Institute of Microstructure Technology, Karlsruhe Institute of Technology, Hermann-von-Helmholtz-Platz 1, D-76344 Eggenstein-Leopoldshafen, Germany.
Department of Biology, CH-8093 Zürich, Switzerland.
Anal Chem. 2024 Jun 18;96(24):9859-9865. doi: 10.1021/acs.analchem.4c00333. Epub 2024 Jun 3.
In drug discovery, ligands are sought that modulate the (mal-)function of medicinally relevant target proteins. In order to develop new drugs, typically a multitude of potential ligands are initially screened for binding and subsequently characterized for their affinity. Nuclear magnetic resonance (NMR) is a well-established and highly sensitive technology for characterizing such interactions. However, it has limited throughput, because only one sample can be measured at a time. In contrast, magnetic resonance imaging (MRI) is inherently parallel and MR parameters can conveniently be encoded in its images, potentially offering increased sample throughput. We explore this application using a custom-built 9-fold sample holder and a F-MRI coil. With this setup, we show that ligand binding can be detected by -weighted F-MRI using 4-(trifluoromethyl)benzamidine (TFBA) and trypsin as the reporter ligand and target protein, respectively. Furthermore, we demonstrate that the affinity of nonfluorinated ligands can be determined in a competition format by monitoring the dose-dependent displacement of TFBA. By comparing F--weighted MR images of TFBA in the presence of different benzamidine (BA) concentrations-all recorded in parallel-the affinity of BA could be derived. Therefore, this approach promises parallel characterization of protein-ligand interactions and increased throughput of biochemical assays, with potential for increased sensitivity when combined with hyperpolarization techniques.
在药物发现中，人们寻求能够调节具有医学相关性的靶蛋白（异常）功能的配体。为了开发新药，通常会对大量潜在的配体进行结合筛选，然后对其亲和力进行特征描述。核磁共振（NMR）是一种成熟且高度灵敏的技术，可用于对这类相互作用进行特征描述。然而，它的通量有限，因为一次只能测量一个样本。相比之下，磁共振成像（MRI）本质上是并行的，并且可以方便地在其图像中对 MR 参数进行编码，这可能会提高样本通量。我们使用定制的 9 倍样本架和 F-MRI 线圈来探索这种应用。使用这种设置，我们证明了使用 4-（三氟甲基）苯甲脒（TFBA）和胰蛋白酶分别作为报告配体和靶蛋白，可以通过加权 F-MRI 检测配体结合。此外，我们还证明了可以通过监测 TFBA 的剂量依赖性置换，以竞争格式确定非氟化配体的亲和力。通过比较存在不同苯甲脒（BA）浓度的 TFBA 的 F--加权 MR 图像——这些图像都是并行记录的——可以推导出 BA 的亲和力。因此，这种方法有望实现蛋白质-配体相互作用的并行特征描述，并提高生化分析的通量，与极化技术结合时具有提高灵敏度的潜力。
