Chronic pain resulting from nerve injury, inflammation, or cancer infiltration poses a significant health challenge. Despite the availability of treatments such as non-steroidal anti-inflammatory drugs and opioids, chronic pain remains inadequately managed, with limited efficacy or serious side effects [1], [2], [3]. G-protein-coupled receptors, including the seven transmembrane receptors that play a major role in pain modulation, represent important targets for pain treatment [2], [4]. Many previous studies have suggested a possible relationship between the pathogenesis of inflammatory/immune-mediated neuropathies, G protein-coupled receptor 7 (GPR7) expression, and pain transmission [5], [6]. The GPR7 was deorphanized with the identification of endogenous ligands neuropeptide B (NPB) and neuropeptide W (NPW), and was subsequently reclassified as the neuropeptide B/W receptor-1 (BGNPBWR1) [7], [8]. However, the mechanisms by which G-protein-coupled receptor 7 contributes to inflammatory pain remain unclear. Therefore, clarifying its activation mechanism and inventing better-targeted peptides are crucial for the understanding of activation behavior and application.

NPB and NPW belong to the neuropeptide family as endogenous ligands for GPR7. Although NPB and NPW share 61 % sequence homology at the amino acid level, NPB has a higher affinity for GPR7 [9], [10]. Furthermore, NPB mRNA was detected in the thalamus and hypothalamus of the human brain, suggesting the important role of NPB in the brain [11]. Systematic SAR studies indicated that the Trp1 residues of NPB play critical roles for ligand–receptor interactions. Previous observations have shown that NPB has analgesic activity as an endogenous ligand of GPR7 in the central nervous system (CNS). Given the distribution of NPB and GPR7 mRNAs outside the CNS, it is clear that these peptides may also function as paracrine or endocrine factors in peripheral organs. NPB has high homology and the same key action domain in some organisms [12], [13]. Previous studies have shown that BgNPB screened from toad venom protein has anti-inflammatory and analgesic activity [14]. It has significant medicinal value and can be exploited for therapeutic purposes. To optimize the efficacy of BgNPB and elevate its activation activity on GPR7, we conducted transdermal modification.

Studies have shown that the third alpha-helix of the homeodomain of Drosophila antennapedia proteins (ANTP; 16 amino acid residues from 43 to 58) was the minimal peptide that retained transduction functions. This peptide can be used as an internalization vector for oligopeptides or oligonucleotides for transfer into the cytoplasm and nucleus of all cell types in a receptor-, channel-, energy-, and endocytosis-independent fashion. ANTP can act directly on the lipid bilayer to complete transmembrane movement and enter cells [15], [16], [17]. Thus, BgNPB can be made to enter keratinocyte cells easily by generating a fusion peptide by attaching the N-terminal tryptophan of BgNPB to a C-terminal lysine of the 16 amino acid peptides. Therefore, we used the ANTP protein as the guiding peptide to synthesize the ANTP-BgNPB fusion peptide. Meanwhile, the optimal expression conditions of the fusion peptide were found.

In this study, we aimed to explore the expression and analgesic mechanism of a fusion peptide. To achieve this goal, we meticulously optimized the conditions for inducing the expression of the fusion peptide, including the induction temperature, inducer concentration, and induction time. Western blot analysis confirmed the soluble expression of the recombinant protein. In addition, we utilized molecular docking and molecular dynamics simulation to investigate the molecular interactions between peptides and GPR7 active sites. To further understand, we also evaluated the analgesic activity of the polypeptides in pain model mice. Given the potential benefits of ANTP-BgNPB, further research is necessary to determine the optimal molecular modification and mechanism of action. This study not only lays a solid foundation for future investigations into the therapeutic potential of ANTP-BgNPB in inflammatory pain treatment but also provides valuable insights for clinical translation and drug development.