Institute of Molecular Virology, Ulm University Medical Center, 89081, Ulm, Germany.
Core Facility Functional Peptidomics, Ulm University Medical Center, 89081, Ulm, Germany.
Sci Rep. 2020 Sep 29;10(1):16036. doi: 10.1038/s41598-020-73012-4.
C-X-C chemokine receptor type 4 (CXCR4) is involved in several intractable disease processes, including HIV infection, cancer cell metastasis, leukemia cell progression, rheumatoid arthritis, asthma and pulmonary fibrosis. Thus, CXCR4 represents a promising drug target and several CXCR4 antagonizing agents are in preclinical or clinical development. Important parameters in drug lead evaluation are determination of binding affinities to the receptor and assessment of their stability and activity in plasma or blood of animals and humans. Here, we designed a microtiter plate-based CXCR4 antibody competition assay that enables to measure inhibitory concentrations (IC values) and affinity constants (K values) of CXCR4 targeting drugs. The assay is based on the observation that most if not all CXCR4 antagonists compete with binding of the fluorescence-tagged CXCR4 antibody 12G5 to the receptor. We demonstrate that this antibody-competition assay allows a convenient and cheap determination of binding affinities of various CXCR4 antagonists in living cells within just 3 h. Moreover, the assay can be performed in the presence of high concentrations of physiologically relevant body fluids, and thus is a useful readout to evaluate stability (i.e. half-life) of CXCR4 ligands in serum/plasma, and even whole human and mouse blood ex vivo. Thus, this optimized 12G5 antibody-competition assay allows a robust and convenient determination and calculation of various important pharmacological parameters of CXCR4 receptor-drug interaction and may not only foster future drug development but also animal welfare by reducing the number of experimental animals.
C-X-C 趋化因子受体 4（CXCR4）参与多种难治性疾病过程，包括 HIV 感染、癌细胞转移、白血病细胞进展、类风湿关节炎、哮喘和肺纤维化。因此，CXCR4 代表了一个有前途的药物靶点，几种 CXCR4 拮抗药物正在临床前或临床开发中。药物先导评估中的重要参数是确定与受体的结合亲和力，并评估其在动物和人类血浆或血液中的稳定性和活性。在这里，我们设计了一种基于微孔板的 CXCR4 抗体竞争测定法，可用于测量 CXCR4 靶向药物的抑制浓度（IC 值）和亲和力常数（K 值）。该测定法基于这样的观察结果，即大多数（如果不是全部）CXCR4 拮抗剂与荧光标记的 CXCR4 抗体 12G5 与受体的结合竞争。我们证明，这种抗体竞争测定法允许在 3 小时内方便且廉价地测定各种 CXCR4 拮抗剂在活细胞中的结合亲和力。此外，该测定法可在生理相关体液的高浓度下进行，因此是评估 CXCR4 配体在血清/血浆中稳定性（即半衰期）的有用指标，甚至可在体外检测全人血和鼠血。因此，这种优化的 12G5 抗体竞争测定法可用于稳健、方便地测定和计算 CXCR4 受体-药物相互作用的各种重要药理学参数，不仅可以通过减少实验动物的数量来促进未来的药物开发，还可以促进动物福利。
