Calcaterra Andrea, Iovine Valentina, Botta Bruno, Quaglio Deborah, D'Acquarica Ilaria, Ciogli Alessia, Iazzetti Antonia, Alfonsi Romina, Lospinoso Severini Ludovica, Infante Paola, Di Marcotullio Lucia, Mori Mattia, Ghirga Francesca
a Department of Chemistry and Technology of Drugs , Sapienza University of Rome , Rome , Italy.
b Department of Molecular Medicine , Sapienza University of Rome , Rome , Italy.
J Enzyme Inhib Med Chem. 2018 Dec;33(1):349-358. doi: 10.1080/14756366.2017.1419221.
This work aims at elucidating the mechanism and kinetics of hydrolysis of GANT61, the first and most-widely used inhibitor of the Hedgehog (Hh) signalling pathway that targets Glioma-associated oncogene homologue (Gli) proteins, and at confirming the chemical nature of its bioactive form. GANT61 is poorly stable under physiological conditions and rapidly hydrolyses into an aldehyde species (GANT61-A), which is devoid of the biological activity against Hh signalling, and a diamine derivative (GANT61-D), which has shown inhibition of Gli-mediated transcription. Here, we combined chemical synthesis, NMR spectroscopy, analytical studies, molecular modelling and functional cell assays to characterise the GANT61 hydrolysis pathway. Our results show that GANT61-D is the bioactive form of GANT61 in NIH3T3 Shh-Light II cells and SuFu mouse embryonic fibroblasts, and clarify the structural requirements for GANT61-D binding to Gli1. This study paves the way to the design of GANT61 derivatives with improved potency and chemical stability.
这项工作旨在阐明GANT61的水解机制和动力学，GANT61是首个也是使用最广泛的靶向胶质瘤相关癌基因同源物（Gli）蛋白的刺猬信号通路（Hh）抑制剂，同时也旨在确认其生物活性形式的化学性质。GANT61在生理条件下稳定性较差，会迅速水解为一种醛类物质（GANT61-A），其对Hh信号通路无生物活性，以及一种二胺衍生物（GANT61-D），该衍生物已显示出对Gli介导的转录的抑制作用。在此，我们结合化学合成、核磁共振光谱、分析研究、分子建模和功能性细胞检测来表征GANT61的水解途径。我们的结果表明，在NIH3T3 Shh-Light II细胞和SuFu小鼠胚胎成纤维细胞中，GANT61-D是GANT61的生物活性形式，并阐明了GANT61-D与Gli1结合的结构要求。这项研究为设计具有更高效力和化学稳定性的GANT61衍生物铺平了道路。
