Studies of FSH,FSH-analogues and the FSH-receptor

描述(申请人提供)：修饰hFSH的碳水化合物含量对体内激素的生理特性有显著影响。到目前为止，已经研究了一种这样的修饰的初步特征：hFSH从hCG的β-亚基连接到CTP上(作为单链多肽或作为与α-亚基相关的二聚体)。这种修饰增加了激素的半衰期和体内生物活性。O-连接碳水化合物的数量减少或增加的影响，对这些部分间距的影响，以及N-连接糖在这种能力中的潜在作用，尚未得到彻底的研究。这些研究将产生有关碳水化合物生理学一般原理的重要数据。通过确定差异糖基化的作用，可以通过碳水化合物操作开发治疗衍生物，这种操作可能适用于所有糖蛋白激素以及一般的重组蛋白。此外，我们有初步数据表明，生产由hFSH与生长因子连接的双功能hFSH类似物是可行的。在体外和体内卵巢水平的双功能都得到了证实。这使得对各种靶向生长因子在卵巢生理上的作用的持续和扩大的研究成为可能。对卵泡发育的协同作用也可能对需要卵巢刺激的不孕症妇女有治疗意义。FSH及其受体之间的界面结构知识对于开发干扰受体活性的方法是必不可少的。我们计划确定hFSH受体的胞外域(ECD)的结构，该受体是G蛋白偶联受体超家族的成员。糖蛋白激素受体结合区的三维表示将首次被阐明。配体结合后受体的结构动力学和构象变化将为我们理解受体激活所需的条件提供至关重要的数据。总之，本申请中描述的研究将极大地加深我们对促性腺激素生物学、各种辅助因子在卵巢生理中的作用的了解，并引发改变重组蛋白的药效学和药动学的新方法。

DESCRIPTION (provided by applicant): Modification of the carbohydrate content of hFSH has significant effect on the physiologic properties of the hormone in vivo. To date, preliminary characterization of one such modification has been investigated: hFSH tethered to the CTP from the beta-subunit of hCG (either as a single-chain peptide or as a dimer associated with alpha-subunit). This modification increases the half-life and in vivo bioactivity of the hormone. The effect of fewer or greater numbers of O-linked carbohydrates, the impact on spacing of these moieties, and the potential role of N-linked sugars in this capacity have yet to be thoroughly investigated. These studies will generate important data as to general principles for carbohydrate physiology. By defining the role of differential glycosylation, therapeutic derivatives may be developed through carbohydrate manipulations that may be applicable to all the glycoprotein hormones as well as recombinant proteins in general. In addition, we have preliminary data indicating the feasibility of producing bifunctional hFSH analogues consisting of hFSH tethered to a growth factor. Bifunctionality, both in vitro and in vivo at the level of the ovary was confirmed. This allows for continued and expanded studies on the role of various targeted growth factors on ovarian physiology. Synergistic action on follicular development may also have therapeutic implications for women requiring ovarian stimulation for infertility. Structural knowledge of the interface between FSH and its receptor is essential to developing methods to perturb receptor activity. We plan to determine the structure of the extracellular domain (ECD) of the hFSH receptor, a member of the G protein- coupled receptor superfamily. A three-dimensional representation of the binding region of a glycoprotein hormone receptor will be elucidated for the first time. The structural dynamics and conformational changes in the receptor following ligand binding will provide data crucial to our understanding of the conditions requisite for receptor activation. Together, the studies described in this application will greatly enhance our understanding of gonadotropin biology, the role of various cofactors in ovarian physiology and elicit new methods for altering pharmacodynamics and pharmacokinetics of recombinant proteins.