Evaluation of cyclooxygenase-2 inhibitors using pulsed ultrafiltration mass spectrometry
Evaluation of cyclooxygenase-2 inhibitors using pulsed ultrafiltration mass spectrometry
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DOI：
10.1021/ac0000980
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发表时间：
2000-08-15
影响因子：
7.4
通讯作者：
van Breemen, RB
中科院分区：
文献类型：
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作者：
Nikolic, D;Habibi-Goudarzi, S;van Breemen, RB
由于选择性抑制环氧合酶（考克斯-2）的可诱导形式可以保留经典非甾体类抗炎药的所有益处，同时避免与抑制组成型异构体考克斯-1相关的主要副作用，因此考克斯-2已成为发现和开发新抗炎药的重要靶点。为了帮助这种选择性抑制剂的发现和表征，我们应用了基于质谱的筛选技术，脉冲超滤质谱，使用考克斯-2作为目标。在盲法研究中，评价了18个富含一种或多种考克斯-2抑制剂的样品。供试品的基质由DMSO、植物提取物或细菌发酵液提取物的DMSO溶液组成。在试验期间，样品的组成未知，考克斯-2抑制剂的浓度也未知。将可溶性重组形式的人考克斯-2与各样品一起孵育，然后将各混合物的等分试样注入装有30 000 MW截留超滤膜的搅拌超滤室中。洗去未结合和弱结合的化合物后，使用酸化的10%甲醇溶液破坏配体-受体复合物。将释放的配体捕获在Cls柱上，然后使用液相色谱-负离子电喷雾质谱法以捕获柱作为HPLC柱进行鉴定。植物基质和发酵液提取物均未发现干扰测定。在每个样品中鉴定了两个或三个考克斯-2的配体，其包括极性和非极性化合物以及IC 50值范围为100 μ M至10 nM的抑制剂。
Since selective inhibition of the inducible form of cyclooxygenase (COX-2) might retain all the benefits of classical nonsteroidal antiinflammatory agents while avoiding the major side effects associated with inhibition of the constitutive isoform COX-1, COX-2 has become an important target for the discovery and development of new antiinflammatory drugs. To aid in the discovery and characterization of such selective inhibitors, we have applied a mass spectrometry-based screening technique, pulsed ultrafiltration mass spectrometry, using COX-2 as the target. In a blind study, 18 samples enriched with one or more inhibitors of COX-2 were evaluated. The matrixes for the test samples consisted of DMSO, r DMSO solutions of a plant extract, or a bacterial fermentation broth extract. The composition of the samples was unknown during the assays, as were the concentrations of the COX-2 inhibitors. A soluble recombinant form of human COX-2 was incubated with each sample, and then an aliquot of each mixture was injected into the stirred ultrafiltration chamber fitted with a 30 000 MW cutoff ultrafiltration membrane. After the unbound and weakly bound compounds were washed away, the ligand-receptor complexes were disrupted using an acidified 10% methanol solution. The released ligands were trapped on a Cls cartridge and then identified using liquid chromatography-negative ion electrospray mass spectrometry with the trapping cartridge as the HPLC column. Neither the plant matrix nor the fermentation broth extract were found to interfere with the assay. Two or three ligands for COX-2 were identified in each sample, which included polar and nonpolar compounds and inhibitors with IC50 values ranging from 100 mu M to 10 nM.