*8.2. New Molecular Entities for the Study of Anti-Inflammatory Diseases*

Lopap is the most studied lipocalin isolated from *L. obliqua*, and the activity of its derived peptides has been the aim of several studies.

Lipocalins are known by their role as protective factors with participation in development, regeneration, and tissue repair [95–97]. Moreover, several lipocalins have been involved in the mediation of cell regulation, such as cell growth and differentiation [98,99]. Lipocalins share three main structures: structurally conserved region 1 (SCR1) is the highest conserved region with the 3–10 helix and adjacent strand leading into a loop. SCR2 is formed by two strands, and the loop that connects them varies across proteins; however, the essential features of the loop are retained, despite the unfavorable main chain. Lastly, SCR3 contains a strand with arginine as the last residue and part of the loop linking it to the C-terminal α-helix [100].

Synthetic peptides based on these three conserved motifs found in Lopap—pM1, pM2a/pM2b, and pM3 (the numbers are related to the motif)—were studied to determine their capability to promote cell survival. Motif 2-related peptides showed a cytoprotective activity, in which pM2b was found to be the shortest peptide with similar activity to Lopap, despite not containing the residues supposedly involved in the catalytic activity of Lopap. This peptide triggers cell survival of neutrophils and endothelial cells via nitric oxide [82]. The effect of peptide pM2b was also evaluated in the modulation of extracellular matrix (ECM) proteins using both in vitro and in vivo approaches and showed an increased production of ECM, as well as a modulation of mediators involved in apoptosis, antiapoptosis, and proliferation in human fibroblasts. Additionally, an increased production of collagen was observed in vivo [83]. Peptide pM2b also displayed modulating effects in an in vivo model of wound healing. Furthermore, the increased production of collagen, glycosaminoglycans, and metalloproteinases along with improvement of wound closure was observed [84]. Those studies suggest an involvement of motif 2 in cell protection. Moreover, using a peptide mapping approach and tertiary structure comparison, a lipocalin sequence signature able to modulate cell survival was identified [82]. A computational analysis of the peptide sequence signature YAIGYSC, later called pM2c, revealed great similarity with antiapoptotic lipocalins [85]. Alignment of the Lopap sequence with other lipocalins showed that this peptide sequence was highly conserved, and yet few different patterns could be observed. Ten aligned peptides, along with pM2c, were modeled in 3D structures, followed by the analysis of molecular descriptors. This study showed that, even with the amino-acid modifications, the calculated molecular properties were generally maintained, especially the molecular shape and electronic density distribution, highlighting the importance of these two properties for molecular recognition process, while the lipophilicity was more related to the pharmacokinetic profile, validating the lipocalin sequence signature previously reported [85].

Other peptides have been isolated from Lopap (Table 3). The peptide CNF011.05D, along with three peptides comprising an amino-acid sequence of at least 70% identity, was described and patented for being capable of stimulating the production of ECM proteins in human fibroblast cells (Patent No. P4US88374OB2 in the USA and No. EP2245149B1 in Europe). The ECM proteins evaluated were fibronectin, tenascin, procollagen, and collagen. In addition, it was demonstrated that this peptide induced the production of nitric oxide, and a wound healing model in rats and pigs showed a faster rate of reduced wound size.

Regarding Losac, the structural analysis revealed that it shares the conserved motifs and immunoglobulin-like domains scattered throughout their four domains (D1 to D4) with hemolins from different Lepidoptera species [43]: *N*-glycosylation and protein kinase C in D3 and cAMP/cGMP in D2; furthermore, the conserved Lys–Gly–Asp (KDG) motif is found in domains D1 and D3 [101]. The KDG motif is related to cell adhesion, and previous studies in hemocytes revealed that hemolin could be involved in cell differentiation or regeneration of wounded tissue, acting as a cell attachment component, suggesting its participation in cellular immune response and morphogenesis [102,103]. This also resembles the function of cell adhesion molecules such as selectins and cadherins; they are important in lymphocyte homing during the inflammatory response and morphogenesis, as well as in the development of neural systems [103–105].


**Table 3.** Patents applied and granted regarding *Lonomia* toxins and derived peptides.

Despite advances involving the pathogenesis of inflammatory diseases and new therapeutic strategies, the progression of inflammatory diseases has become a public health problem in recent years, mainly due to new viruses and resistant bacteria [106], along with inflammatory diseases related to aging [107]. Therefore, studies on new molecular targets are needed for development of more effective drugs and therapeutic approaches that can benefit the health and quality of life of patients. Accordingly, allied with the promising results from *Lonomia* peptides, several other peptides were engineered and assayed in several in vitro models of inflammatory diseases, such as arthritis. Some of these peptides had anti-inflammatory effects on several cellular models developed at CENTD (Table 3), reversing the expression of molecules associated with the inflammatory mechanism of cells, in addition to decreasing pain markers in the neuron model and inducing regenerative proteins in different cell models. Those peptides were chosen as a powerful tool for the discovery of new inflammatory targets against arthritis, associating omics approaches with the developed cell models and LOCBE.
