In fact, any molecular entity may undergo structural modifications due to the exposure to different cellular environments, metabolic enzymes, reactions with redox species, and so on. One of the most challenging tasks in drug discovery is the identification of the active species responsible for the observed biological activity of a drug or a drug candidate (thereafter referred as the bioactive form).
This study paves the way to the design of GANT61 derivatives with improved potency and chemical stability. 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. Here, we combined chemical synthesis, NMR spectroscopy, analytical studies, molecular modelling and functional cell assays to characterise the GANT61 hydrolysis pathway. 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. 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.
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