Hu Bin, Xiong Bing, Qiu Bei-ying, Li Xin, Yu Hai-ping, Xiao Kun, Wang Xin, Li Jia, Shen Jing-kang
State Key Laboratory of Drug Research and National Center for Drug Screening, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China.
Acta Pharmacol Sin. 2006 Dec;27(12):1586-93. doi: 10.1111/j.1745-7254.2006.00432.x.
To develop probes for detecting the binding specificity between beta-secretase and substrate, and provide reliable biological activity data for further researching encircling substrate-based inhibitors.
To prepare the inhibitors, the hydroxyethylene (HE) segment including P1 and P1'was synthesized after multi-step reactions; the combination of all segments was then completed through solid phase synthesis. Recombinant human beta-secretase ectodomain (amino acid residues 1-460) was expressed as a secreted protein with a C-terminal His tag in insect cells using baculovirus infection, and all compounds were evaluated in this beta-secretase enzyme assay. In order to understand the interaction in detail, the theoretical methods, namely molecular dynamics (MD) simulation and molecular mechanics-generalized-born surface area (MM-GBSA) analysis, were performed on the complex of beta-secretase and OM99-2 to obtain the geometrical and energetical information.
We designed and constructed a positional scanning combinatorial library including 16 compounds; all members of the library were synthesized based on HE dipeptide isostere. Structure-activity relationship studies at the P4-P1 and P1' -P4'positions led to the discoveries of P and P'sides binding specificity and potent inhibitors 14, 18, and 22. The binding free energy on the whole system and every residue were compared to the biological assay result.
The removal of P4' yielded inhibitor 22 (A3 *B2) with high potency; further truncation of P3'gave inhibitor 18 (A3 *B1) with equal activity, implying that the right side of the inhibitors play a less important role and could be easily simplified, while change on the P side may cause substantial results.
开发用于检测β-分泌酶与底物之间结合特异性的探针，并为进一步研究基于底物的抑制剂提供可靠的生物活性数据。
为制备抑制剂，经过多步反应合成了包含P1和P1'的羟乙烯（HE）片段；然后通过固相合成完成所有片段的组合。使用杆状病毒感染在昆虫细胞中表达重组人β-分泌酶胞外域（氨基酸残基1-460），其为带有C末端组氨酸标签的分泌蛋白，并在该β-分泌酶酶分析中评估所有化合物。为了详细了解相互作用，对β-分泌酶与OM99-2的复合物进行了理论方法，即分子动力学（MD）模拟和分子力学-广义Born表面积（MM-GBSA）分析，以获得几何和能量信息。
我们设计并构建了一个包含16种化合物的位置扫描组合文库；文库的所有成员均基于HE二肽等排体合成。在P4-P1和P1'-P4'位置的构效关系研究导致发现了P和P'侧的结合特异性以及强效抑制剂14、18和22。将整个系统和每个残基上的结合自由能与生物学分析结果进行了比较。
去除P4'得到高效抑制剂22（A3B2）；进一步截短P3'得到活性相当的抑制剂18（A3B1），这意味着抑制剂的右侧作用较小且易于简化，而P侧的变化可能会产生实质性结果。
