Wroblewski Victor J, Witcher Derrick R, Becker Gerald W, Davis Kelly A, Dou Shenshen, Micanovic Radmila, Newton Christy M, Noblitt Timothy W, Richardson John M, Song Ho Yeong, Hale John E
Department of Drug Disposition Development/Commercialization, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285, USA.
Biochem Pharmacol. 2003 Feb 15;65(4):657-67. doi: 10.1016/s0006-2952(02)01612-x.
Fas ligand (FasL) and Fas receptor are members of the tumor necrosis factor (TNF) receptor and ligand family that play an important role in regulating apoptosis in normal physiology. Decoy receptor 3 (DcR3) is a novel member of the TNF receptor superfamily, which binds to and blocks the activities of the ligands FasL and LIGHT. We have demonstrated that DcR3 was degraded rapidly to a major circulating metabolic fragment after subcutaneous administration in primates and mice. This fragment was also generated in subcutaneous tissue homogenate in vitro. Mass spectrometry and N-terminal sequencing indicated that DcR3 was proteolytically cleaved between R218 and A219 in the primary sequence to yield the fragment DcR3(1-218). While retaining its ability to bind LIGHT and inhibit LIGHT-mediated activities, DcR3(1-218) no longer bound FasL and did not inhibit FasL-mediated apoptosis in vitro. The primary sequence of DcR3 was molecularly engineered, changing the arginine residue at position 218 to glutamine to generate an analog, DcR3(R218Q), which we termed FLINT (LY498919). We demonstrated that FLINT was more stable to proteolytic degradation in vitro and in vivo and maintained its activity against both soluble FasL and soluble LIGHT in vitro. As a result, the modification in the sequence of DcR3 to produce FLINT (LY498919) should result in a pharmacologically superior molecule in the therapeutic intervention of diseases in which the pathogenesis is linked to FasL-mediated apoptotic or inflammatory events.
Fas配体（FasL）和Fas受体是肿瘤坏死因子（TNF）受体和配体家族的成员，在正常生理过程中调节细胞凋亡方面发挥着重要作用。诱骗受体3（DcR3）是TNF受体超家族的一个新成员，它能结合并阻断配体FasL和LIGHT的活性。我们已经证明，在灵长类动物和小鼠皮下给药后，DcR3迅速降解为一种主要的循环代谢片段。这种片段在体外皮下组织匀浆中也会产生。质谱分析和N端测序表明，DcR3在一级序列中的R218和A219之间被蛋白水解切割，产生片段DcR3(1 - 218)。虽然保留了结合LIGHT和抑制LIGHT介导活性的能力，但DcR3(1 - 218)不再结合FasL，并且在体外不抑制FasL介导的细胞凋亡。对DcR3的一级序列进行分子工程改造，将第218位的精氨酸残基替换为谷氨酰胺，生成一种类似物DcR3(R218Q)，我们将其命名为FLINT（LY498919）。我们证明，FLINT在体外和体内对蛋白水解降解更稳定，并且在体外对可溶性FasL和可溶性LIGHT均保持其活性。因此，对DcR3序列进行改造以产生FLINT（LY498919），应该会在治疗发病机制与FasL介导的凋亡或炎症事件相关的疾病中产生一种药理学上更优越的分子。
