Cleavage of disulfide-linked fetuin-bisphosphonate conjugates with three physiological thiols.
An effective therapeutic agent for treatment of bone diseases is expected to exhibit a high affinity to bone. Conjugating proteins to bisphosphonates (BPs), a class of molecules with an exceptional affinity to bone mineral hydroxyapatite (HA), is a feasible means to impart such a bone affinity. Protein-BP conjugates with cleavable linkages, which allow protein release from the mineral, are preferable over conjugates with stable linkages. To this end, 2-(3-mercaptopropylsulfanyl)-ethyl-1,1-bisphosphonic acid (thiolBP) was conjugated onto fetuin, a model protein, using N-succinimidyl-3-(2-pyridyldithio)propionate to create disulfide-linked conjugates. Although the fetuin-thiolBP conjugates were stable under aqueous conditions, the disulfide linkage was readily cleaved in the presence of the physiological thiols l-cysteine, dl-homocysteine, and l-glutathione. dl-Homocysteine exhibited the highest cleavage of the disulfide linkage among these thiols. The imparted bone affinity as a result of thiolBP conjugation, as assessed by HA binding in vitro, was eliminated upon cleavage of the disulfide linkage. The cleavage of the conjugates bound to HA was as effective as the conjugate cleavage in solution, and even more so at high concentrations of l-glutathione. In conclusion, disulfide-linked fetuin-thiolBP conjugates exhibited a high affinity to HA, which was readily lost upon cleavage with thiols found in physiological milieu.
Zhang
Sufeng
S
Department of Chemical and Materials Engineering, Faculty of Engineering, University of Alberta, Edmonton, AB T6G 2G6, Canada.
Wright
Jennifer E I
JE
Bansal
Geeti
G
Cho
Philip
P
Uludag
Hasan
H
D001672:Biocompatible Materials / Q000737:chemistry; D001847:Bone Diseases / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D004164:Diphosphonates / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004337:Drug Carriers / Q000737:chemistry; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism; D006710:Homocysteine / Q000737:chemistry; D006801:Humans; D006882:Hydroxyapatites / Q000737:chemistry; D008956:Models, Chemical; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013997:Time Factors; D014302:Trinitrobenzenesulfonic Acid / Q000737:chemistry; D000509:alpha-Fetoproteins / Q000737:chemistry*
D001672:Biocompatible Materials; D003432:Cross-Linking Reagents; D004164:Diphosphonates; D004220:Disulfides; D004337:Drug Carriers; D006882:Hydroxyapatites; D013438:Sulfhydryl Compounds; D000509:alpha-Fetoproteins; D006710:Homocysteine; D014302:Trinitrobenzenesulfonic Acid; D004228:Dithionitrobenzoic Acid; D005978:Glutathione; D003545:Cysteine