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

J Enzyme Inhib Med Chem. 2018 Dec;33(1):349-358. doi: 10.1080/14756366.2017.1419221.

Abstract

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.

Keywords: GANT61; Gli inhibitor; Hedgehog pathway; bioactive form; chemical stability.

MeSH terms

  • Animals
  • Dose-Response Relationship, Drug
  • Fibroblasts / drug effects
  • Hedgehog Proteins / antagonists & inhibitors*
  • Hedgehog Proteins / metabolism
  • Hydrolysis
  • Kinetics
  • Mice
  • Models, Molecular
  • Molecular Structure
  • NIH 3T3 Cells
  • Pyridines / chemical synthesis
  • Pyridines / chemistry
  • Pyridines / pharmacology*
  • Pyrimidines / chemical synthesis
  • Pyrimidines / chemistry
  • Pyrimidines / pharmacology*
  • Signal Transduction / drug effects
  • Structure-Activity Relationship

Substances

  • GANT 61
  • Hedgehog Proteins
  • Pyridines
  • Pyrimidines

Grants and funding

Authors gratefully acknowledge Professor Francesco Gasparrini from Sapienza University of Rome for his precious support with HILIC measurements. We thank the financial support from Associazione Italiana Ricerca Cancro (AIRC) Grant #IG14723 and #IG20801, PRIN 2012–2013 (2012C5YJSK002), Progetti di Ricerca di Università Sapienza di Roma, Pasteur Institute/Cenci Bolognetti Foundation. We would like to acknowledge networking contribution by the COST Action CM-1407 “Challenging organic syntheses inspired by nature – from natural products chemistry to drug discovery”.