Rehan Shahid, Ashok Yashwanth, Nanekar Rahul, Jaakola Veli-Pekka
Oulu Biocenter and Faculty of Biochemistry and Molecular Medicine, University of Oulu, P.O. Box 3000, FI-90014 Oulu, Finland.
Oulu Biocenter and Faculty of Biochemistry and Molecular Medicine, University of Oulu, P.O. Box 3000, FI-90014 Oulu, Finland.
Biochem Pharmacol. 2015 Dec 15;98(4):681-9. doi: 10.1016/j.bcp.2015.09.019. Epub 2015 Sep 30.
Many nucleoside transport inhibitors are in clinical use as anti-cancer, vasodilator and cardioprotective drugs. However, little is known about the binding energetics of these inhibitors to nucleoside transporters (NTs) due to their low endogenous expression levels and difficulties in the biophysical characterization of purified protein with ligands. Here, we present kinetics and thermodynamic analyses of inhibitor binding to the human equilibrative nucleoside transporter-1 (hENT1), also known as SLC29A1. Using a radioligand binding assay, we obtained equilibrium binding and kinetic rate constants of well-known NT inhibitors--[(3)H]nitrobenzylmercaptopurine ribonucleoside ([(3)H]NBMPR), dilazep, and dipyridamole--and the native permeant, adenosine, to hENT1. We observed that the equilibrium binding affinities for all inhibitors decreased whereas, the kinetic rate constants increased with increasing temperature. Furthermore, we found that binding is enthalpy driven and thus, an exothermic reaction, implying that the transporter does not discriminate between its inhibitors and substrates thermodynamically. This predominantly enthalpy-driven binding by four chemically distinct ligands suggests that the transporter may not tolerate diversity in the type of interactions that lead to high affinity binding. Consistent with this, the measured activation energy of [(3)H]NBMPR association was relatively large (20 kcal mol(-1)) suggesting a conformational change upon inhibitor binding. For all three inhibitors the enthalpy (ΔH°) and entropy (ΔS°) contributions to the reaction energetics were determined by van't Hoff analysis to be roughly similar (25-75% ΔG°). Gains in enthalpy with increasing polar surface area of inhibitors suggest that the binding is favored by electrostatic or polar interactions between the ligands and the transporter.
许多核苷转运抑制剂作为抗癌、血管舒张和心脏保护药物正在临床使用。然而，由于核苷转运蛋白（NTs）的内源性表达水平较低，以及在对纯化的配体蛋白进行生物物理表征时存在困难，人们对这些抑制剂与核苷转运蛋白的结合能了解甚少。在此，我们展示了抑制剂与人平衡核苷转运蛋白-1（hENT1，也称为SLC29A1）结合的动力学和热力学分析。使用放射性配体结合测定法，我们获得了著名的核苷转运抑制剂——[³H]硝基苄基巯基嘌呤核糖核苷（[³H]NBMPR）、双嘧达莫和潘生丁——以及天然通透剂腺苷与hENT1的平衡结合和动力学速率常数。我们观察到，随着温度升高，所有抑制剂的平衡结合亲和力降低，而动力学速率常数增加。此外，我们发现结合是由焓驱动的，因此是一个放热反应，这意味着转运蛋白在热力学上无法区分其抑制剂和底物。这四种化学性质不同的配体主要由焓驱动的结合表明，转运蛋白可能无法容忍导致高亲和力结合的相互作用类型的多样性。与此一致的是，[³H]NBMPR结合的测量活化能相对较大（20 kcal mol⁻¹），表明抑制剂结合后发生了构象变化。对于所有三种抑制剂，通过范特霍夫分析确定反应能量学中的焓（ΔH°）和熵（ΔS°）贡献大致相似（25 - 75% ΔG°）。随着抑制剂极性表面积增加，焓的增加表明配体与转运蛋白之间的静电或极性相互作用有利于结合。
