In Vitro and In Silico Study on the Impact of Chlorogenic Acid in Colorectal Cancer Cells: Proliferation, Apoptosis, and Interaction with ß-Catenin and LRP6.

Colorectal cancer mortality rate and highly altered proteins from the Wnt/ß-catenin pathway increase the scientific community's interest in finding alternatives for prevention and treatment. This study aims to determine the biological effect of chlorogenic acid (CGA) on two colorectal cancer cell lines, HT-29 and SW480, and its interactions with ß-catenin and LRP6 to elucidate a possible modulatory mechanism on the Wnt/ß-catenin pathway. These effects were determined by propidium iodide and DiOC6 for mitochondrial membrane permeability, MitoTracker Red for mitochondrial ROS production, DNA content for cell distribution on cell cycle phases, and molecular docking for protein-ligand interactions and binding affinity. Here, it was found that CGA at 2000 µM significantly affects cell viability and causes DNA fragmentation in SW480 cells rather than in HT-29 cells, but in both cell lines, it induces ROS production. Additionally, CGA has similar affinity and interactions for LRP6 as niclosamide but has a higher affinity for both ß-catenin sites than C2 and iCRT14. These results suggest a possible modulatory role of CGA over the Wnt/ß-catenin pathway in colorectal cancer.

Inhibitory Effects of Varespladib, CP471474, and Their Potential Synergistic Activity on Bothrops asper and Crotalus durissus cumanensis Venoms.

Snakebite is a neglected tropical disease that causes extensive mortality and morbidity in rural communities. Antivenim sera are the currently approved therapy for snake bites; however, they have some therapeutic limitations that have been extensively documented. Recently, small molecule toxin inhibitors have received significant attention as potential alternatives or co-adjuvant to immunoglobulin-based snakebite therapies. Thus, in this study, we evaluated the inhibitory effects of the phospholipase A2 inhibitor varespladib and the metalloproteinase inhibitor CP471474 and their synergistic effects on the lethal, edema-forming, hemorrhagic, and myotoxic activities of Bothrops asper and Crotalus durissus cumanensis venoms from Colombia. Except for the preincubation assay of the lethal activity with B. asper venom, the mixture showed the best inhibitory activity. Nevertheless, the mix did not display statistically significant differences to varespladib and CP471474 used separately in all assays. In preincubation assays, varespladib showed the best inhibitory activity against the lethal effect induced by B. asper venom. However, in independent injection assays, the mix of the compounds partially inhibited the lethal activity of both venoms (50%). In addition, in the assays to test the inhibition of edema-forming activity, the mixture exhibited the best inhibitory activity, followed by Varespladib, but without statistically significant differences (p > 0.05). The combination also decreased the myotoxic activity of evaluated venoms. In these assays, the mix showed statistical differences regarding CP471474 (p < 0.05). The mixture also abolished the hemorrhagic activity of B. asper venom in preincubation assays, with no statistical differences to CP471474. Finally, the mixture showed inhibition in studies with independent administration in a time-dependent manner. To propose a mode of action of varespladib and CP471474, molecular docking was performed. PLA2s and SVMPs from tested venoms were used as targets. In all cases, our molecular modeling results suggested that inhibitors may occupy the substrate-binding cleft of the enzymes, which was supported by specific interaction with amino acids from the active site, such as His48 for PLA2s and Glu143 for the metalloproteinase. In addition, varespladib and CP471474 also showed interaction with residues from the hydrophobic channel in PLA2s and substrate binding subsites in the SVMP. Our results suggest a synergistic action of the mixed inhibitors and show the potential of varespladib, CP471474, and their mixture to generate new treatments for snakebite envenoming with application in the field or as antivenom co-adjuvants.

Synthetic Inhibitors of Snake Venom Enzymes: Thioesters Derived from 2-Sulfenyl Ethylacetate.

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.