Department of Systems Biology, Harvard Medical School, Boston, MA 02115;
Center for Systems Biology, Massachusetts General Hospital, Boston, MA 02114.
Proc Natl Acad Sci U S A. 2018 Nov 27;115(48):E11406-E11414. doi: 10.1073/pnas.1800047115. Epub 2018 Nov 14.
Drug receptor site occupancy is a central pharmacology parameter that quantitatively relates the biochemistry of drug binding to the biology of drug action. Taxanes and epothilones bind to overlapping sites in microtubules (MTs) and stabilize them. They are used to treat cancer and are under investigation for neurodegeneration. In cells, they cause concentration-dependent inhibition of MT dynamics and perturbation of mitosis, but the degree of site occupancy required to trigger different effects has not been measured. We report a live cell assay for taxane-site occupancy, and relationships between site occupancy and biological effects across four drugs and two cell lines. By normalizing to site occupancy, we were able to quantitatively compare drug activities and cell sensitivities independent of differences in drug affinity and uptake/efflux kinetics. Across all drugs and cells tested, we found that inhibition of MT dynamics, postmitotic micronucleation, and mitotic arrest required successively higher site occupancy. We also found interesting differences between cells and drugs, for example, insensitivity of the spindle assembly checkpoint to site occupancy. By extending our assay to a mouse xenograft tumor model, we estimated the initial site occupancy required for paclitaxel to completely prevent tumor growth as 80%. The most important cellular action of taxanes for cancer treatment may be formation of micronuclei, which occurs over a broad range of site occupancies.
药物受体占据是一个重要的药理学参数，它将药物结合的生物化学与药物作用的生物学定量联系起来。紫杉烷类和埃坡霉素类药物结合到微管（MTs）的重叠部位并稳定它们。它们被用于治疗癌症，并正在进行神经退行性疾病的研究。在细胞中，它们会导致 MT 动力学的浓度依赖性抑制和有丝分裂的扰动，但引发不同效应所需的占据程度尚未被测量。我们报告了一种用于紫杉烷类药物占据的活细胞检测方法，并报告了四种药物和两种细胞系之间药物占据与生物学效应之间的关系。通过将占据程度归一化，我们能够定量比较药物活性和细胞敏感性，而不受药物亲和力和摄取/流出动力学差异的影响。在所有测试的药物和细胞中，我们发现 MT 动力学抑制、有丝分裂后微核形成和有丝分裂阻滞需要逐渐增加的占据程度。我们还发现了细胞和药物之间有趣的差异，例如，纺锤体组装检查点对占据程度不敏感。通过将我们的检测方法扩展到小鼠异种移植肿瘤模型，我们估计紫杉醇完全阻止肿瘤生长所需的初始占据程度为 80%。紫杉烷类药物治疗癌症的最重要的细胞作用可能是形成微核，这种现象发生在占据程度较宽的范围内。
