Exploring the therapeutic potential of an antinociceptive and anti-inflammatory peptide from wasp venom

Abstract

Animal venoms are rich sources of neuroactive compounds, including anti-inflammatory, antiepileptic, and antinociceptive molecules. Our study identified a protonectin peptide from the wasp Parachartergus fraternus' venom using mass spectrometry and cDNA library construction. Using this peptide as a template, we designed a new peptide, protonectin-F, which exhibited higher antinociceptive activity and less motor impairment compared to protonectin. In drug interaction experiments with naloxone and AM251, Protonectin-F's activity was decreased by opioid and cannabinoid antagonism, two critical antinociception pathways. Further experiments revealed that this effect is most likely not induced by direct action on receptors but by activation of the descending pain control pathway. We noted that protonectin-F induced less tolerance in mice after repeated administration than morphine. Protonectin-F was also able to decrease TNF-α production in vitro and modulate the inflammatory response, which can further contribute to its antinociceptive activity. These findings suggest that protonectin-F may be a potential molecule for developing drugs to treat pain disorders with fewer adverse effects. Our results reinforce the biotechnological importance of animal venom for developing new molecules of clinical interest.

Figure 1

(a) High performance liquid chromatography fractionation (HPLC) of low molecular weight compounds found in the venom of Parachartergus fraternus social wasp. Ten fractions numbered 1–10 were collected for this work. (b) MALDI-TOF mass spectra of HPLC fraction 9, revealing the presence of ions [M + H]⁺  = 1209.94, [M + Na]⁺  = 1231.21 and [M + K]⁺  = 1247.79. (C) MALDI-TOF/TOF mass spectra in LIFT mode of the compound was obtained from fraction 9. Molecular mass analysis revealed a ten amino acid residues-long peptide with seven leucine/isoleucine ambiguities.

Figure 2

(a) The nucleotide sequences of precursors encoding Protonectin. (b) ClustalW sequence alignment of precursor peptides found in four wasp species: Parachartergus fraternus, Orancistrocerus drewseni, Vespa magnifica and Vespa affinis. represented above the blue line while mature peptides are represented above the red line. (c) ClustalW sequence alignment of mature peptides found in six wasp species: Parachartergus fraternus (P. fraternus), Agelaia pallipes pallipes (A. pallipes pallipes), Protonectarina sylveirae (P. sylveirae), Polybia paulista (P. paulista), Orancistrocerus drewseni (O. drewseni) and Vespa magnifica (V. magnifica).

Figure 3

(a) Antinociception index obtained from the hot plate assay after i.c.v. injection of natural protonectin at 8 nmol/animal or at 16 nmol/animal. Control groups received either morphine at 16 nmol per animal or vehicle solution. Data were analyzed with Two-Way ANOVA followed by Bonferroni post-hoc test. (*) indicates statistical difference when compared morphine to vehicle control (* = p < 0.05). (#) indicates difference when compared Protonectin-F 16 nmol to vehicle control with p < 0.05. (b)- Area under curve obtained from the antinociception index results. Data were analyzed by ANOVA followed by Tukey’s post-hoc test. (*) indicates statistical difference (**** = p < 0.0001; ** = p < 0.01; * = p < 0.05).

Figure 4

(a) Antinociception index obtained from the hot plate assay after i.c.v. injection of the modified protonectin-F at 16, 8 or 4 nmol/animal. Control groups received either morphine at 16 nmol per animal or vehicle solution. Data were analyzed with Two-Way ANOVA followed by Bonferroni post-hoc test. (*) indicates statistical difference when compared morphine to vehicle control. (#) indicates difference when compared protonectin-F 16 nmol to vehicle control with p < 0.05. ( +) indicates difference when compared to the group treated with protonectin-F at 16 nmol/animal with p < 0.05. (b) Area under curve obtained from the antinociception index results. Data were analyzed by ANOVA followed by Tukey’s post-hoc test. ( +) indicates statistical difference when compared to vehicle control (⁺⁺⁺  = p < 0.001). (#) indicates difference when compared to morphine control with p < 0.05 (### = p < 0.001; ## = p < 0.01). (*) indicates difference when compared to the group treated with protonectin-F at 16 nmol/animal (**** = p < 0.0001; ** = p < 0.01).

Figure 5

(a) TNF-α production after LPS insult. Every concentration of protonectin-F tested was able to reduce TNF-α production of peritoneal macrophages. Data were analyzed by One-Way ANOVA followed by Tukey’s post-hoc test. Statistically significant differences are indicated when compared to M1 + LPS control (*). (b) Demonstration of time dependence of protonectin-F immunomodulation. Data were analyzed by One-Way ANOVA followed by Tukey’s post-hoc test. Statistically significant differences are indicated when compared to M1 + LPS (12 h) control (*).

Figure 6

(A) Antinociception index obtained from the hot plate assay exploring the pharmacological antagonism of naloxone. The antinociceptive effect of protonectin-F (16 nmol) and morphine (16 nmol) was evaluated after administration of naloxone, as well as protonectin-F or morphine alone. Control groups were treated with vehicle alone or with naloxone and vehicle. Data were analyzed by Two-Way ANOVA followed by Bonferroni’s post-hoc test. (***) indicates statistical difference when compared to vehicle control p < 0.001. (###) indicates difference when compared the group treated with naloxone and morphine 16 nmol with p < 0.001. (+ + +) indicates difference when compared to the control group treated with naloxone and protonectin- F with p < 0.001. (B) Antinociception index obtained from the hot plate assay exploring the pharmacological antagonism of AM251. The antinociceptive effect of protonectin-F was evaluated after administration of AM251, as well as protonectin-F alone. Control groups were treated with vehicle or with AM251. Data were analyzed by Two-Way ANOVA followed by Tukey’s post-hoc test. (***) indicates statistical difference when compared to vehicle control with p < 0.001. (###) indicates difference when compared the group treated with AM251 and protonectin-F with p < 0.001.

Figure 7

Antinociception index obtained from the hot plate assay after i.c.v injection of Protonectin-F (16 nmol) or morphine (16 nmol). Control group was treated with vehicle alone. All experimental groups were treated for five consecutive days and the antinociception was measured 120 min after injection. Data were analyzed by Two-Way ANOVA followed by Tukey’s post-hoc test. (*) statistically significant differences (p < 0.05) are indicated when compared to vehicle control, (#) statistically significant differences (p < 0.05) are indicated when compared to morphine group.