MRT‐92 inhibits Hedgehog signaling by blocking overlapping binding sites in the transmembrane domain of the Smoothened receptor

Hoch, Lucile; Faure, Hélène; Roudaut, Hermine; Schoenfelder, Angèle; Mann, André; Girard, Nicolas; Bihannic, Laure; Ayrault, Olivier; Petricci, Elena; Taddei, Maurizio; Rognan, Didier; Ruat, Martial

doi:10.1096/fj.14-267849

Cited by 49 publications

(59 citation statements)

References 50 publications

(132 reference statements)

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“…Furthermore, the phenomenon of allosteric regulation in the SMO binding site was also inferred from studies on other SMO ligands Hoch et al, 2015).…”

Section: Discussionmentioning

confidence: 99%

A NanoBRET-based binding assay for Smoothened allows real time analysis of small-molecule ligand binding and distinction of two separate ligand binding sites for BODIPY-cyclopamine

Kozielewicz

Bowin

Schulte

2019

Preprint

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Background and Purpose: Smoothened (SMO) is a GPCR that mediates hedgehog signaling. Hedgehog binds the Patched, which in turn regulates SMO activation.Overactive SMO signaling is oncogenic and is therefore a clinically established drug target. Here, we establish a nanoluciferase bioluminescence resonance energy transfer (NanoBRET)-based ligand binding assay for SMO providing a sensitive and high throughput-compatible addition to the toolbox of GPCR pharmacologists.Experimental Approach: In the NanoBRET-based binding assay, SMO is N terminally tagged with nanoluciferase (Nluc) and binding of BODIPY-cyclopamine is assessed by quantifying resonance energy transfer between receptor and ligand. The assay allows kinetic analysis of ligand-receptor binding in living HEK293 cells and competition binding experiments using commercially available SMO ligands (SANT-1, cyclopamine-KAAD, SAG1.3 and purmorphamine).Key Results: The NanoBRET binding assay for SMO is sensitive and superior to purely fluorescence-based binding assays. BODIPY-cyclopamine showed complex binding parameters suggesting separate binding sites.Conclusions and Implications: The NanoBRET ligand binding assay for SMO provides a fast, sensitive and reliable alternative to assess SMO ligand binding.Furthermore, this assay is sufficiently sensitive to dissect a SANT-1-sensitive and a SANT-1-insensitive cyclopamine binding site in the 7TM core, and will be important to further dissect and understand the molecular pharmacology of Class F receptors.

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“…Furthermore, the phenomenon of allosteric regulation in the SMO binding site was also inferred from studies on other SMO ligands Hoch et al, 2015).…”

Section: Discussionmentioning

confidence: 99%

A NanoBRET-based binding assay for Smoothened allows real time analysis of small-molecule ligand binding and distinction of two separate ligand binding sites for BODIPY-cyclopamine

Kozielewicz

Bowin

Schulte

2019

Preprint

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“…Particularly, 99 (MRT‐83) was showed to display potent inhibition against both human Smo‐WT and Smo‐D473H mutant ( K i = 4.6 and 5.5 nM, respectively). Further elongation of the biaryl moiety of compound 99 led to the discovery of 100 with a phenylethylphenyl tail . Compound 100 (MRT‐92) showed higher Hh inhibitory activity than 99 in the SHh light II Gli‐luc assay (IC 50 = 2.8 nM) and the alkaline phosphatase assay stimulation by SAG (IC 50 = 5.6 nM).…”

Section: Hh Signaling Pathway Inhibitorsmentioning

confidence: 99%

Strategies to target the Hedgehog signaling pathway for cancer therapy

Xin

Cruz

et al. 2018

Medicinal Research Reviews

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Hedgehog (Hh) signaling is an essential pathway in the human body, and plays a major role in embryo development and tissue patterning. Constitutive activation of the Hh signaling pathway through sporadic mutations or other mechanisms is explicitly associated with cancer development and progression in various solid malignancies. Therefore, targeted inhibition of the Hh signaling pathway has emerged as an attractive and validated therapeutic strategy for the treatment of a wide range of cancers. Vismodegib, a first-in-class Hh signaling pathway inhibitor was approved by the US Food and Drug Administration in 2012, and sonidegib, another potent Hh pathway inhibitor, received FDA's approval in 2015 as a new treatment of locally advanced or metastatic basal cell carcinoma. The clinical success of vismodegib and sonidegib provided strong support for the development of Hh signaling pathway inhibitors via targeting the smoothened (Smo) receptor. Moreover, Hh signaling pathway inhibitors aimed to target proteins, which are downstream or upstream of Smo, have also been pursued based on the identification of additional therapeutic benefits. Recently, much progress has been made in Hh singling and inhibitors of this pathway. Herein, medicinal chemistry strategies, especially the structural optimization process of different classes of Hh inhibitors, are comprehensively summarized. Further therapeutic potentials and challenges are also discussed.

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“…36 Crystal structures of various SMO/ligand complexes have revealed a narrow hydrophobic cavity extending from the extracellular loops into the heptahelical bundle. 26,37–39 These studies suggest that SMO ligands can be grouped into at least three general classes: compounds that primarily bind to the extracellular entrance of the cavity (e.g., cyclopamine; see Figure 3C), those that penetrate deeply into the transmembrane cavity (e.g., SANT-1), and others that engage both sites (e.g., MRT-92; 10 , Figure 4A). …”

Section: Targeted Anticancer Therapiesmentioning

confidence: 99%

I only have eye for ewe: the discovery of cyclopamine and development of Hedgehog pathway-targeting drugs

Chen

2016

Nat. Prod. Rep.

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During the 1950s, sheep ranchers in the western United States experienced episodic outbreaks of cyclopic lambs. In this highlight I describe how these mysterious incidents were traced to the grazing of Veratrum californicum wildflowers by pregnant ewes, leading to the discovery of cyclopamine (1) as a plant-derived teratogen. The precise mechanism of cyclopamine action remained enigmatic for 30 years, until this steroid alkaloid was found to be the first specific inhibitor of Hedgehog (Hh) signalling and a direct antagonist of the transmembrane receptor Smoothened (SMO). In addition to being a valuable probe of Hh pathway function, cyclopamine has been used to demonstrate the therapeutic potential of Hh pathway inhibitors. I discuss the development of SMO antagonists as anticancer therapies and emerging challenges.

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MRT‐92 inhibits Hedgehog signaling by blocking overlapping binding sites in the transmembrane domain of the Smoothened receptor

Cited by 49 publications

References 50 publications

A NanoBRET-based binding assay for Smoothened allows real time analysis of small-molecule ligand binding and distinction of two separate ligand binding sites for BODIPY-cyclopamine

A NanoBRET-based binding assay for Smoothened allows real time analysis of small-molecule ligand binding and distinction of two separate ligand binding sites for BODIPY-cyclopamine

Strategies to target the Hedgehog signaling pathway for cancer therapy

I only have eye for ewe: the discovery of cyclopamine and development of Hedgehog pathway-targeting drugs

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