Gaus Hans, Miller Colton M, Seth Punit P, Harris Edward N
Department of Medicinal Chemistry , Ionis Pharmaceuticals , Carlsbad , California 92010 , United States.
Department of Biochemistry , University of Nebraska , Lincoln , Nebraska 68588 , United States.
Biochemistry. 2018 Apr 10;57(14):2061-2064. doi: 10.1021/acs.biochem.8b00126. Epub 2018 Mar 30.
The Stabilin receptors are systemic clearance receptors for some classes of chemically modified nucleic acid therapeutics. In this study, the recombinant human secreted ecto-domain of the small isoform of Stabilin-2 (s190) was purified from cell culture and evaluated for direct binding with a multitude of antisense oligonucleotides (ASOs) using a fluorescence polarization-based assay. The tested ASOs varied in their backbone composition, modification of the ribose 2' position, overall length of the oligo, and sequence of the nucleotide bases. A fully phosphorothioate (PS) ASO with a 5-10-5 pattern of flanking 2'- O-methoxyethyl modifications was then used to test the effects of pH and salt concentration on receptor binding. These tests concluded that the PS backbone was the primary determinant for ASO binding and that decreasing pH and increasing salt generally increased the rate of ligand dissociation and fit within the biological parameters expected of a constitutive recycling receptor. These results will be useful in the rational design of therapeutic oligonucleotides for enhancing their affinity or avoidance of the Stabilin receptors.
Stabilin受体是某些化学修饰核酸治疗剂的全身清除受体。在本研究中，从细胞培养物中纯化了重组人分泌型Stabilin-2小异构体的胞外域（s190），并使用基于荧光偏振的测定法评估其与多种反义寡核苷酸（ASO）的直接结合。所测试的ASO在其骨架组成、核糖2'位修饰、寡核苷酸的全长和核苷酸碱基序列方面各不相同。然后使用具有5-10-5模式的侧翼2'-O-甲氧基乙基修饰的全硫代磷酸酯（PS）ASO来测试pH和盐浓度对受体结合的影响。这些测试得出结论，PS骨架是ASO结合的主要决定因素，降低pH和增加盐浓度通常会增加配体解离速率，并且符合组成型循环受体预期的生物学参数。这些结果将有助于合理设计治疗性寡核苷酸，以增强其对Stabilin受体的亲和力或避免与该受体结合。
