Schullek J R, Butler J H, Ni Z J, Chen D, Yuan Z
Affymax Research Institute, 4001 Miranda Avenue, Palo Alto, California, 94304, USA.
Anal Biochem. 1997 Mar 1;246(1):20-9. doi: 10.1006/abio.1996.9958.
A novel, miniaturized high-throughput screening format is described for assay of combinatorial libraries generated on beads. This approach, which is ideally suited to encoded libraries synthesized on beads, utilizes the photolytic cleavage of individual compounds into a high-density well array (>6500 wells within a standard 96-well microtiter plate footprint) with well volumes as low as 0.37 microl. As a model study, an encoded dipeptide library (324 members) acylated with isobutyl succinate was assayed using this format to search for potential inhibitors of matrilysin, a member of the matrix metalloproteinase superfamily. In situ release of compounds from solid support was accomplished by photochemical cleavage after beads and enzyme were distributed to the wells. After the addition of a fluorogenic substrate to the array, the extent of enzyme inhibition and identification of active compounds was quantitated by imaging of the fluorescence emission upon uv irradiation. The structure-activity relationship data generated from the identified inhibitors in this study corroborate previous findings, thus validating the utility of this approach as a means of high-throughput screening of bead-based libraries.
本文描述了一种新型的、小型化的高通量筛选方法，用于分析在珠子上生成的组合文库。这种方法非常适合于在珠子上合成的编码文库，它利用光解作用将单个化合物裂解到高密度孔阵列中（在标准96孔微量滴定板的面积内有>6500个孔），孔体积低至0.37微升。作为一个模型研究，使用这种方法对一个用异丁基琥珀酸酰化的编码二肽文库（324个成员）进行分析，以寻找基质金属蛋白酶超家族成员基质溶素的潜在抑制剂。在珠子和酶被分配到孔中后，通过光化学裂解实现化合物从固体支持物上的原位释放。在向阵列中加入荧光底物后，通过对紫外线照射下的荧光发射进行成像来定量酶抑制程度和鉴定活性化合物。本研究中从鉴定出的抑制剂生成的构效关系数据证实了先前的发现，从而验证了这种方法作为基于珠子的文库高通量筛选手段的实用性。
