Studies on cassava extracts have received increased attention due to their high content of cyanogenic compounds which have significant anti-cancer activities. In cassava, linamarin is hydrolyzed to form hydrogen cyanide, HCN, by a β-glucosidase, linamarase. Targeted HCN treatment can be used to destroy cancer cells in the human body. However, linamarin is not readily hydrolysable in human cells and its hydrolysis mechanism is not well understood. In this study we investigate the mechanism of enzymatic hydrolysis of linamarin and identify structural analogs of linamarin with better hydrolysis potential. The proposed hydrolysis pathway of linamarin follows a S<sub>N</sub>2 like mechanism to yield a sugar molecule and HCN. A human β-glucosidase with ~ 93% structural homology to the linamarase enzyme was selected for this study. Density functional theory (DFT) was used to evaluate the hydrolysis mechanism of linamarin by human β-glucosidase and compare the hydrolysability rates of its analogs. Each analog was found to pass through a half-chair like transition state (TS) in both mechanistic steps. Based on transition state theory (TST), we found that alpha anomers result in low activation barriers for hydrolysis while bulkiness and chemical nature of leaving groups define hydrolysabilities. Out of the linamarin derivatives studied, LIN7 (a β-analog) was found to have the lowest reaction barrier, thus explaining the feasibility to liberate hydrogen cyanide. Furthermore, the dissociation of the product was evaluated in the presence of water. Results suggested the spontaneous breakdown of compound into ketone and hydrogen cyanide. Meanwhile, molecular dynamic simulations validated the stability of linamarin within the reaction site and its strong interactions with the reacting residues. In the long term, this novel study will contribute valuable insight towards the drug discovery process for cancer treatment.

Identification of novel linamarin analogs as potent anticancer drugs: A computational approach

ACS Fall 2021

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