Identification of Relaxin Receptor Antagonists

描述（由申请人提供）：松弛素是一种异二聚体肽激素，由怀孕和非怀孕女性的黄体、蜕膜和胎盘产生，在怀孕期间达到最高血浆水平。除了它在抑制子宫收缩活动中的作用外，松弛素已被证明可以诱导胶原重塑并随之软化产道（子宫颈和阴道）组织、乳腺的生长和分化以及血管的扩张。因此，松弛素在妊娠期生理适应的整体调节中起着重要作用。基于一级和二级结构特征，松弛素与胰岛素和胰岛素样生长因子（IGFs）作为结构同源物分组。与胰岛素类似，松弛素的前体形式前松弛素具有与胰岛素和IGF前体相似的结构域排列；成熟的松弛素似乎通过转化酶从前松弛素产生两链异二聚体。在人类中，除了两个几乎相同的松弛素基因外，还有五个松弛素家族基因，包括编码INSL3/RLF、INSIA/EPIL、INSL5/RIF2、INSL6/RIF1和relaxin3的基因。孕前期被动免疫抗松弛素抗体可减少宫颈的生长和延伸性，并扰乱啮齿动物的生育。此外，大量研究表明，血清松弛素是人类妊娠多个阶段早产风险的独立预测因子。这些早期的研究推动了这样一个命题，即通过受体拮抗剂或免疫降低松弛素的活性，与针对其他信号通路的传统方法相结合，对早产和分娩的治疗可能很重要。我们最近的研究已经证实松弛素可以激活两个孤儿G蛋白偶联受体LGR7和LGR8 （Hsu et al., 2002）。Science 295:671-4)以及靶细胞中camp依赖通路，而密切相关的relaxin3和INSL3分别是LGR7和LGR8的选择性激动剂。为了利用松弛素信号传导的这些新发现为生殖健康研究提供益处，我们建议筛选突变型重组松弛素拮抗剂，这些拮抗剂保留受体结合活性，但缺乏受体激活活性。一旦获得仅具有受体结合活性的突变肽，将在共处理实验中测试这些肽的拮抗作用，以确定突变肽是否能够拮抗LGR7和LGR8表达细胞中松弛素诱导的cAMP产生，并延缓孕鼠体内的分娩。这些拮抗剂可用于阻断早产时的松弛作用，为进一步研究LGR7和LGR8在生殖道生理中的作用提供了工具。

DESCRIPTION (provided by applicant): Relaxin is a heterodimeric peptide hormone produced by the corpus luteum, decidua, and placenta in pregnant and nonpregnant females attaining the highest plasma levels during pregnancy. Other than its role in the inhibition of uterine contractile activity, relaxin has been shown to effect the induction of collagen remodeling and the consequent softening of the tissues of the birth canal (cervix and vagina), growth and differentiation of the mammary gland, and dilation of the blood vessels. Thus, relaxin plays a significant role in the overall regulation of physiological adaptation during pregnancy. Based on: primary and secondary structural characteristics, relaxin was grouped with insulin and insulin-like growth factors (IGFs) as a structural homolog. Analogous to insulin, prorelaxin, the precursor form of relaxin, has a domain arrangement similar to insulin and IGF precursors; mature relaxin appears to be processed by convertases to generate a two-chain heterodimer from the prorelaxin. In addition to two almost identical relaxin genes in human, there are five additional relaxin family genes including those encoding INSL3/RLF, INSIA/EPIL, INSL5/RIF2, INSL6/RIF1, and relaxin3. Passive immunization with anti-relaxin antibodies during the anteparmm period reduces cervical growth and extensibility as well as disrupts birth in rodents. In addition, numerous studies have shown that serum relaxin is an independent predictor of the risk of preterm delivery at multiple stages of pregnancy in humans. These earlier studies have propelled the proposition that a reduction of relaxin activity through receptor antagonists or immunization could be important for the treatment of preterm labor and birth in conjunction with traditional methods targeting other signaling pathways. Our recent studies have established that relaxin activates two orphan G protein-coupled receptors, LGR7 and LGR8 (Hsu et al., 2002. Science 295:671-4) as well as the cAMP-dependent pathway in target cells, whereas the closely related relaxin3 and INSL3 are selective agonists for LGR7 and LGR8, respectively. To take advantage of these new findings in relaxin signaling for the benefit of reproductive health research, we propose to screen for mutant recombinant relaxin antagonists that retain receptor-binding activity while being devoid of receptor-activation activity. Once mutant peptides with only the receptor binding activity are obtained, the antagonistic effect of these peptides will be tested in co-treatment assays to determine whether the mutant peptides are capable of antagonizing the relaxin-induced cAMP production in LGR7- and LGR8- expressing cells as well as delaying parturition in pregnant rats in vivo. These antagonists could be used to block relaxin action during preterm labor as well as provide a tool for further research on the role of LGR7 and LGR8 in reproductive tract physiology.