Avaliação de potenciais agentes anti-inflamatórios tendo como alvo a inibição do Fator de Necrose Tumoral pela interação com a metaloproteinase BmooMP-alfa-I

Abstract

Inflammatory disorders are common among population probably leading to autoimmune disease or cancer. These diseases are debilitating for the patient and also difficult to treat. Agents that reduce the activity of the cytokine TNF (tumor necrosis factor) have been used to improve the clinical signs of autoimmune diseases, since the exacerbated production of TNF is considered the main cause of inflammatory disorders appearance. Although widely used, current anti-TNF agents are not effective for all patients. Regarding this, our group intend to identify a product that can reduce the immune response and help in the treatment of anti-inflammatory disorders. Following this aim, we first isolated a snake metalloprotease named BmooMP-alpha-I and verified that this metalloprotease was able to improve clinical aspects, such as weight loss and animal survival, in an animal model of Crohn's Disease induced by oral infection of. Toxoplasma gondii cysts. Furthermore, we observed a three-fold reduction in TNF amount in the group treated with the protein when compared to the control. Corroborating with previous result, there was a reduction in the activity of immune cells, especially macrophages. Considering the effectiveness of the interaction between metalloprotease and TNF, we decided to develop peptides that could interact with TNF based on the interaction between TNF and BmooMP-alpha-I. Initially, we selected six peptides based on the interaction between the two proteins. After analysing the physicochemical characteristics and similarity with other ligands in silico, we selected two peptides named pep1 and pep 4. After selection, we docked the peptides with human and murine TNF and we verified that the peptides present similarity in their interactions with TNF, especially by Arg32 and ALA33. We also verified the pharmacological properties by analysis of Absorption, Distribution, Metabolism and Excretion (ADME) in silico and found that the peptides have a paracellular absorption and do not present high cytotoxicity, in addition peptides do not have first-pass metabolism and have good intestinal absorption. After computational analysis, we synthesized the peptides and the FTIR analysis performed demonstrated a change in chemical interactions indicating that both peptides interact with TNF. Furthermore, we determined that both peptides were able to reduce the interaction between TNF and TNF detection antibody in 20% after 1 h of incubation and more than 80% after 24 h of incubation. Based on these results, we can conclude that both peptides are capable of interacting with TNF, even though in vivo studies are necessary to know if peptides present biological activity and how it manipulates the immune system.