Search

Citrobacter freundii methionine γ-lyase (MGL), in addition to the physiological reaction, catalyzes the β-elimination reaction of S-alk(en)yl-L-cysteine sulfoxides to yield thiosulfinates, which have antibacterial activity. We have obtained the mutant form C115H MGL, which cleaves S-alk(en)yl-L-cysteine sulfoxides more effectively than the wild type enzyme does. The binary system MGL/S-alk(en)yl-L-cysteine sulfoxides may be considered as a new pharmacological pair in enzyme prodrug therapy (EPT). Despite of the successful application of this pair in antibacterial studies in vitro, in vivo experiments may lead to several problems typical of therapeutic proteins including a relatively short-lasting biological activity. To circumvent these problems, we have investigated several approaches to improve safety and efficacy of the enzyme component of the pharmacological pair. This included covalent attachment of poly(ethylene glycol) to the enzyme, its encapsulation in liposomes and polymeric vesicles (PICsomes). The steady-state and pharmacokinetic parameters of modified/encapsulated enzyme were determined. It was demonstrated that the encapsulation in PICsomes prolongs in vivo stability of C115H MGL to over 42 h compared to PEGylated enzyme (3 h). Antibacterial activity of binary system ("pharmacological pair") modified/encapsulated enzyme/S-alk(en)yl-L-cysteine sulfoxides was tested and remained the same as for the naked enzyme. Thus, the usage of MGL-loaded PICsomes as enzymatic nanoreactors in ETP to produce antimicrobial thiosulfinates is promising., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Human ovarian cancer cells specifically bind the isoflavone daidzein. A chemical conjugate between daidzein and the garlic enzyme alliinase was prepared. The conjugate specifically bound to ovarian cancer cells and upon addition of the prodrug alliin, it effectively produced cytotoxic allicin molecules which killed the cancer cells. In vivo targeting and antitumor effect was confirmed by NIR and bioluminescence imaging using daidzein-alliinase-CyTE-777 conjugates and luciferase-expressing ovarian cancer cells. Co-localization of the fluorescent conjugate with bioluminescence was observed for intraperitoneal tumors while nonconjugated alliinase did not accumulate. Biodistribution studies with Europium-labeled conjugate revealed a five fold higher uptake in tumors as compared to other tissues. Treatment of tumor bearing mice with daidzein-alliinase and alliin effectively attenuated tumor progression during the first 12 days while a 5-fold increase in bioluminescence was detected in placebo-treated animals. Autopsy revealed only small individual foci of luminescence at the site of tumor cells inoculation. Histological examination of organs and tissues did not reveal any additional foci of carcinoma or signs of toxicity. These results suggest that the targeted alliinase conjugates in the presence of alliin, generated therapeutically effective levels of allicin which were capable of suppressing tumor progression of intraperitoneal ovarian cancer in an animal model.