Most of the snakebite envenomations in Central and South America are caused by species belonging to Bothrops genus. Their venom is composed mainly by zinc-dependent metalloproteinases, responsible of the hemorrhage characteristic of these envenomations. The aim of this study was to determine the inhibitory ability of ten flavonoids on the in-vitro proteolytic activity of Bothrops atrox venom and on the hemorrhagic, edema-forming and myonecrotic activities of Batx-I, the most abundant metalloproteinase isolated from this venom. Myricetin was the most active compound, exhibiting an IC ${}_{50}$ value of 150 $\mathsf{\mu }$ M and 1021 $\mathsf{\mu }$ M for the inhibition of proteolytic and hemorrhagic activity, respectively. Independent injection experiments, with a concentration of 1600 $\mathsf{\mu }$ M of myricetin administered locally, immediately after toxin injection, demonstrated a reduction of $28\pm 6\%$ in the hemorrhagic lesion. Additionally, myricetin at concentrations 800, 1200 and 1600 $\mathsf{\mu }$ M promoted a reduction in plasma creatine kinase activity induced by Batx-I of $21\pm 2\%$ , $60\pm 5\%$ and $63\pm 2\%$ , respectively. Molecular dynamics simulations coupled with the adaptive biasing method suggest that myricetin can bind to the metalloproteinase active site via formation of hydrogen bonds between the hydroxyl groups 3’, 4’ and 5’ of the benzyl moiety and amino acid Glu143 of the metalloproteinase. The hydroxyl substitution pattern of myricetin appears to be essential for its inhibitory activity. Based on this evidence, myricetin constitutes a candidate for the development of inhibitors to reduce local tissue damage in snakebite envenomations.

Snake bite envenoming is a public health problem that was recently included in the list of neglected tropical diseases of the World Health Organization. In the search of new therapies for the treatment of local tissue damage induced by snake venom metalloproteinases (SVMPs), we tested the inhibitory activity of peptidomimetic compounds designed as inhibitors of matrix metalloproteinases on the activities of the SVMP Batx-I, from Bothrops atrox venom. The evaluated compounds show great potential for the inhibition of Batx-I proteolytic, hemorrhagic and edema-forming activities, especially the compound CP471474, a peptidomimetic including a hydroxamate zinc binding group. Molecular dynamics simulations suggest that binding of this compound to the enzyme is mediated by the electrostatic interaction between the hydroxamate group and the zinc cofactor, as well as contacts, mainly hydrophobic, between the side chain of the compound and amino acids located in the substrate binding subsites S1 and S1 ${}^{\prime }$ . These results show that CP471474 constitutes a promising compound for the development of co-adjuvants to neutralize local tissue damage induced by snake venom metalloproteinases.

Myotoxic phospholipases A₂ (PLA₂) are responsible for many clinical manifestations in envenomation by Bothrops snakes. A new myotoxic acidic Asp49 PLA₂ (BaCol PLA₂) was isolated from Colombian Bothrops asper venom using reverse-phase high performance liquid chromatography (RP-HPLC). BaCol PLA₂ had a molecular mass of 14,180.69 Da (by mass spectrometry) and an isoelectric point of 4.4. The complete amino acid sequence was obtained by cDNA cloning (GenBank accession No. MF319968) and revealed a mature product of 124 amino acids with Asp at position 49. BaCol PLA₂ showed structural homology with other acidic PLA₂ isolated from Bothrops venoms, including a non-myotoxic PLA₂ from Costa Rican B. asper. In vitro studies showed cell membrane damage without exposure of phosphatidylserine, an early apoptosis hallmark. BaCol PLA₂ had high indirect hemolytic activity and moderate anticoagulant action. In mice, BaCol PLA₂ caused marked edema and myotoxicity, the latter seen as an increase in plasma creatine kinase and histological damage to gastrocnemius muscle fibers that included vacuolization and hyalinization necrosis of the sarcoplasm.

Snakebite envenomings are a global public health issue. The therapy based on the administration of animal-derived antivenoms has limited efficacy against the venom-induced local tissue damage, which often leads to permanent disability. Therefore, there is a need to find inhibitors against toxins responsible for local damage. This work aimed to synthesize thioesters derived from 2-sulfenyl ethylacetate and to evaluate the inhibitory effects on two snake venom toxins. Ethyl 2-((4-chlorobenzoyl)thio)acetate (I), Ethyl 2-((3-nitrobenzoyl)thio)acetate (II) and Ethyl 2-((4-nitrobenzoyl)thio)acetate (III) were synthesized and spectroscopically characterized. Computational calculations were performed to support the study. The inhibitory capacity of compounds (I–III) was evaluated on a phospholipase A₂ (Cdcum6) isolated from the venom of the Colombian rattlesnake Crotalus durissus cumanensis and the P-I type metalloproteinase Batx-I isolated from Bothrops atrox. I–III inhibited PLA₂ with IC₅₀ values of 193.2, 305.4 and 132.7 µM, respectively. Otherwise, compounds II and III inhibited the proteolytic activity of Batx-I with IC₅₀ of 2774 and 1879 µM. Molecular docking studies show that inhibition of PLA₂ may be due to interactions of the studied compounds with amino acids in the catalytic site and the cofactor Ca²⁺. Probably, a blockage of the hydrophobic channel and some amino acids of the interfacial binding surface of PLA₂ may occur.