Structure-guided synthesis of melittin analogs with reduced hemolysis and enhanced anti-inflammatory activity

2.1. General remarks

All the amino acid derivatives and reagents were purchased from InnoChem, the side chain protecting groups were t-Bu for Ser and Thr, Trt for Gln, Boc for Lys and Trp, and Pbf for Arg. Rink-Amide resin (100-200 mesh, 0.42 mmol/g) and Wang resin (100-200 mesh, 0.58 mmol/g) were purchased from Hecheng Science & Technology Co., Ltd, and the solvent was purchased from Sinopharm. Dulbecco’s modified Eagle’s medium (DMEM) (1X), fetal calf serum (FCS), phosphate buffer solution (PBS), and penicillin/streptomycin used for cell culture were from Gibco, cell counter kit - 8 (CCK-8) was from TargetMol, and lipopolysaccharide (LPS) was from Solarbio. Mouse mononuclear macrophage cells (RAW264.7) were cultured by the Beijing Institute of Radiation Medicine, and rabbit blood cells (RBC) were purchased from Hongquan Biological Technology Co., Ltd.

Analytical reverse phase-high performance liquid chromatography (RP-HPLC) was performed on an Agilent 1100 analytical instrument using a Venusil ASB C18 column (0.46 × 15 cm, 5 μm, 100 A): flow 1 mL/min, detection at 220 nm and eluents (A) 0.1% trifluoroacetic acid (TFA) in water and (B) acetonitrile with a gradient application over 30 min.

All peptides were characterized by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The purification of peptides was performed by preparative RP-HPLC on an FL-H100G instrument with an HP-Q-UV 100Z detector (220 nm), on a Venusil ASB C18 column (21.2 × 250 mm, 5 μm): flow rate 16 mL/min, with an equivalence application over 30 min, using the same solvent system as for analytical RP-HPLC.

Male BALB/c and Kunming (KM) mice (5-6 weeks old) were procured from SPF (Beijing) Biotechnology Co., Ltd., with BALB/c strains employed in sepsis models and KM strains utilized for acute inflammatory edema evaluation. All animals were maintained under controlled conditions (25°C, 50% ± 10% relative humidity) with a 12h:12h light-dark cycle to simulate natural circadian rhythms. Standard chow and water were provided ad libitum throughout the study. Experimental protocols strictly adhered to the ethical guidelines approved by the Committee on the Ethics of Animal Experiments of the Animal Center at the Beijing Institute of Radiation Medicine (IACUC of DWZX 2024-P548).

2.2. Amino acid synthesis

Unless otherwise noted, all commercially available compounds were used as provided without further purification. Solvents for chromatography were technical grade. Flash column chromatography was done using silica gel (100-200 mesh). Melting points were determined by an electrothermal melting apparatus and were uncorrected. Chemical yields refer to isolated pure substances.

The amino acid derivatives required for peptide synthesis were prepared following established methodologies: aspartic acid and glutamic acid bearing 3-(2-nitrophenyl)butan-2-ol (Npb-OH) side-chain protecting groups (in Scheme 1) for cyclic peptide construction were synthesized according to reference [23], while olefin-bearing alanine derivatives utilized in stapled peptide synthesis were prepared as described in reference [24,25].

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Scheme 1.

Synthetic routes for aspartic acid and glutamic acid bearing 3-(2-nitrophenyl)butan-2-ol (Npb-OH) side-chain protecting groups.

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2.3. Solid phase peptide synthesis

The synthesis of melittin analogs followed the workflow illustrated in Figure 1(a-d), and the structures of the analogs have been shown in Table 1. Initial synthesis of Mw was performed through manual solid-phase peptide synthesis (SPPS) using Wang resin, commencing with Fmoc-Gln(Trt)-OH immobilization via N,N’-Diisopropylcarbodiimide (DIC)/DMAP. Residual hydroxyl groups on the resin were acetylated using acetic anhydride/pyridine/DMF (2:2:1, v/v/v). Subsequent analogs were synthesized on Rink Amide resin through sequential Fmoc-based coupling cycles. Each amino acid derivative (3eq) was activated with HOBt/DIC, with incomplete couplings identified by the Kaiser test requiring repeat reactions. Deprotection of the Fmoc groups was achieved using 20% piperidine/DMF. Special handling was implemented for CM-D and CM-E analogs containing Fmoc-Asp(Npb)-OH or Fmoc-Glu(Npb)-OH, where subsequent couplings were conducted under light-protected conditions in Scheme 2.

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Figure 1.

Synthetic route for the melittin analogues. (a) Replace ILE2, LEU6, and TRP19 by Glu or Thr; (b) Hydrophobic amino acid at the i/i+4 site in the prototype peptide chain was replaced with olefin-carrying amino acids. The synthesis of the stapled peptide was completed using the ring-closing metathesis cyclisation strategy; (c) Side-chain photolabile protecting groups Npb-OH were deprotected by 365 nm UV irradiation and cyclized with the terminal amino group; (d) The condensation of long-chain fatty acids with terminal amino groups results in the formation of lipopeptides.

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Scheme 2.

Synthetic routes for (i) cyclic peptide and (ii) stapled peptide with the SPPS strategy.

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Lipidated derivatives C16 and C18 were generated through HOBt/DIC-mediated conjugation of long-chain fatty acids to peptide termini in Figure 1(d). Final cleavage from resin employed a mixture solution of TFA/1,2-Ethylenedithiol (EDT)/m-Cresol/Triisopropylsilane (TIPS)/H₂O (90:2.5:2.5:2.5:2.5, v/v/v/v/v) followed by precipitation with anhydrous ether. This was then dried under argon atmosphere. Crude peptides underwent purification via preparative RP-HPLC, lyophilization, and quantification prior to quality control. Analytical characterization included purity assessment by RP-HPLC and structural confirmation through MALDI-TOF mass spectrometry.

2.4. Peptide cyclization

Following the completion of peptide synthesis via Fmoc-based solid-phase peptide synthesis (SPPS) using the sequence outlined in Figure 1(c), the photolabile protecting groups Npb-OH on CM-D and CM-E were site-specifically removed under irradiation at 500 W UV LED (λ=365 nm) in DMF/methanol (3:1, v/v), followed by 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU)/HOBt/4-Methylmorpholine (NMM) -mediated esterification to achieve cyclization (Scheme 2(i)).

For stapled peptide synthesis in Scheme 2(ⅱ), following Fmoc deprotection and chain assembly, N-terminal acylation was performed using anhydride reagents. Subsequent cyclization was accomplished via Grubbs-II catalyst-driven ring-closing metathesis (RCM) in anhydrous 1,2-dichloroethane, following established methodology in Scheme 2(ⅱ) [26,27].

2.5. Cell culture

RAW264.7 murine macrophages were grown in Dulbecco’s MEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37°C in 5% CO₂.

2.6. RAW264.7 cell viability

RAW 264.7 were seeded in 96-well plates at approximately 3 × 10⁴ cells per well and treated with melittin analogues at varying concentrations (1.25, 2.5, 5, 10, 20 μM) for 24 h at 37°C. Untreated cells in complete medium served as the negative control, while cell-free medium provided background absorbance. Following incubation, 10 μL CCK-8 reagent was added to each well and incubated for 2 h. Absorbance was measured at 450 nm using a microplate reader. Cell viability (%) was calculated using the formula (Eq. 1):

2.7. Determination of nitric oxide (NO)

RAW 264.7 were seeded in 96-well plates at approximately 5 × 10⁴ cells per well and incubated overnight at 37°C. The culture medium was replaced with DMEM containing melittin analogues, and cells were pre-treated for 1 h prior to LPS stimulation (500 ng/mL). After 24 h incubation, supernatants were collected for nitric oxide quantification using a commercial assay kit (Beyotime Biotechnology) according to manufacturer protocols.

2.8. Evaluation of cytokine release assays

The cell supernatants were obtained as above. To assess the inhibitory effect of melittin analogues on LPS-induced cytokine release, the levels of Interleukin-6 (IL-6) and Tumour Necrosis Factor-α (TNF-α) in cell supernatants were quantified using a QuantiCyto mouse ELISA kit (Neo Bioscience), in accordance with the manufacturer’s instructions.

2.9. Hemolytic assay

The 4% rabbit erythrocyte suspension was centrifuged at 450 × g for 5 min, washed twice, and adjusted to a 1% hematocrit in PBS. Aliquots (160 μL) of the erythrocyte suspension were dispensed into 96-well plates and combined with 40 μL PBS containing melittin analogues. Controls included 0.1% Triton X-100 in PBS (positive control, 100% hemolysis) and PBS alone (negative control, 0% hemolysis). After 30 min incubation at 37°C, plates were centrifuged at 450 × g for 10 min and supernatants were analyzed at 540 nm using a Multiskan FC enzyme marker (Thermo Fisher Scientific). Hemolysis percentage was calculated as the following formula (Eq. 2):

2.10. Proteolytic stability assay

Melittin analogues were individually dissolved in PBS (1 mM final concentration), while α-chymotrypsin was prepared in PBS supplemented with 2 mM calcium chloride (5 ng/μL final concentration). For enzymatic stability assessment, 100 μL peptide solutions were combined with 1 mL enzyme solution to initiate digestion at room temperature. Aliquots (100 μL) were withdrawn at specified intervals (0, 5, 10, 15, 20, 30, 40, 60 min) and immediately quenched with 20 μL 1 M HCl. Enzymatic degradation kinetics were monitored via reverse-phase HPLC (220 nm detection wavelength) by quantifying intact peptide remaining at each time point.

2.11. Circular dichroism (CD) spectroscopy analysis

CD spectra were recorded on a Jasco-J-1500 spectropolarimeter in PBS (pH 7.4) at room temperature. A cell length of 10 mm was used, and concentrations of the solutions were 0.1 mg/mL. Each spectrum represents the average of at least four scans. Ellipticity was reported as the optical rotation (mdeg) and used to calculate the content of each secondary structure from [http://dichroweb.cryst.bbk.ac.uk] [28].

2.12. Survival analysis in sepsis

BALB/c mice were randomly allocated into six experimental groups (n=10/group): (1) saline control, (2-3) dexamethasone (Dex) at 2 mg/kg and 5 mg/kg doses, and (4-8) peptides (Mel, E1, S15-1, S15-3, C16) administered at 1 mg/kg (low) or 2 mg/kg (high). Following intraperitoneal injection of treatments or saline, mice received LPS (20 mg/kg, i.p.) 1 h post-treatment. Animals were monitored for 7 days with ad libitum access to food/water, during which survival rates and body weights were recorded daily.

2.13. Acute inflammatory oedema assay

KM mice were randomly allocated into five experimental groups (n=10/group): model control, dexamethasone (Dex, 5 mg/kg), melittin (Mel, 2 mg/kg), and analogs E1 and S15-1 (2 mg/kg). One hour post-intraperitoneal administration of treatments or saline, 20 μL xylene was evenly applied to the right auricle, with the left ear serving as an untreated control. Mice were euthanized 1 h post-stimulation, and 6-mm punch biopsies from standardized regions of both ears were weighed. Ear swelling (Eq. 3) and inhibition rates (Eq. 4) were calculated as:

2.14. Statistical analysis

Differences between variables were assessed by a two-tailed Student’s t-test or one-way analysis of variance (ANOVA). All statistical analyses were calculated by SPSS 13.0 or GraphPad Prism 9.0. The statistical data were expressed as the mean ± SD. In all assays, a P value < 0.05 was considered statistically significant.

3.1. Synthesis

This investigation systematically developed 17 melittin analogs through four structural modification strategies: amino acid substitutions, hydrocarbon-stapled constraints, cyclization, and lipid conjugation. Notably, for cyclic peptide synthesis, the third-dimensional protection strategy utilizing photolabile protection groups for side-chain carboxyl groups in SPPS was employed. By modifying the side chains, the carboxyl groups of glutamic acid and aspartic acid were protected using photolabile protection groups. Once the peptide assembly was completed sequentially, the photolabile protection groups were selectively removed using 365 nm UV LED. Subsequently, the exposed side-chain carboxyl groups were reacted with the N-terminal amino group in situ. This process resulted in the formation of cyclic peptides, thereby enhancing synthetic efficiency. All analogs were rigorously characterized by MALDI-TOF mass spectrometry, with experimental molecular masses matching theoretical values. Corresponding analytical RP-HPLC chromatograms and mass spectral data have been provided in the supplementary materials (Figures S1-S35).

3.2. Cytotoxicity of melittin analogues on RAW264.7 cells

Cytotoxicity assessment of melittin analogs proved critical for validating their therapeutic potential in subsequent anti-inflammatory evaluations. As illustrated in Figure 2 and Table 2, RAW264.7 cells maintained >90% viability when treated with M, Mw, or S1 at 1.25 μM, though S1 retained residual cytotoxicity at this concentration. Remarkably, modified analogs achieved comparable cytocompatibility (≥90% viability) at 5 μM concentrations, a threshold where native melittin exhibits significant toxicity. Notably, analogs CM-D, CM-E, E2, T1, and T3 demonstrated no concentration-dependent cytotoxicity across the tested ranges.

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Figure 2.

Cell viability of melittin analogs on RAW264.7 cells was measured by CCK-8. Data points are mean ± SE from three experiments performed in quadruplicate.

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3.3. Hemolytic activity

The hemolysis assay serves as a critical biocompatibility assessment, quantifying erythrocyte membrane disruption to evaluate the hemolytic potential of melittin analogues. As shown in Figure 3, with the exception of the analogs Mw, S1, and S2, which exhibited low hemolytic activity against rabbit erythrocytes compared to melittin, even at maximum tested concentrations (20 μM), demonstrating >90% reduction in hemolytic toxicity relative to the melittin.

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Figure 3.

Hemolytic assay. Controls for 0 and 100% hemolysis were determined by PBS buffer and 1‰ Triton-X, respectively. Percentage hemolysis (Heme/%) is expressed as mean ± SE (n = 3). *P <0.05, ***P <0.001 compared with melittin group (Mel); ns means P >0.05, compared with control group.

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3.4. In vitro anti-inflammatory activity assay

Nitric oxide (NO) quantification served as the primary screening parameter for inflammatory response, with analogs exhibiting significant NO suppression (Figure 4a) selected for subsequent cytokine profiling. While E2, T1/T3, CM-D/CM-E, and S15-2 showed limited anti-inflammatory potential, the other active analogues demonstrated dose-dependent inhibition of LPS-induced TNF-α and IL-6 secretion in ELISA analyses (Figures 4b and c). Notably, these modified peptides maintained therapeutic efficacy at concentrations exceeding melittin’s cytotoxic threshold, confirming preserved bioactivity post-structural optimization. The concentration-response relationship observed across analogues suggests a direct correlation between dosing and anti-inflammatory potency.

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Figure 4.

(a). The impact of melittin analogues on LPS-induced. NO expression levels in RAW264.7 cells were quantified using a nitric oxide assay kit. (b). The names under each group of data represent the analogue designation - administered concentration (μM). The effect of varying concentrations of melittin analogues on the release of inflammatory factors. (c). The impact of melittin analogs on LPS-induced IL-6 The impact of melittin analogs on LPS-induced TNF-α from LPS-induced RAW264.7 cells was determined by ELISA. The data are presented as the mean ± SEM (n = 3). ***p < 0.001, ns means P >0.05, compared with Mod group, ns: no significance.

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3.5. Proteolytic stability assay with α-chymotrypsin

α-Chymotrypsin-mediated proteolytic stability was assessed for anti-inflammatory analogues, given the enzyme’s preferential cleavage at the carboxyl side of positively charged amino acids (such as Trp/Leu). As shown in Figure 5, structural modifications significantly enhanced resistance to enzymatic degradation. While native melittin exhibited rapid hydrolysis (t₁/₂ = 3 min), all analogs except E3 and Mw demonstrated extended half-lives. Strikingly, the stapled derivative S15-3 displayed exceptional stability, retaining 60% intact peptide after 60 min incubation – a 20-fold improvement over the melittin.

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Figure 5.

Proteolytic stability of the melittin and analogs at the final concentration of 0.1 mM in a-chymotrypsin solution (5 ng/mL in 50 mM PBS buffer). Data points are presented as the mean ± SE of duplicate independent experiments (n = 3). *P <0.05, **P <0.01, ***P <0.001 compared with Melittin group (M). The percentage of residual peptide was monitored by analytical RP-HPLC.

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3.6. CD spectroscopy analysis

Secondary structure analysis via circular dichroism (CD) spectroscopy (190-250 nm) revealed significant conformational variations among melittin analogs (Figure 6, Table 3). Native melittin exhibited 36.1% α-helical content, while E3 and Mw analogues displayed reduced α-helix proportions. In contrast, stapled (S1, S2, S15-1, S15-3) and lipidated (C16, C18) variants demonstrated 2.2-fold enhanced α-helical content relative to the parent peptide.

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Figure 6.

CD spectra of melittin analogues.

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3.7. In vivo assay of anti-inflammatory activity of melittin analogues

The in vivo anti-inflammatory efficacy of analogs E1, S15-1, S15-3, and C16 was first assessed through a murine sepsis model, with melittin (Mel) and dexamethasone (Dex) serving as positive controls. As shown in Figure 7(a), model group mice succumbed within 48 h post-LPS challenge, confirming model validity. Both peptide-treated and dexamethasone groups exhibited significantly improved survival rates (P < 0.05 vs. control). At high-dose E1(2 mg/kg), with survival rates of 40% demonstrated therapeutic efficacy comparable to Mel (2 mg/kg) (P > 0.05 vs. positive controls), and superior to the low-dose group Dex (2 mg/kg) with survival rates of 38%. While, the high-dose S15-1(2 mg/kg) demonstrated a survival rate of 50%, comparable to the high-dose Dex (5 mg/kg), and prior to both the low-dose Dex(2 mg/kg), which had a survival rate of 38%, and the high and low-dose Mel(2 and 1 mg/kg), with survival rates of 45% and 20% respectively. This indicated that S15-1 possessed significant anti-inflammatory activity in vivo. Concurrently, body weight recovery initiated on day 4 and normalized to pre-molding levels by day 7 (Figure 7b).

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Figure 7.

(a) Alterations in the survival rate of male BALB/c mice at 7 days after treated with saline (control), dexamethasone (Dex: H-5mg/kg; L-2mg/kg), melittin (Mel: H-2mg/kg; L-1mg/kg) and analogs (S15-1, S15-3, C16, E1: H-2mg/kg; L-1mg/kg) and intraperitoneal injection of LPS (30 mg/kg). 10 mice in per group. The survival rates were estimated by the Kaplan–Meier method and compared by using the log-rank test. *P <0.05, **P <0.01 compared with control group, #P >0.05 compared with Dex(H) and Mel(H) group, ns means P >0.05 compared with control group. (b) The body weights of male BALB/c mice (n = 10 per group) were measured 7 days. Data points are presented as the mean ± SE.

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Based on the above findings, E1 and S15-1 were further evaluated in an acute auricular edema assay (Figure 8). At 2 mg/kg, both analogs achieved anti-inflammatory potency equivalent to 5 mg/kg dexamethasone and 2 mg/kg melittin, with inhibition rates of 46.3% (E1) and 47.5% (S15-1), respectively.

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Figure 8.

Alterations in treatment with Dex (5 mg/kg), Mel (2 mg/kg), E1 (2 mg/kg), and S15-1 (2 mg/kg) on xylene-induced ear swelling in BALB/c mice (n=10 per group). ***P <0.01 compared with control group; ns mean no significant difference (P > 0.05).

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3.8. Discussion

As a natural molecule with pronounced anti-inflammatory properties, melittin has been demonstrated to elicit anti-inflammatory effects at concentrations below 3 μM [29,30], but faces clinical translation challenges due to its inherent hemolytic and cytotoxic properties. The hemolytic activity of melittin is primarily attributed to the hydrophobic regions GLY1-LEU9 and LEU13-ILE20. Deletion of any amino acid within this region could result in a reduction in hemolytic activity. The VAL8, THR11, PRO14, and LEU16 are the key residues responsible for the bioactivity of melittin. TRP19 is the only aromatic residue in melittin, which has been demonstrated to be pivotal for its hemolytic activity [20,31,32]. Additionally, the hemolytic activity of melittin is contingent upon its intrinsic hydrophobicity, the number of positive charges, and the α-helical structure, among other factors. Modifying the hydrophobicity of melittin or reducing the number of positive charges it carries could effectively mitigate its hemolytic activity.

In light of the aforementioned considerations, we initially opted to substitute ILE2, LEU6, and TRP19 of melittin with the polar amino acid threonine or the acidic amino acid glutamine. Table 4 demonstrated that the hydrophobicity and the number of cationic charges carried by the analogs were reduced. Furthermore, cytotoxicity and hemolytic activity assays revealed that these substitutions successfully attenuated hemolytic activity by 97-99% and cytotoxicity by 52-100% compared to native melittin, indicating that the modification method and modification site are feasible. However, analogs E2, T1, and T3 exhibited diminished NO inhibition capacity compared with melittin in vitro. The common point of these three analogs was the substitution of LEU6, which led to the hypothesis that the leucine zipper motif (LEU6-LEU13-ILE20) was one of the main sources of melittin’s bioactivity [33]. The substitution of LEU6 to glutamine or threonine disrupts this leucine zipper motif; although it can reduce the hemolytic activity of melittin, it may also affect the anti-inflammatory activity. This suggests LEU6’s dual role in maintaining both structural integrity for target engagement and amphipathicity for membrane penetration.

Guided by these structure-activity relationships, we hypothesized that anti-inflammatory efficacy and cytotoxic potential in melittin analogs share mechanistic dependencies on amphipathic α-helical stabilization. To test this, hydrocarbon-stapled variants S1 and S2 were rationally designed to enforce helical topology while increasing hydrophobicity. As predicted, both stapled peptides exhibited enhanced α-helical content and elevated hemolytic/cytotoxic profiles compared to native melittin. Crucially, at equimolar concentrations, S1/S2 maintained anti-inflammatory potency equivalent to melittin in LPS-challenged macrophages (NO inhibition: 84/92% vs. 62% for melittin). The parallel retention of bioactivity and enhanced toxicity substantiates our hypothesis that the structural determinants responsible for therapeutic effects and adverse effects in melittin-derived peptides are intrinsically linked.

In the course of developing a modification method for melittin, we discovered that Yu [35] had isolated an active fragment of melittin comprising 15 amino acid residues by removing the hydrophobic portion [36]. This not only reduced the hemolytic activity but also retained a good anti-inflammatory activity. Building upon this discovery, we employed this truncated peptide as a structural scaffold for multimodal optimization. Through strategic incorporation of (i) macrocyclization via side-chain carboxyl activation, (ii) C-terminal lipid conjugation, and (iii) all-hydrocarbon stapling, we aimed to enhance proteolytic stability and pharmacokinetic properties while preserving therapeutic efficacy.

The modified analogs retained the advantages of low hemolytic activity and low cytotoxicity and inhibited the expression of NO, IL-6, and TNF-α at doses exceeding the toxic concentrations of natural melittin. According to Pareek [33], melittin has been shown to effectively inhibit the onset and progression of inflammation in the skin, liver, aorta, neural, and joint tissues. The principal mechanism through which melittin mediated its anti-inflammatory effects involved the inhibition of signaling pathways associated with Nuclear Factor kappa-B (NF-κB), Mitogen-Activated Protein Kinase (MAPK), and Janus Kinase (JAK)/ Signal Transducers and Activators of Transcription (STAT) [37]. The NF-κB signaling pathway was widely recognized as a classical pro-inflammatory pathway [38]. Under inflammatory conditions, NF-κB was aberrantly activated, inducing the expression of pro-inflammatory cytokines such as TNF-α, Interleukin-1 beta (IL-1β), and IL-6. The upregulation of these cytokines further amplified NF-κB activation through positive feedback regulation, exacerbating inflammatory responses. Moreover, melittin reduced inflammatory responses by simultaneously inhibiting Toll receptors, JNK, and p38 phosphorylation, and suppressing the activation of both MAPK and NF-κB pathways. Additionally, melittin alleviated synovial inflammation by interfering with STAT transcription factor activity, inducing synovial cell apoptosis, and downregulating inflammatory cytokine secretion. Notably, melittin also inhibited neuroinflammation by obstructing Akt phosphorylation and suppressing the expression of inflammatory proteins [39]. In this study, we hypothesized S1, S2, E1, T2, E3, S15-1, S15-3, C16, and C18 might inhibit JNK phosphorylation, simultaneously suppress NF-κB signaling pathways. As a result, IKK and the phosphorylation of IκB were decreased, the binding ability of NF-κB to DNA was reduced, leading to downregulation of proinflammatory genes, which significantly contributed to the suppression of pro-inflammatory mediators, including NO, IL-6, and TNF-α, demonstrating the therapeutic potential of the above 9 melittin analogs in managing sepsis and other inflammatory conditions (Figure 9).

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Figure 9.

The possible mechanism of 9 analogues’ anti-inflammatory action. c-Jun N-terminal Kinase (JNK), protein kinase B (Akt), Inhibitor of Kappa B Kinase (IKK), Inhibitor of NF-κB(IκB), Toll-like receptors (TLRs).

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In an in vitro enzymatic stability assay, S15-1, S15-3, C16, and C18 demonstrated markedly improved α-chymotrypsin resistance. That may be attribute to the stapled (S15-1, S15-3) and lapidated (C16, C18) peptides, which exhibited a 2.2-fold increasing in α-helical content compared to the parent peptide. This enhancement substantially increased their enzymatic stability, subsequently elevating the anti-inflammatory activity. However, cyclized derivatives CM-D/CM-E and stapled peptide S15-2 showed a complete loss of bioactivity, suggesting structural constraints. By comparing the modification sites, we hypothesized that the ILE9 of the active fragment of melittin constitutes a leucine zipper with LEU2, and that the ILE9 may also be the key amino acid for binding to the target site. The substitution of amino acid residues and cyclization might disrupt the conserved LEU2-ILE9 hydrophobic staple critical for target engagement, while proline hinge-mediated conformational flexibility, essential for receptor binding [40,41], may be compromised by rigid cyclic architectures. These structure-activity relationships highlight two key design principles: (i) The LEU2-ILE9 zipper motif governs both membrane interaction and target recognition, with ILE9 substitutions destabilizing this functional epitope; (ii) Preserving conformational plasticity in the proline hinge region proves essential for maintaining pharmacological activity during structural optimization.

Based on comprehensive in vitro characterization, analogs E1, S15-1, S15-3, and C16 were advanced to in vivo anti-inflammatory experiments. In the LPS-induced sepsis model, a lethal condition driven by organ dysfunction caused by a dysregulated host response to infection [42]. High-dose E1 (2 mg/kg) and S15-1 (2 mg/kg) demonstrated survival rates of 40% and 50%, respectively, comparable to dexamethasone (5 mg/kg, 45%) and melittin (2 mg/kg, 38%) (P > 0.05). Treated mice exhibited rapid clinical improvement, resolving lethargy, piloerection, and motor deficits by day 3 post-challenge, with full weight recovery to pre-molding levels by day 7. To decouple LPS-neutralizing effects from broader anti-inflammatory mechanisms, we employed a xylene-induced auricular edema model in KM mice. Both E1 and S15-1 (2 mg/kg) achieved edema inhibition rates of 46.3% and 47.5%, respectively, matching the efficacy of 5 mg/kg dexamethasone (48.1%) and 2 mg/kg melittin (45.9%) (P > 0.05). This means E1 and S15-1 exhibited excellent anti-inflammatory activity in vivo, compared to dexamethasone and melittin. These data strongly suggest that structural optimization preserves melittin’s core anti-inflammatory pharmacology while mitigating toxicity via distinct inflammatory models.