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

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.

Synergistic inhibition of the Hedgehog pathway by newly designed Smo and Gli antagonists bearing the isoflavone scaffold

“…However, drug-resistant mutations occurring at the Smo receptor as a consequence of pharmacological pressure, coupled with Hh activation downstream or independently of Smo, have raised the need to identify novel strategy to inhibit Hh signaling [12,29]. In this regard, one of the most promising Hh target is the final and most powerful effector Gli1, whose druggability has been assessed by means of the isoflavone GlaB [22], and the synthetic molecule GANT-61, although this latter suffers from chemical instability as substantiated recently [23,56].…”

“…Therefore, here we designed a smallsize focused library of isoflavones bearing different substituents in different positions of the ring B. Molecules were then tested in silico for the interaction with the heptahelical bundle of Smo or the DNA binding site of Gli1. To this aim, the computational protocols already established and validated in previous works were used [17,18,22,23,50]. Results unequivocally show that, for most of tested compounds, the O-substitution at para position of ring B is preferred for the interaction with Smo, whereas the same substitution at meta position is preferred to interact with Gli1.…”

GLI1 Inhibitor SRI-38832 Attenuates Chemotherapeutic Resistance by Downregulating NBS1 Transcription in BRAFV600E Colorectal Cancer

“…While GANT61 is useful for in vitro pharmacological inhibition assays, it becomes hydrolyzed and loses biological activity in vivo (29). Indeed, an analysis of the absorption, distribution, metabolism, and excretion (ADME) properties determined that solubility and stability values could not be determined ( Supplemental Table 1A).…”

<p>Self-Renewal Signalling Pathway Inhibitors: Perspectives on Therapeutic Approaches for Cancer Stem Cells</p>

“…77 Meanwhile, it is the most extensively used antagonist that targets Gli proteins to specifically block Hh signalling. 76 GANT61 has a strong inhibitory influence on cell proliferation and migration, which has been confirmed in both in vivo and in vitro experiments in breast cancer, pancreatic cancer and other malignancies. [78][79][80] Because basal cell carcinoma patients were previously reported to develop resistance to Smo inhibitors, the Gli inhibitor GANT61 is one of the most promising drugs for inhibiting Hh signalling and has been studied in various cancers.…”

“…Drug-resistant Smo mutations and abnormal activation of Hh signalling downstream of Smo have been observed clinically, suggesting that alternative approaches are urgently needed 76. …”