonic cells (Schwartz et al. 2010). As observed in Table 1, both KTP derivatives were found to be active against Gram-negative bacteria E. coli at concentration below 35 µM. This concentration against E. coli had a 90% inhibition, at least. The assay with S. pneumoniae showed a 50% inhibition with Indol-KTP-NH2 at 101.8 µM concentration, this was the better value of inhibition with this derivative in the MIC test. As to P. aeruginosa, all derivatives were found inactive up to 250 µM. The Indol-KTP-NH2 did not inhibit yeast growth, for this reason only the GABA-KTP-NH2 activity was tested against the Candida species. The GABA-KTP-NH2 derivative It showed an efficacy of 50% inhibition on Candida species in a concentration range of 5.86 a 23.97 uM, as seen in Table 1.

Table 1 Quantification of the inhibition activity of KTP derivatives tested in MIC assay. Percentage of inhibition for Indol-KTP-NH2 and GABA-KTP-NH2, as determined by absorbance at 530 nm
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In the test of biofilm formation Indol-KTP-NH2 and GABA-KTP-NH2 has almost 95% of inhibition in the biofilm formation for E. coli and S. pneumoniae, respectively, at the 62.5 µM and 250 µM, as presented in Fig. 1 A and 1 B. Moreover, biofilms of C. krusei were weakly inhibited by the two KTP-derivatives tested (below 30% inhibition) (Fig. 1C). The biofilm inhibition mean values and standard deviation are presented in Table 2 (see Supplementary Material).

Fig. 1
figure 1

Percentage of biofilm inhibition of KTP derivatives. A Bacteria Gram-negative E. coli; B Bacteria Gram-positive S. pneumoniae; C Fungi C. krusei. Percentage of inhibition for Indol-KTP-NH2 and GABA-KTP-NH2, Chloramphenicol (positive control for bacteria) and Amphotericin (positive control for fungi). These values were determined by absorbance at 590 nm. p  < 0.05 ‘*’

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The efficacy of Indol-KTP-NH2 and GABA-KTP-NH2 against preformed (24 h) biofilms of microorganisms was evaluated by CFU count after 24 h of treatment. The number of CFU/mL of E. coli sample decreased significantly compared to the control group at the highest concentrations of the two KTP- derivatives (Fig. 2A). The result of the number of viable cells obtained by CFU corroborates with previous results on biofilm inhibition. The results of the CFU test for the biofilm of S. pneumoniae didn’t have the same satisfactory result (Fig. 2B). There was no significant difference between the number of cells in the negative control group and the groups with KTP- derivatives, although the percentage of inhibition was significant. The results for the C. krusei (Fig. 2C) demonstrated a significant decrease of CFU/mL compared to the control group in all concentrations of the derivatives tested, even though the percentage of biofilm inhibition was low. The biofilm viability mean values and standard deviation are presented in Table 3 (see Supplementary Material).

Fig. 2
figure 2

Quantification of viable cells from A Gram-negative bacteria E. coli, B Gram-positive bacteria S.pneumoniae and C Fungi C. krusei. Biofilm inhibition test by Indol-KTP-NH2 and GABA-KTP-NH2 derivatives. p value < 0.05 ‘*’

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The MTT test showed that Indol-KTP-NH2 are cytotoxic to the MEF cell since the viability is below 60% in the lowest concentration. However, GABA-KTP-NH2 did not show cytotoxicity for the cells. Instead, the cell viability was 80% to 98% in the highest and lowest concentration, respectively, as seen in Fig. 3A. Also, in this test the derivatives showed no effect in tumor cells B16. The tumor cells had a high percentage of viability, as seen in Fig. 3B. After the analysis, the IC50 values were calculated for both peptides. In MEF cells, the concentrations for Indol-KTP-NH2 and GABA-KTP-NH2 were 164 µM ± 41.87 and 1042 µM ± 210.8, respectively.

Fig. 3
figure 3

Quantification of cell viability in MTT test with KTP derivatives. A MEF cells; B B16-F10-Nex2 cells. Percentage of cell viability with Indol-KTP-NH2, GABA-KTP-NH2 and DMSO 50% (positive control), as determined by absorbance at 540 nm. The different concentrations of peptides were compared with the positive control group. *p < 0.0001; **p  = 0.0251; ***p  = 0.0014 and ****p  = 0.0004

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The G. mellonella model was used to carry out the toxicity tests of the peptides Indol-KTP-NH2 and GABA-KTP-NH2. This test exhibited a high survival percentage on both derivatives, similar to the survival percentage of PBS (positive control), as presented in Fig. 4. The lethal concentration of E. coli, S. pneumoniae and C.krusei for the larvae of G. mellonella was evaluated using the microorganism strain standardization assay (supplementary material). For microorganisms E. coli and C. krusei the concentration of 108 cells/mL was chosen and for the S. pneumoniae the concentration of 105 cells/mL was chosen for subsequent trials.

Fig. 4
figure 4

Results of toxicity test in G. mellonella with the KTP derivatives A Indol-KTP-NH2 and B GABA-KTP-NH2, and PBS as positive control, as determined by the count of live larvae. Values with p value < 0.05

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The infection test showed that the peptides were efficient in the treatment against infection caused by all microorganisms evaluated and the survival remained above 80% (Fig. 5). For E. coli the two derivatives demonstrated an effective action of the three tested concentrations, GABA-KTP-NH2 (200 µM) and Indol-KTP-NH2 (200 µM and 100 µM) presented total effectiveness (Fig. 5A). Moreover, both derivatives in all tested concentrations showed total efficacy against the S. pneumoniae (Fig. 5B). For the C. krusei we evaluated only the peptide GABA-KTP-NH2, once the Indol-KTP-NH2 did not inhibit fungus growth in MIC assay. The results demonstrated an effective action of the three tested concentrations (200, 100 and 50 µM). The rate of survival of infected larvae was 100% in the 200 µM concentration, followed by 90% for the 100 µM concentration and 80% for the 50 µM concentration (Fig. 5C).

Fig. 5
figure 5

Survival of G. mellonella infected with E. coli (108 cells/mL), S. pneumoniae (105 cells/mL) and C. krusei (108 cells/mL) and treated with GABA-KTP-NH2 (200, 100 and 50 µM), Indol-KTP-NH2 (200,100 and 50 µM) and PBS (used as a negative control). Results are the average of three independent experiments. Statistically significant results when compared to the control group (PBS). Statistical significance was calculated using Gehan-Breslow-Wilcoxon test p ≤ 0.05. The null hypothesis assumes that the survival curves are identical in the overall populations

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Acute toxicity of KTP derivatives, based on mortality of embryos, was evaluated each 24 h until 96 h using 0 hpf zebrafish embryos exposed to three doses (100, 50 and 25 μM) (Fig. 6). Four parameters were evaluated as indicators of acute lethality: (1) coagulation of fertilized eggs; (2) absence of somite formation; (3) lack of separation between the tail and the yolk sac and (4) the absence of a heartbeat. Our results in Fig. 6 demonstrated that KTP derivatives presented embryo mortality from 48 h for the Indol-KTP-NH2 and 72 h for the GABA-KTP-NH2. The GABA-KTP-NH2 derivative showed mortality rate statistically significant at the concentration of 100 µM in the periods of 72 hpf (p = 0.0119) and 96 hpf (p = 0.0119). The Indol-KTP-NH2 derivative showed mortality rate statistically significant at the three concentrations evaluated in the periods of 72 hpf (100 µM p < 0.0001; 50 µM p = 0.0033; 25 µM p  = 0.0005) and 96 hpf (100 µM p < 0.0001; 50 µM p = 0.0033; 25 µM p = 0.0005). In the period of 48 hpf, only the concentrations of 100 µM (p = 0.0005) and 25 µM (p = 0.0183) showed a statistically significant mortality rate.

Fig. 6
figure 6

Mortality percentage of zebrafish embryos exposed for 96 h to both KTP derivative. The embryos were immersed 0 hpf to different GABA-KTP-NH2 or Indol-KTP-NH2 concentrations diluted in E2 medium, and the effects were counted overtime every 24 h. The experiments were performed four times, using 5 embryos/concentration (final n = 20). The tested concentration was considered statistically significant compared with control (E2 medium) when p > 0.05

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In the search for the phenotype-based malformations induced by non-lethal doses of KTP derivatives, embryos were analyzed for sub-lethality (yolk sac edema, absent eyes and pericardial edema) and teratogenicity (retarded growth, spinal deformity and curved tail) after 4 days of exposure (96 hpf) without renewal of the E2 0.5 × medium (Fig. 7). All tested concentrations of the two KTP derivatives caused some type of malformation after 96 h of exposure. The GABA-KTP-NH2 showed more malformations at the highest tested concentration (100 μM), mainly in relation to teratogenicity, and the Indol-KTP-NH2 showed more malformations at the lowest tested concentration (25 μM).

Fig. 7
figure 7

Malformations induced by non-lethal doses of KTP derivatives, embryos were analyzed for sub-lethality (yolk sac edema, absent eyes and pericardial edema) and teratogenicity (retarded growth, spinal deformity and curved tail) after 4 days of exposure (96 hpf) without renewal of the E2 0.5 × medium

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We documented zebrafish individuals exposed to KTP derivatives in all concentrations tested (100, 50 and 25 μM). Three defects were observed in the both KTP derivatives (GABA-KTP-NH2 and Indol-KTP-NH2) pericardial edema, yolk sac edema and curved tail (Fig. 8).

Fig. 8
figure 8

Representative embryo morphological changes observed after 96 h of GABA-KTP-NH2 (A, B and C) or Indol-KTP-NH2 (D, E and F) exposition. Embryos presenting malformation in the tail (curved tail—arrow 3), edema in the cardiac cavity (arrow 1), and edema in the yolk sac (arrow 2). The control showed no morphological abnormalities (G)

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Discussion

The discovery of novel strategies to combat bacterial resistance to antibiotics highlights the renewed significance of bioactive peptides. Our hypothesis revolves around the antibiotic and antifungal potential of two KTP-derived compounds, namely GABA-KTP-NH2 and Indol-KTP-NH2, recognized for their multifunctional peptide properties. In the scope of this investigation, we assessed the efficacy of these two KTP derivatives against three microbial strains: S. pneumoniae (Gram-positive bacteria), E. coli (Gram-negative bacteria), and Candida krusei (fungi).

The results demonstrate the efficacy of KTP derivatives in inhibiting biofilm formation when tested against both E. coli and S. pneumoniae. Upon closer examination of the MIC values of these derivatives, it becomes evident that all derivatives exhibited significant growth inhibition of E. coli. GABA-KTP-NH2 displayed the highest potency against this Gram-negative bacteria. Intriguingly, this efficacy did not extend to P. aeruginosa, another gram-negative bacteria. Despite both bacteria sharing a Gram-negative classification and bacilli-shaped morphology, it is plausible that differences in zeta potential within their respective membranes could account for this variation in peptide interaction (Alves et al. 2010).

Multiple Candida species possess the capacity to infect critical organs, including the CNS (Li et al. 2017). This is attributed to their remarkable ability to cross the blood–brain barrier (Jong et al. 2001; Wu et al. 2019). Furthermore, infections stemming from yeast species within the Candida genus exhibit alarmingly high mortality rates (Gudlaugsson et al. 2003). Additionally, the limited repertoire of antifungal agents available for treating such infections underscores the pressing necessity for the development of novel antifungal compounds. Amphotericin B stands out as one of the foremost choices in treating these infections. However, it is not without its drawbacks, such as the potential for nephrotoxicity and the occurrence of fever accompanied by chills (Goodman and Gilman 1996). Consequently, our research embarked on evaluating alternative antifungal options for addressing the current shortcomings in medical treatment.

In a previous study conducted by our research group (Martins de Andrade et al. 2020), it was suggested that kyotorphins, due to their cationic characteristics, could potentially demonstrate significant antifungal properties. Indeed, Martins de Andrade et al. (2020) confirmed their hypothesis by demonstrating that IbKTP-NH2, a kyotorphin derivative, displayed both antifungal and antibiofilm activities against Candida species, including C. albicans and non-albicans strains. Remarkably, IbKTP-NH2 demonstrated robust efficacy against C. krusei, with a 93% inhibition rate and complete eradication at a concentration of 1000 mM, and the CFU count after 24 h of treatment were weakly inhibited by 500 mM but strongly inhibited by 1000 mM. It's worth noting that C. krusei is recognized as one of the most pathogenic Candida species (Sullivan et al. 2004; Turner and Butler 2014). Therefore, IbKTP-NH2 emerges as a promising candidate with substantial antifungal potential against C. krusei and other Candida species.

GABA-KTP-NH2 and Ib-KTP-NH2 are synthetized KTP-derivatives based in the amidation (-KTP-NH2) and conjugation of residues, besides that they are membrane-active peptides (Ribeiro et al. 2011a, b; Serrano et al. 2015). Few synthetic membrane-active peptides targeting pathogenic fungi have been investigated. In our current investigation, GABA-KTP-NH2 demonstrated a significantly decrease in CFU/mL compared to the control group across all tested concentrations when challenged with C. krusei. Although the percentage of biofilm inhibition may have been relatively modest, these results indicate the potential of GABA-KTP-NH2 as a promising antifungal candidate.

As previously observed, both Indol-KTP-NH2 and GABA-KTP-NH2 exhibited a 50% inhibition of the MIC at concentrations exceeding 50 µM. In the context of the biofilm growth tests, it's important to note that the concentrations employed were higher. This difference can be attributed to the higher cell density within the biofilm tests compared to the MIC tests. It was observed that an intermediate concentration yielded a more favorable percentage of biofilm inhibition compared to the highest concentration, suggesting that a simple dose–response mechanism was not at play here. This observation aligns with a prior study conducted by Overhage et al. (2008), which revealed that even lower concentrations of LL-37 peptide could exhibit enhanced efficacy against bacterial biofilm formation. This phenomenon is attributed to the fact that antimicrobial peptides often target various elements within bacterial cells, including cell membranes, cellular proteins, DNA, and RNA (Powers and Hancock 2003). Consequently, it is plausible that KTP derivatives interact with some of these targets to inhibit biofilm formation, and this interaction may reach a saturation point at which smaller doses prove to be more effective. This trend is further corroborated by our findings in the case of S. pneumoniae, observed in both the MIC and biofilm inhibition assays.

The CFU analysis for E. coli corroborates the findings from previous experiments. The data suggests that the concentration of the most prominent GABA-KTP-NH2 derivative falls between the concentrations observed in MIC and biofilm tests, specifically at 35 µM and 62.5 µM. This assay assesses the count of viable cells in both planktonic and biofilm forms (Huang et al. 2017). In contrast, the results of the S. pneumoniae test indicate no reduction in the count of viable cells, despite a noticeable inhibition in biofilm growth. The biofilm is a set of microorganisms entangled in an organic polymer matrix, which provides greater resistance to antimicrobial molecules. In a study conducted by Dias et al. (2022), the effectiveness of a spider-derived cyclic AMP gomesin analogue, [G1K,K8R]cGm, was investigated against bacterial cells embedded within S. aureus biofilms. The three-dimensional structure of [G1K, K8R]cGm was found to be crucial for its antibacterial and antibiofilm properties. Mechanistic examinations indicate that the peptide possesses the capability to permeate the biofilm and eradicate bacteria by selectively targeting and disrupting their cell membranes. This approach, involving antimicrobial peptides (AMPs) that can penetrate complex polymeric matrices and eliminate bacteria without relying on their metabolic activity, offers a promising alternative strategy for combating biofilm infections.

G. mellonella, commonly known as the greater wax moth larva, has emerged as a prominent model organism for investigating toxicity and infection dynamics. Over recent years, it has gained increasing popularity as a valuable tool in studies related to antibiotics and molecular antimicrobial potential (Tsai et al. 2016; Serrano et al. 2023). Recently this animal model has found a novel role in immunological research. Its significance lies in its capacity to facilitate the exploration of various facets of host–pathogen interactions, the elucidation of innate immune mechanisms, the assessment of virulence factors employed by human pathogens, the in vivo evaluation of the antimicrobial efficacy of emerging drugs, and the discovery of bioactive molecules with diverse applications, ranging from antimicrobial, antiviral, and anticancer properties to their potential use as biopesticides (Wojda et al. 2020; Serrano et al. 2023).

Firstly an in vivo assay with G. mellonella was conducted to evaluate the KTP derivatives toxicity. Indol-KTP-NH2 and GABA-KTP-NH2 exhibited good pharmacological potential by demonstrating non-toxic properties and yielding results akin to the positive control (PBS). This outcome holds significant importance due to the structural and functional resemblance of this model organism's innate immune system to that of mammals. Consequently, this larval model is ethically accepted as a viable alternative for toxicity research, offering valuable insights into predicting toxicity outcomes in mammalian systems (Wojda et al. 2020; Serrano et al. 2023). Subsequently, we conducted another in vivo assay involving the infection of G. mellonella larvae with E. coli, S. pneumoniae, and C. krusei to assess the therapeutic potential of Indol-KTP-NH2 and GABA-KTP-NH2. The results demonstrated the efficacy of both KTP derivatives against these infections. GABA-KTP-NH2 exhibited a significant antifungal effect against C. krusei, underscoring its potential as an antifungal candidate. In contrast, Indol-KTP-NH2 did not exhibit inhibition of yeast growth in the MIC assay; therefore, this derivative was not further evaluated against the fungus C. krusei.

Previous studies of our group (Martins de Andrade et al. 2020) demonstrated that IbKTP-NH2, a derivative of kyotorphin, reduced the yeast cell count in the larval hemolymph. The survival curves not only revealed that IbKTP-NH2 prevented the mortality of 80% of the sample but also extended the lifespan of infected larvae compared to those treated with PBS, and 20% of the treated larvae remained alive at the conclusion of the experimental period. Furthermore, the evaluation of IbKTP-NH2's toxicity using G. mellonella larvae demonstrated that the drug's toxicity remained low up to 2 days post-treatment, aligning with the treatment results. These discoveries underscore the substantial therapeutic potential of IbKTP-NH2 as an antifungal agent. On the other hand, the study by Ridyard et al. (2023) presents data indicating high toxicity for the LL-32 peptide at a concentration of 32 µg/mL (½ MIC), resulting in the death of 100% of the larvae after 24 h of the experiment. Our findings regarding the GABA-KTP-NH2 peptide align with those of Martins de Andrade et al. (2020) and suggest that this peptide represents a compelling candidate, particularly as an antifungal agent, within the realm of antimicrobial drug development.

The data obtained from treating E. coli infections with KTP derivatives can be correlated with the results obtained from in vitro tests. Both Indol-KTP-NH2 and GABA-KTP-NH2 exhibited significant efficacy against this infection. However, it's worth noting that these derivatives did not yield the same level of effectiveness against S. pneumoniae. In the G. mellonella infection test, both derivatives, across all tested concentrations, demonstrated complete efficacy against S. pneumoniae. Nonetheless, it's noteworthy that in the CFU test for S. pneumoniae biofilm, there was no significant difference observed between the number of cells in the negative control group and the groups treated with KTP derivatives, despite a notable percentage of inhibition. In general, antimicrobial peptides have demonstrated notably potent antimicrobial activity against biofilms formed by certain bacteria and fungi, as evidenced in the study conducted by Ribeiro and collaborators (Ribeiro et al. 2012). Our results have shown significant activity of Indol-KTP-NH2 and GABA-KTP-NH2. Given that E. coli, S. pneumoniae, and C. krusei are responsible for a range of common infections, these derivatives hold promise for further development as therapeutic antimicrobials.

The zebrafish (Danio rerio) is widely used in environmental toxicity assessment of chemicals. More recently, however, this animal model, particularly zebrafish embryos, has gained substantial traction in the realm of new drug development (Barros et al. 2008; Kalueff et al. 2014; Kanungo et al. 2014; Raldúa and Piña 2014; Augustine-Rauch et al. 2016; Jia et al. 2019; Batista-Filho et al. 2020). The validated alternative zebrafish OECD #236 test embryo model to assess fish acute toxicity (OECD, 2013) originally designed for identifying the toxicity of toxins, particulate matter, and nanoparticles (Ahkin Chin Tai and Freeman 2020; Rahman et al. 2020), and monitoring of various environmental contaminants including pesticides, ethanol, and pharmaceuticals (Vargas and Ponce-Canchihuaman 2017; Disner et al. 2021), has evolved into a versatile tool. It is now being explored as a potential substitute for one of the regulatory in vivo mammalian embryo fetal developmental toxicity studies for human pharmaceuticals. Recent studies have used this animal model, particularly zebrafish embryos, for drug discovery, early drug development, and toxicological screening (Barros et al. 2008; Kalueff et al. 2014; Kanungo et al. 2014; Raldúa and Piña 2014; Augustine-Rauch et al. 2016; Jia et al. 2019; Batista-Filho et al. 2020).

In our current investigation, we conducted the FET test to investigate the impact of Indol-KTP-NH2 and GABA-KTP-NH2 on the survival and morphological development of zebrafish embryos. The results showed that Indol-KTP-NH2 displayed a pronounced association with lethality. All tested doses of Indol-KTP-NH2 induced high mortality rates in the embryos after both 72 h and 96 h of exposure. Furthermore, after the 48-h mark, lethality was evident at concentrations of 100 μM and 25 μM. Conversely, malformations were predominantly observed in embryos exposed to the 25 μM concentration. These malformations, indicative of sub-lethality and teratogenic effects, occurred at relatively low rates, underscoring the heightened toxicity of Indol-KTP-NH2 at higher concentrations. This finding aligns with the outcomes of the MTT assay, which demonstrated cytotoxicity of Indol-KTP-NH2 to MEF cells across all tested concentrations. In contrast, when subjected to toxicity testing using the model organism G. mellonella, Indol-KTP-NH2 exhibited no discernible toxicity.

On the other hand, the GABA-KTP-NH2 derivative exhibited a lower degree of lethality, with lethality being observed only at the 100 μM concentration after 72 h and 96 h of exposure. Nevertheless, it displayed elevated rates of malformation at this same concentration, particularly concerning teratogenic effects. Moreover, these KTP derivatives also demonstrated substantial malformation rates at lower concentrations. Additionally, in the MTT assay, GABA-KTP-NH2 did not exhibit cytotoxicity towards the two tested cell types and in the toxicity assessment, involving the model organism G. mellonella, GABA-KTP-NH2 displayed no discernible toxicity. Batista-Filho et al. 2020 conducted investigative toxicology assessments on the patented anti-inflammatory peptide TnP using zebrafish as the model organism. The results indicated that elevated concentrations of the peptide were associated with increased lethality and the induction of dose-dependent abnormalities in the developing embryos, in addition, lower concentrations of the peptide exhibited a reduced propensity to induce such abnormalities. Based on this premise, it is prudent to contemplate future tests at concentrations equal to or less than 25 μM for the GABA-KTP-NH2 peptide, particularly in the context of its effectiveness against the fungus C. krusei.

Our results emphasize the notable antifungal effectiveness of the GABA-KTP-NH2 peptide in the infection test, and the toxicity evaluations indicate a comparatively lower toxicity profile with a reduced likelihood of inducing abnormalities. Nevertheless, it's noteworthy that our results caution against the use of these derivatives at higher tested concentrations, as they led to both mortality and abnormalities in the developing embryos. Furthermore, this study's outcomes emphasize the pivotal role of the zebrafish as a precise model for investigative toxicology, particularly in evaluating the acute toxicity of molecules during the preclinical development phase. They also underscore the importance of employing a multifaceted approach involving various techniques for a comprehensive assessment of toxicity.

Research Involving Human and Animal Participants

The authors declare that all animals was kept according to animal welfare standards. The experiments were carried out under the laws of the National Council for Animal Experiment Control (CONCEA) and approved by the UNIFESP Animal Use Ethics Commission (CEUA nº 8861040323).