Department of Chemistry, The Scripps Research Institute, Scripps Florida, 130 Scripps Way, Jupiter, FL 33458, USA.
Bioorg Med Chem Lett. 2011 Sep 1;21(17):4960-4. doi: 10.1016/j.bmcl.2011.06.011. Epub 2011 Jun 16.
Synthetic equivalents of phosphoprotein-specific antibodies would be valuable reagents for biological research, since these antibodies can often be difficult to produce. Protein phosphorylation is thought to result in significant conformational changes in most substrate proteins. Therefore, one approach might be to simply screen combinatorial libraries for ligands to the phosphorylated state in the hope of isolating a ligand that binds to a pocket created by the conformational shift. In this study, we probe this strategy by screening a peptoid library for ligands to the phosphorylated form of the Brd4 chromatin adaptor and transcriptional coactivator protein. We find that peptoids with high selectivity for binding to the phosphorylation form of Brd4 can indeed be isolated in this screen. Moreover, these ligands do not bind promiscuously to other phospho-proteins. However, attempts to employ these reagents as antibody substitutes in an immunoaffinity purification-like application showed that they do not perform as well as bona fide antibodies and that significant optimization will be required. This study highlights the potential and current limitations of a naïve library screening strategy for phosphoprotein-specific antibody surrogates.
合成的磷酸化蛋白特异性抗体等价物将是生物研究中非常有价值的试剂，因为这些抗体通常很难生产。人们认为蛋白质磷酸化会导致大多数底物蛋白发生显著的构象变化。因此，一种方法可能是简单地筛选组合文库以寻找磷酸化状态的配体，希望能分离出与构象变化产生的口袋结合的配体。在这项研究中，我们通过筛选肽库来探测这种策略，以寻找 Brd4 染色质衔接子和转录共激活蛋白磷酸化形式的配体。我们发现，在这种筛选中确实可以分离出对 Brd4 磷酸化形式具有高选择性结合的肽。此外，这些配体不会随意结合其他磷酸化蛋白。然而，在免疫亲和纯化样应用中尝试将这些试剂用作抗体替代物的尝试表明，它们的性能不如真正的抗体，需要进行重大优化。这项研究突出了一种针对磷酸化蛋白特异性抗体替代物的简单文库筛选策略的潜在优势和当前局限性。
