Kubacka Dorota, Kozarski Mateusz, Baranowski Marek R, Wojcik Radoslaw, Panecka-Hofman Joanna, Strzelecka Dominika, Basquin Jerome, Jemielity Jacek, Kowalska Joanna
Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Pasteura 5, 02-093 Warsaw, Poland.
Centre of New Technologies, University of Warsaw, Banacha 2c, 02-097 Warsaw, Poland.
Pharmaceuticals (Basel). 2022 Apr 29;15(5):554. doi: 10.3390/ph15050554.
Cytosolic nucleotidases (cNs) catalyze dephosphorylation of nucleoside 5'-monophosphates and thereby contribute to the regulation of nucleotide levels in cells. cNs have also been shown to dephosphorylate several therapeutically relevant nucleotide analogues. cN-IIIB has shown in vitro a distinctive activity towards 7-mehtylguanosine monophosphate (mGMP), which is one key metabolites of mRNA cap. Consequently, it has been proposed that cN-IIIB participates in mRNA cap turnover and prevents undesired accumulation and salvage of mGMP. Here, we sought to develop molecular tools enabling more advanced studies on the cellular role of cN-IIIB. To that end, we performed substrate and inhibitor property profiling using a library of 41 substrate analogs. The most potent hit compounds (identified among mGMP analogs) were used as a starting point for structure-activity relationship studies. As a result, we identified several 7-benzylguanosine 5'-monophosphate (BnGMP) derivatives as potent, unhydrolyzable cN-IIIB inhibitors. The mechanism of inhibition was elucidated using X-ray crystallography and molecular docking. Finally, we showed that compounds that potently inhibit recombinant cN-IIIB have the ability to inhibit mGMP decay in cell lysates.
胞质核苷酸酶（cNs）催化核苷5'-单磷酸的去磷酸化反应，从而有助于调节细胞内的核苷酸水平。cNs还被证明能够使几种具有治疗意义的核苷酸类似物去磷酸化。cN-IIIB在体外已显示出对7-甲基鸟苷单磷酸（mGMP）具有独特的活性，mGMP是mRNA帽的关键代谢产物之一。因此，有人提出cN-IIIB参与mRNA帽的周转，并防止mGMP的不必要积累和挽救。在这里，我们试图开发分子工具，以便能够更深入地研究cN-IIIB在细胞中的作用。为此，我们使用包含41种底物类似物的文库进行了底物和抑制剂特性分析。最有效的命中化合物（在mGMP类似物中鉴定）被用作构效关系研究的起点。结果，我们鉴定出几种7-苄基鸟苷5'-单磷酸（BnGMP）衍生物作为有效的、不可水解的cN-IIIB抑制剂。使用X射线晶体学和分子对接阐明了抑制机制。最后，我们表明，能有效抑制重组cN-IIIB的化合物具有抑制细胞裂解液中mGMP降解的能力。
