Desenvolvimento de peptídeo cíclico derivado do ativador do plasminogênio do tipo uroquinase (uPA19-31): inibição do seu receptor (uPAR) e avaliação dos processos relacionados à invasividade

Abstract

Cancer is considered a complex disease characterized by a set of genetic and/or epigenetic conditions, with disordered and proliferative development of abnormal cells throughout the body, which may lead to metastasis. Thus, alternative therapies based on the development of synthetic inhibitory peptides capable of competing with the Urokinase-type Plasminogen Activator (uPA) for the binding site on its receptor (uPAR) are at an advanced stage of research. This is because inhibiting the formation of the uPA–uPAR complex through these peptides can prevent the dissemination and proliferation of highly invasive tumor cell lines.. Quantum-level computational simulations have been employed in recent years to understand the various binding mechanisms and the physicochemical properties of biological systems. Thus, the application of Molecular Fractionation with Conjugated Caps (MFCC) allows to obtain important results for the development of more specific therapeutic agents regarding their actions in the body. The present work aimed to develop and quantify an inhibitor peptide of the Urokinase-type Plasminogen Activator Receptor (uPAR) and evaluate the synthetic peptide in processes related to invasiveness and metastasis formation. The input data for the simulations were the 2.5 Å resolution structure of the human uPA-uPAR complex (PDB ID: 3BT2), which underwent computational simulations and quantum analyses, with a binding energy of the uPA-uPAR complex of -143.45 kcal/mol. Based on the interaction sequence of uPA19-31, the design of peptides was proposed through in silico mutagenic alterations. Two new cyclic peptides were developed; the test cyclic peptide (Ser19-Lys20-Trp21-Asp22-Arg23-Trp24-Phe25-Asn26-Lys27-Leu28-Trp29-Trp30) presented a binding energy with the receptor of -280.34 kcal/mol, and the control cyclic peptide (Phe19-Trp20-Lys21-Arg22-Leu23-Lys24-Trp25-Trp26-Asn27-Lys28-Trp29-Asp30-Ser31) had a binding energy of -184.10 kcal/mol. The test and control cyclic peptides showed partial action in processes involving metastasis formation (related to invasiveness) in B16F10 tumor cells, such as adhesion, migration, invasion, and expression of Plaur and uPAR – in vitro –, where they presented the following results: Cell Adhesion - The test peptide significantly reduced B16F10 cell adhesion to type I collagen by 18.27%, while the control peptide caused a reduction of 10.06%, suggesting that both interfere with cell adhesion. Cell Migration - Treated with the test peptide, B16F10 cells showed a significant 49.52% inhibition in migration after 24 hours compared to the negative control, whereas the control peptide inhibited about 19.68%. Cell Invasion - The test peptide inhibited B16F10 cell invasion through Matrigel by 73.7%, while the control peptide caused an inhibition of 43.4%, indicating a more pronounced anti-invasive effect of the test peptide. Expression of Plaur and uPAR - PCR analysis revealed a significant increase in Plaur gene expression in cells treated with the test peptide, indicating that this peptide may influence uPAR expression. Thus, the test and control peptides presented fluorescence densities of 6.63% and 11.95%, respectively, while the negative control showed a fluorescence density of 14.68% in B16F10 tumor cells. In conclusion, the synthetic test and control peptides demonstrated participation in the inhibition of processes associated with invasiveness and metastasis formation, where the use of tools such as MFCC and molecular dynamics for the development of synthetic peptides is essential and highly promising.