α-MSH, as an endogenous neuropeptide (Fetissov et al., 2019; Hidese et al., 2023), regulates nuclear factor kappa-B (NF-κB) (Ichiyama et al., 2000), interleukin-1 (IL-1) (Machado et al., 2010), interleukin-6 (IL-6) (Nieto et al., 2020), tumor necrosis factor-α (TNF-α) (Rajora et al., 1997), cytokine synthesis inhibitor interleukin-10 (IL-10) (Kleiner et al., 2021) and neutrophil chemotaxis (Catania et al., 1996). These studies have indicated that α-MSH exhibits significant potential as a therapeutic medicines for inflammatory treatment (Dinparastisaleh and Mirsaeidi, 2021). The clinical application of α-MSH is limited due to its short half-life (Palazzi et al., 2020). Thus, the extension of α-MSH's half-life holds significant importance for its clinical application (Hruby, 2016). In our previous study, we successfully addressed the issue of short half-life by fusing it with the carrier HSA. And the protein transduction domain (PTD) was fused to enhance the efficiency of penetrating the BBB (Wang et al., 2016). However, we believe that the anti-inflammatory effect of the fusion protein falls short of meeting clinical requirements. Therefore, further study is necessary to improve the anti-inflammatory activity in the CNS.

The fusion proteins containing various domains are required to undergo the consequences of structural disorder (Lu and Feng, 2008). Convincing evidence has indicated that the direct binding of domains in fusion proteins leads to the disruption of their respective spatial structures and impairs their biological function (Yangyang et al., 2022). Therefore, reducing the structural interference between domains is one of the crucial challenges in developing fusion protein medicines (Monterrey et al., 2022). In recent years, the linker peptide technology has been proposed to solve this problem (Li et al., 2016; Patel et al., 2022). Linker peptides are commonly classified into three categories (Baghbeheshti et al., 2021). In addition to being commonly used in tumor-targeted in vivo cleavable linker peptides (Alas et al., 2021), they also have flexible and rigid ones. The flexible linker peptides, because of their flexible properties, are in favor of correct folding and reduced steric hindrance between domains (Gong et al., 2016). On the contrary, the rigid linker peptides can help maintain a distance and reduce interactions between domains (Yang et al., 2022). The rational design of linker peptide can significantly improve the stability and biological activities of fusion proteins (Ren et al., 2021; Wriggers et al., 2005; Zane et al., 2023; Zhao et al., 2008). We expected to improve the half-life and activity of α-MSH fusion protein by optimizing the linker peptide to get closer to clinical requirements.

In our previous research, we identified two promising candidates from a pool of nine α-MSH fusion proteins with various linker peptides. This selection was made through computer aided drug design (CADD), as well as analyses of expression, purification, stability, and in vitro activity. These candidates include a fusion protein with a flexible linker peptide (FPFL) and a fusion protein with a rigid linker peptide (FPRL). FPFL shows high expression levels in yeast and demonstrates in vitro anti-inflammatory activity. FPRL has better stability and outstanding anti-inflammatory activity in vitro compared to FPFL. Based on the in vitro study of these α-MSH fusion proteins, further investigation in vivo is necessary. Therefore, we carried out further study of FPFL and FPRL by intravenous administration, including the metabolism, the efficiency to penetrate BBB, the extended half-life, and the regulation of related inflammatory cytokines.