title: Cytosolic beta-glycosidases for activation of glycoside prodrugs of daunorubicin.
abstract: Human cytosolic beta-glycosidase is a small monomeric enzyme that is active under physiological conditions, which might be ideal for enzyme-prodrug therapy. We have previously reported the synthesis of a galactoside (DNR-GlA3) and a glucoside (DNR-GsA3) prodrug of daunorubicin. In the present study, we established that cellular uptake of DNR-GlA3 and DNR-GsA3 was low in contrast to that of daunorubicin. Recombinant human beta-glycosidase converted both prodrugs to daunorubicin as shown by liquid chromatography. The kinetics of the conversion of DNR-GlA3 and DNR-GsA3 by human beta-glycosidase, however, was unfavorable as the K(m) values were, respectively, 3- and 6-fold higher than those of another mammalian beta-glycosidase of bovine origin. The V(max) values were, respectively, 3.3 and 8.5nmol/hr/mg as compared to 158.3 and 147.8nmol/hr/mg of the bovine enzyme. Treatment of OVCAR-3 cells with human beta-glycosidase (0.5U/mL) and 0.5 microM DNR-GlA3 or DNR-GsA3 resulted in, respectively, 86 and 81% cell growth inhibition, while the prodrugs alone inhibited growth to only 19 and 1%. Treatment of cells with the bovine enzyme and the prodrugs inhibited cell growth more efficiently. We conclude that the endogenous intracellular beta-glycosidase is not available for extracellular prodrug activation. Thus, the incorporation of the enzyme in enzyme-prodrug therapy might be an elegant approach to achieve tumor-specific prodrug conversion. The efficiency of glycoside prodrug conversion might be improved by design of a prodrug that is more readily activated by human beta-glycosidase or by evolution of the enzyme into a mutant form that displays high activity towards these prodrugs.
authors.affiliation: Department of Medical Oncology, Vrije Universiteit Medical Center, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands.
authors.forename: Michelle
authors.initials: M
authors.lastname: de Graaf
authors.forename: Herbert M
authors.initials: HM
authors.lastname: Pinedo
authors.forename: Razi
authors.initials: R
authors.lastname: Quadir
authors.forename: Hidde J
authors.initials: HJ
authors.lastname: Haisma
authors.forename: Epie
authors.initials: E
authors.lastname: Boven
mesh_terms: D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000378:metabolism / Q000494:pharmacology*; D001711:Biotransformation; D016466:CHO Cells; D019556:COS Cells; D002455:Cell Division / Q000187:drug effects; D006224:Cricetinae; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D003630:Daunorubicin / Q000378:metabolism / Q000494:pharmacology*; D005697:Galactosides / Q000378:metabolism; D005960:Glucosides / Q000378:metabolism; D006027:Glycosides / Q000378:metabolism*; D006801:Humans; D011355:Prodrugs / Q000378:metabolism / Q000494:pharmacology*; D001617:beta-Glucosidase / Q000378:metabolism*
chemical_list: D000903:Antibiotics, Antineoplastic; D005697:Galactosides; D005960:Glucosides; D006027:Glycosides; D011355:Prodrugs; D001617:beta-Glucosidase; D003630:Daunorubicin