Fan Xiaomin, Venegas Ruben, Fey Robert, van der Heyde Henri, Bernard Mark A, Lazarides Elias, Woods Catherine M
AvantGen, Inc., 9924 Mesa Rim Rd, San Diego, California 92121, USA.
Pharm Res. 2007 May;24(5):868-79. doi: 10.1007/s11095-007-9238-z. Epub 2007 Mar 22.
The goals in this study were several-fold. First, to optimize the in vivo phage display methodology by incorporating phage pharmacokinetic properties, to isolate peptides that target the brain microvasculature, and then to build focused libraries to obtain structure activity relationship information in vivo to identify the optimal targeting motif.
The blood pharmacokinetics of filamentous and T7 phage were evaluated to choose the optimal platform. A randomized peptide library with a motif CX(10)C was constructed in T7 phage and used for in vivo panning. Focused peptide libraries around each structural element of the brain-specific peptide were constructed to perform kinetic structure activity relationship (kSAR) analysis in vivo. To determine potential function, sepsis was induced in mice by LPS administration and four hours later the effect of GST-peptide on adhesion of rhodamine-labelled lymphocytes or CFDA-labelled platelets to pial microvasculature was observed by intravital microscopy.
The blood phamacokinetics of T7 was rapid (half-life of 12 min) which aids the clearance of non-specific phage. In vivo panning in brain enriched for isolates expressing the motif CAGALCY. Kinetic analysis of focused libraries built around each structural element of the peptide provided for rapid pharmacophore mapping. The computer modeling data suggested the peptide showed similarities to peptide mimetics of adhesion molecule ligands. GST-CAGALCY but not GST control protein was able to inhibit the rolling and adhesion of labeled platelets to inflamed pial vasculature. GST-CAGALCY had no effect on lymphocyte adhesion.
Incorporating normal blood phamacokinetics of T7 phage into in vivo phage display improves the ability to recover targeting peptide motifs and allows effective lead optimization by kSAR. This approach led to the isolation of a brain-specific peptide, CAGALCY, which appears to function as an effective antagonist of platelet adhesion to activated pial microvasculature.
本研究有多个目标。首先，通过纳入噬菌体药代动力学特性来优化体内噬菌体展示方法，分离靶向脑微血管的肽，然后构建聚焦文库以在体内获得构效关系信息，从而确定最佳靶向基序。
评估丝状噬菌体和T7噬菌体的血液药代动力学，以选择最佳平台。在T7噬菌体中构建具有CX(10)C基序的随机肽文库，并用于体内淘选。围绕脑特异性肽的每个结构元件构建聚焦肽文库，以在体内进行动力学构效关系（kSAR）分析。为了确定潜在功能，通过给予LPS在小鼠中诱导脓毒症，4小时后通过活体显微镜观察GST-肽对罗丹明标记的淋巴细胞或CFDA标记的血小板与软脑膜微血管粘附的影响。
T7的血液药代动力学很快（半衰期为12分钟），这有助于清除非特异性噬菌体。在脑中进行的体内淘选富集了表达CAGALCY基序的分离株。围绕肽的每个结构元件构建的聚焦文库的动力学分析提供了快速的药效团图谱。计算机建模数据表明该肽与粘附分子配体的肽模拟物相似。GST-CAGALCY而非GST对照蛋白能够抑制标记血小板向炎症软脑膜血管的滚动和粘附。GST-CAGALCY对淋巴细胞粘附没有影响。
将T7噬菌体的正常血液药代动力学纳入体内噬菌体展示可提高回收靶向肽基序的能力，并通过kSAR实现有效的先导优化。这种方法导致分离出一种脑特异性肽CAGALCY，它似乎作为血小板与活化软脑膜微血管粘附的有效拮抗剂发挥作用。
