RESUMO
Quercetin derivatives have already shown their anti-inflammatory potential, inhibiting essential enzymes involved in this process. Among diverse pro-inflammatory toxins from snake venoms, phospholipase A2 is one of the most abundant in some species, such as Crotalus durissus terrificus and Bothrops jararacussu from the Viperidae family. These enzymes can induce the inflammatory process through hydrolysis at the sn-2 position of glycerophospholipids. Hence, elucidating the main residues involved in the biological effects of these macromolecules can help to identify potential compounds with inhibitory activity. In silico tools were used in this study to evaluate the potential of quercetin methylated derivatives in the inhibition of bothropstoxin I (BthTX-I) and II (BthTX-II) from Bothrops jararacussu and phospholipase A2 from Crotalus durissus terrificus. The use of a transitional analogous and two classical inhibitors of phospholipase A2 guided this work to find the role of residues involved in the phospholipid anchoring and the subsequent development of the inflammatory process. First, main cavities were studied, revealing the best regions to be inhibited by a compound. Focusing on these regions, molecular docking assays were made to show main interactions between each compound. Results reveal that analogue and inhibitors, Varespladib (Var) and p-bromophenacyl bromide (BPB), guided quercetins derivatives analysis, revealing that Leu2, Phe5, Tyr28, glycine in the calcium-binding loop, His48, Asp49 of BthTX-II and Cdtspla2 were the main residues to be inhibited. 3MQ exhibited great interaction with the active site, similar to Var results, while Q anchored better in the BthTX-II active site. However, strong interactions in the C-terminal region, highlighting His120, seem to be crucial to decreasing contacts with phospholipid and BthTX-II. Hence, quercetin derivatives anchor differently with each toxin and further in vitro and in vivo studies are essential to elucidate these data.
RESUMO
Snake venom serine protease (SVSP) interferes with the regulation and control of important biological reactions in homeostasis and can be classified as an activator of the fibrinolytic system and platelet aggregation. Our group has recently isolated a new serine protease from Crotalus durissus terrificus total venom (Cdtsp-2). This protein exhibits edematogenic capacity and myotoxic activity. A Kunitz-like EcTI inhibitor protein with a molecular mass of 20 kDa was isolated from Enterolobium contortisiliquum and showed high trypsin inhibition. Thus, the objective of this work is to verify the possible inhibition of the pharmacological activities of Cdtsp-2 by the Kutinz-type inhibitor EcTI. To isolate Cdtsp-2 from total C. d. terrificus venom, we used three-step chromatographic HPLC. Using the mice paw edema model, we observed an edematogenic effect, myotoxicity and hepatotoxicity caused by Cdtsp-2. In vitro and in vivo experiments showed that the alterations in hemostasis caused by Cdtsp-2 are crucial for the development of marked hepatotoxicity and that EcTI significantly inhibits the enzymatic and pharmacological activities of Cdtsp-2. Kunitz-like inhibitor may be a viable alternative for the development of ancillary treatments against the biological activities of venoms.