Non Smoothened inhibitors 321 Hedgehog protein

The extracellular protein Shh that binds to the transmembrane receptor Ptch, reversing its inhibitory effect on Smo, is the target of the macrocycle robotnikinin (24) [59]. A small-molecule microarray-based screen of a bacterially expressed biologically active Shh N-terminal fragment (ShhN) provided a macrocyclic hit. Optimization of this hit resulted in the identification of robotnikinin. The compound binds to ShhN with a Kd of 3.1 p.M and inhibits Hh signaling in a Gli-luciferase reporter gene cell line, in human primary keratinocytes, and in a synthetic model of the human skin in a dose-dependent fashion. The authors suggest a mechanism involving inhibition of the actions of Shh, either directly or indirectly, by interfering with a precursor complex.

3.2.2 Gli-mediated transcription

Two low-molecular weight compounds, 25 (GANT58) and 26 (GANT61), were identified, which inhibit Hh signaling downstream of Smo and SuFu at the level of Gli with an IC50 of ~ 5 p.M in a Gli-luciferase cellular assay [60]. Mechanistically, both inhibitors act at the nucleus to block Gli function, and one of them, 26, interferes with DNA binding of Gli1. Both compounds display selectivity for the Hh pathway over several unrelated signal transduction pathways such as the TNF/NFkB signaling, glucocorticoid receptor gene transactivation, and the Ras-Raf-Mek-Mapk cascade. Subcutaneous application of 26, dosed at 25mg/kg/day for 18 days, in a human prostate cancer xenograft mouse model induced growth regression with concurrent strongly reduced mRNA levels of Ptch.

Screening of natural products in a cell-based reporter assay of Gli1-mediated transcription provided several hits. The physalins F (27) and B (28) were the most potent inhibitors of Gli1-mediated activity (IC50 values of 0.66 p.M and 0.62 p.M, respectively) [61]. Additionally, these two compounds also reduced Gli2-mediated transcription in a separate cell assay (IC50 values of 1.5 p.M and 1.4 p.M, respectively). Compounds 27 and 28 were cytotoxic in a PANC1 human pancreatic cancer cell line (IC50 values of 2.6 and 5.3 p.M) and decreased the mRNA expression of Gli1, 2 and Ptch genes. The authors did not identify the molecular targets but point out that physalins are known modulators of the NFkB cascade, and the mechanism of their Gli-mediated transcriptional inhibition might include a path of PKC inhibition.

27: physalin F

28: physalin B

27: physalin F

28: physalin B

29: JK184

29: JK184

3.2.3 Microtubule depolymerization

JK184 (29) potently inhibited Hh signaling in a Gli-luciferase cellular assay (IC50 = 30 nM); this inhibition was confirmed by measuring the mRNA levels of Gli1 and Ptch1 by quantitative reverse transcription-polymerase chain reaction (RT-PCR) in this cell line [62]. Compound 29 was not found to bind to Smo, but rather binds directly to alcohol dehydrogenase 7 (Adh 7) with a Kd of 348 nM, and was shown to inhibit the oxidation of retinol by this enzyme with similar potency. The oxidation of retinol by Adh7 is part of the retinoic acid (RA) signaling pathway, which in mouse embryos affects Hh signaling. However, recent studies demonstrated that compound 29 exerts its effects on the Hh pathway by destabilizing microtubules, and its inhibition of Adh7 is an ancillary activity. In this model, 29 disrupts microtubule-dependent processes that are required for the conversion of endogenous full-length Gli proteins into functional transcriptional activators [63].

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