Department of Medicinal Chemistry, University of Minnesota , Minneapolis, Minnesota 55455, United States.
Mol Pharm. 2017 Nov 6;14(11):3987-3997. doi: 10.1021/acs.molpharmaceut.7b00664. Epub 2017 Oct 26.
Nucleotide analogues that incorporate a metabolically labile nucleoside phosphoramidate (a ProTide) have found utility as prodrugs. In humans, ProTides can be cleaved by human histidine triad nucleotide binding protein 1 (hHint1) to expose the nucleotide monophosphate. Activation by this route circumvents highly selective nucleoside kinases that limit the use of nucleosides as prodrugs. To better understand the diversity of potential substrates of hHint1, we created and studied a series of phosphoramidate nucleosides. Using a combination of enzyme kinetics, X-ray crystallography, and isothermal titration calorimetry with both wild-type and inactive mutant enzymes, we have been able to explore the energetics of substrate binding and establish a structural basis for catalytic efficiency. Diverse nucleobases are well tolerated, but portions of the ribose are needed to position substrates for catalysis. Beneficial characteristics of the amine leaving group are also revealed. Structural principles revealed by these results may be exploited to tune the rate of substrate hydrolysis to strategically alter the intracellular release of the product nucleoside monophosphate from the ProTide.
核苷酸类似物，其中包含代谢不稳定的核苷膦酸酯（ProTide），已被用作前药。在人体中，ProTides 可以被人类组氨酸三联核苷酸结合蛋白 1（hHint1）切割，暴露出核苷酸单磷酸。通过这种途径激活可以绕过高度选择性的核苷激酶，限制了核苷作为前药的使用。为了更好地理解 hHint1 的潜在底物的多样性，我们创建并研究了一系列膦酸酯核苷。我们使用酶动力学、X 射线晶体学和与野生型和失活突变酶的等温滴定量热法相结合的方法，能够探索底物结合的能量，并为催化效率建立结构基础。不同的碱基都能很好地耐受，但核糖的一部分需要用于定位催化的底物。胺离去基团的有益特性也得到了揭示。这些结果揭示的结构原则可用于调节底物水解的速率，从而从 ProTide 有策略地改变产物核苷酸单磷酸在细胞内的释放。
