Laboratório de Farmacologia Bioquímica e Molecular, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil (P.A.-N., F.N., L.E.M.Q.); Laboratório de Síntese Orgânica e Nanoestruturas, Universidade Federal de São João del-Rei Campus Centro-Oeste Dona Lindu, Divinópolis, Brazil (S.C.S., J.A.F.P.V.); Laboratório de Bioquímica Celular, Universidade Federal de São João del-Rei (UFSJ) Campus Centro-Oeste Dona Lindu, Divinópolis, Brazil (L.B.); and Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts (G.A.O.)
Laboratório de Farmacologia Bioquímica e Molecular, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil (P.A.-N., F.N., L.E.M.Q.); Laboratório de Síntese Orgânica e Nanoestruturas, Universidade Federal de São João del-Rei Campus Centro-Oeste Dona Lindu, Divinópolis, Brazil (S.C.S., J.A.F.P.V.); Laboratório de Bioquímica Celular, Universidade Federal de São João del-Rei (UFSJ) Campus Centro-Oeste Dona Lindu, Divinópolis, Brazil (L.B.); and Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts (G.A.O.).
Mol Pharmacol. 2024 Oct 17;106(5):225-239. doi: 10.1124/molpharm.124.000934.
The antitumor effect of cardiotonic steroids (CTS) has stimulated the search for new methods to evaluate both kinetic and thermodynamic aspects of their binding to Na/K-ATPase (IUBMB Enzyme Nomenclature). We propose a real-time assay based on a chromogenic substrate for phosphatase activity (pNPPase activity), using only two concentrations with an inhibitory progression curve, to obtain the association rate ( ), dissociation rate ( ), and equilibrium ( ) constants of CTS for the structure-kinetics relationship in drug screening. We show that changing conditions (from ATPase to pNPPase activity) resulted in an increase of of the cardenolides digitoxigenin, essentially due to a reduction of In contrast, the of the structurally related bufadienolide bufalin increased much less due to the reduction of its partially compensating the decrease of its When evaluating the kinetics of 15 natural and semisynthetic CTS, we observed that both and correlated with (Spearman test), suggesting that differences in potency depend on variations of both and A rhamnose in C3 of the steroidal nucleus enhanced the inhibitory potency by a reduction of rather than an increase of Raising the temperature did not alter the of digitoxin, generating a ΔH ( ) of -10.4 ± 4.3 kJ/mol, suggesting a complex dissociation mechanism. Based on a simple and inexpensive methodology, we determined the values of , , and of the CTS and provided original kinetics and thermodynamics differences between CTS that could help the design of new compounds. SIGNIFICANCE STATEMENT: This study describes a fast, simple, and cost-effective method for the measurement of phosphatase pNPPase activity enabling structure-kinetics relationships of Na/K-ATPase inhibitors, which are important compounds due to their antitumor effect and endogenous role. Using 15 compounds, some of them original, this study was able to delineate the kinetics and/or thermodynamics differences due to the type of sugar and lactone ring present in the steroid structure.
强心甾类化合物（CTS）的抗肿瘤作用刺激了寻找新方法来评估其与 Na/K-ATPase 结合的动力学和热力学方面的研究（IUBMB 酶命名法）。我们提出了一种基于磷酸酶活性（pNPPase 活性）显色底物的实时测定法，仅使用两个具有抑制进展曲线的浓度，以获得 CTS 的结合速率（）、解离速率（）和平衡（）常数，用于药物筛选中的结构-动力学关系。我们表明，改变条件（从 ATPase 到 pNPPase 活性）会导致卡多辛Digitoxigenin 的增加，这主要是由于的减少。相比之下，结构相关的蟾毒内酯Bufalin 的增加要少得多，这是由于其减少了部分补偿了其减少的，当评估 15 种天然和半合成 CTS 的动力学时，我们观察到和都与（Spearman 检验）相关，这表明效力的差异取决于和的变化。甾核 C3 上的鼠李糖降低了抑制效力，而不是增加了，这表明了增强效力的机制是通过降低而不是增加。提高温度不会改变地高辛的，产生的 ΔH（）为-10.4 ± 4.3 kJ/mol，表明解离机制复杂。基于一种简单且廉价的方法，我们确定了 CTS 的和的值，并提供了 CTS 之间原始动力学和热力学差异，这有助于设计新化合物。意义陈述：本研究描述了一种快速、简单且经济高效的磷酸酶 pNPPase 活性测定方法，可用于测量 Na/K-ATPase 抑制剂的结构-动力学关系，这些化合物由于其抗肿瘤作用和内源性作用而非常重要。使用 15 种化合物，其中一些是原始化合物，本研究能够描绘出由于甾体结构中存在的糖和内酯环的类型而导致的动力学和/或热力学差异。
