Zhang Sufeng, Wright Jennifer E I, Bansal Geeti, Cho Philip, Uludag Hasan
Department of Chemical and Materials Engineering, Faculty of Engineering, University of Alberta, Edmonton, AB T6G 2G6, Canada.
Biomacromolecules. 2005 Sep-Oct;6(5):2800-8. doi: 10.1021/bm050273s.
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.
一种用于治疗骨疾病的有效治疗剂有望对骨表现出高亲和力。将蛋白质与双膦酸盐（BP）偶联，双膦酸盐是一类对骨矿物质羟基磷灰石（HA）具有非凡亲和力的分子，是赋予这种骨亲和力的可行方法。具有可裂解连接键的蛋白质 - BP偶联物，能使蛋白质从矿物质中释放出来，比具有稳定连接键的偶联物更可取。为此，使用N - 琥珀酰亚胺基 - 3 - （2 - 吡啶基二硫代）丙酸酯将2 - （3 - 巯基丙基硫烷基） - 乙基 - 1,1 - 双膦酸（硫醇BP）偶联到胎球蛋白（一种模型蛋白质）上，以创建二硫键连接的偶联物。尽管胎球蛋白 - 硫醇BP偶联物在水性条件下是稳定的，但二硫键在生理硫醇L - 半胱氨酸、DL - 高半胱氨酸和L - 谷胱甘肽存在下很容易被裂解。在这些硫醇中，DL - 高半胱氨酸对二硫键的裂解作用最强。通过体外HA结合评估，硫醇BP偶联所赋予的骨亲和力在二硫键裂解后消失。与HA结合的偶联物的裂解与溶液中的偶联物裂解一样有效，在高浓度L - 谷胱甘肽时甚至更有效。总之，二硫键连接的胎球蛋白 - 硫醇BP偶联物对HA表现出高亲和力，在生理环境中被硫醇裂解后这种亲和力很容易丧失。
