Chemical, computational and functional insights into the chemical stability of the Hedgehog pathway inhibitor GANT61

• Published in: Journal of enzyme inhibition and medicinal chemistry 2018-01, Vol.33 (1), p.349-358
• Main Authors: 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
• Format: Article
• Language: English
• Subjects: bioactive form; chemical stability; GANT61; Gli inhibitor; Hedgehog pathway; Research Paper
• ISSN: 1475-6366; 1475-6374
• 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.