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

The Smoothened (Smo) receptor, a member of class F G protein-coupled receptors, is the main transducer of the Hedgehog (Hh) signaling pathway implicated in a wide range of developmental and adult processes. Smo is the target of anticancer drugs that bind to a long and narrow cavity in the 7-transmembrane (7TM) domain. X-ray structures of human Smo (hSmo) bound to several ligands have revealed 2 types of 7TM-directed antagonists: those binding mostly to extracellular loops (site 1, e.g., LY2940680) and those penetrating deeply in the 7TM cavity (site 2, e.g., SANT-1). Here we report the development of the acylguanidine MRT-92, which displays subnanomolar antagonist activity against Smo in various Hh cell-based assays. MRT-92 inhibits rodent cerebellar granule cell proliferation induced by Hh pathway activation through pharmacologic (half maximal inhibitory concentration [IC50] = 0.4 nM) or genetic manipulation. Using [(3)H]MRT-92 (Kd = 0.3 nM for hSmo), we created a comprehensive framework for the interaction of small molecule modulators with hSmo and for understanding chemoresistance linked to hSmo mutations. Guided by molecular docking and site-directed mutagenesis data, our work convincingly confirms that MRT-92 simultaneously recognized and occupied both sites 1 and 2. Our data demonstrate the existence of a third type of Smo antagonists, those entirely filling the Smo binding cavity from the upper extracellular part to the lower cytoplasmic-proximal subpocket. Our studies should help design novel potent Smo antagonists and more effective therapeutic strategies for treating Hh-linked cancers and associated chemoresistance.

ArgTX-636, a polyamine isolated from spider venom: A novel class of melanogenesis inhibitors.

To discover new molecules with an inhibitory activity of melanogenesis a hundred of scorpions, snakes, spiders and amphibians venoms were screened for their capacity to inhibit mushroom tyrosinase using 3,4-l-dihydroxyphenylalanine (l-DOPA) as substrate. The Argiope lobata spider venom proved to be the most active. HPLC fraction containing Argiotoxine-636 (ArgTX-636), a polyamine known for its numerous biological activities, was found to also show a good regulation activity of melanogenesis by inhibiting DOPA and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) oxidases activities, wore by tyrosinase (TYR) and tyrosinase-related protein 1 (TRP-1), respectively. Our results demonstrate that ArgTX-636 reduced the mushroom tyrosinase activity in a dose-dependent way with a maximal half inhibitory concentration (IC50) value of 8.34µM, when l-DOPA is used as substrate. The Lineweaver-Burk study showed that ArgTX-636 is a mixed type inhibitor of the diphenolase activity. Moreover, ArgTX-636 inhibits DHICA oxydase activity of mushroom tyrosinase activity with IC50 at 41.3µM. ArgTX-636 has no cytotoxicity in B16F10 melanoma cells at concentrations up to 42.1µM. The effect of ArgTX-636 on melanogenesis showed that melanin production in B16F10 melanoma cell decreased by approximatively 70% compared to untreated cells. ArgTX-636 displayed no significant effect on the TYR expression while the protein level of TRP-1 decreased in B16F10 cells. Thus, ArgTX-636 could have particular interest for cosmetic and/or pharmaceutical use in order to reduce important dermatoses in black and mixed skins.

Discovery, molecular and pharmacological characterization of GSA-10, a novel small-molecule positive modulator of Smoothened.

Activation of the Smoothened (Smo) receptor mediates Hedgehog (Hh) signaling. Hh inhibitors are in clinical trials for cancer, and small-molecule Smo agonists may have therapeutic interests in regenerative medicine. Here, we have generated and validated a pharmacophoric model for Smo agonists and used this model for the virtual screening of a library of commercially available compounds. Among the 20 top-scoring ligands, we have identified and characterized a novel quinolinecarboxamide derivative, propyl 4-(1-hexyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxamido) benzoate, (GSA-10), as a Smo agonist. GSA-10 fits to the agonist pharmacophoric model with two hydrogen bond acceptor groups and four hydrophobic regions. Using pharmacological, biochemical, and molecular approaches, we provide compelling evidence that GSA-10 acts at Smo to promote the differentiation of multipotent mesenchymal progenitor cells into osteoblasts. However, this molecule does not display the hallmarks of reference Smo agonists. Remarkably, GSA-10 does not recognize the classic bodipy-cyclopamine binding site. Its effect on cell differentiation is inhibited by Smo antagonists, such as MRT-83, SANT-1, LDE225, and M25 in the nanomolar range, by GDC-0449 in the micromolar range, but not by cyclopamine and CUR61414. Thus, GSA-10 allows the pharmacological characterization of a novel Smo active site, which is notably not targeted to the primary cilium and strongly potentiated by forskolin and cholera toxin. GSA-10 belongs to a new class of Smo agonists and will be helpful for dissecting Hh mechanism of action, with important implications in physiology and in therapy.

Hedgehog signaling controls fibroblast activation and tissue fibrosis in systemic sclerosis.

OBJECTIVE: Hedgehog signaling not only plays crucial roles during human development but also has been implicated in the pathogenesis of several diseases in adults. The aim of the present study was to investigate the role of the hedgehog pathway in fibroblast activation in systemic sclerosis (SSc). METHODS: Activation of the hedgehog pathway was analyzed by immunohistochemistry and real-time polymerase chain reaction (PCR). The effects of sonic hedgehog (SHH) on collagen synthesis were analyzed by reporter assays, real-time PCR, and Sircol assays. Myofibroblast differentiation was assessed by quantification of α-smooth muscle actin and stress fiber staining. The role of hedgehog signaling in vivo was analyzed by adenoviral overexpression of SHH and using mice lacking 1 allele of the gene for inhibitory receptor Patched homolog 1 (Ptch(+/-) mice). RESULTS: SHH was overexpressed and resulted in activation of hedgehog signaling in patients with SSc, with accumulation of the transcription factors Gli-1 and Gli-2 and increased transcription of hedgehog target genes. Activation of hedgehog signaling induced an activated phenotype in cultured fibroblasts, with differentiation of resting fibroblasts into myofibroblasts and increased release of collagen. Adenoviral overexpression of SHH in the skin of mice was sufficient to induce skin fibrosis. Moreover, Ptch(+/-) mice with increased hedgehog signaling were more sensitive to bleomycin-induced dermal fibrosis. CONCLUSION: We demonstrated that the hedgehog pathway is activated in patients with SSc. Hedgehog signaling potently stimulates the release of collagen and myofibroblast differentiation in vitro and is sufficient to induce fibrosis in vivo. These findings identify the hedgehog cascade as a profibrotic pathway in SSc.

Hedgehog trafficking, cilia and brain functions.

The primary cilium has recently emerged as an important center for transduction of the Sonic Hedgehog (Shh) signal. Genetic studies have shown that Shh signaling at the level of primary cilia is essential for patterning the ventral neural tube and regulating adult stem cells. Some defects observed in human diseases and resulting from mutations affecting the organization of the primary cilium have been attributed to defective Shh signaling. The recent development of Shh pathway inhibitors for treating tumors linked to perturbations of Shh signaling has fostered studies to understand their mechanism of action in Shh receptor complex trafficking at the primary cilium.

Identification and mechanism of action of the acylguanidine MRT-83, a novel potent Smoothened antagonist.

There is a clear need to develop novel pharmacological tools to improve our understanding of Smoothened (Smo) function in normal and pathological states. Here, we report the discovery, the mechanism of action, and the in vivo activity of N-(2-methyl-5-(3-(3,4,5-trimethoxybenzoyl)guanidino)phenyl)biphenyl-4-carboxamide (MRT-83), a novel potent antagonist of Smo that belongs to the acylguanidine family of molecules. MRT-83 fits to a proposed pharmacophoric model for Smo antagonists with three hydrogen bond acceptor groups and three hydrophobic regions. MRT-83 blocks Hedgehog (Hh) signaling in various assays with an IC50 in the nanomolar range, showing greater potency than the reference Smo antagonist cyclopamine. MRT-83 inhibits Bodipy-cyclopamine binding to human and mouse Smo but does not modify Wnt signaling in human embryonic kidney 293 transiently transfected with a Tcf/Lef-dependent Firefly luciferase reporter together with a Renilla reniformis luciferase control reporter. MRT-83 abrogates the agonist-induced trafficking of endogenous mouse or human Smo to the primary cilium of C3H10T1/2 or NT2 cells that derive from a pluripotent testicular carcinoma. Stereotaxic injection into the lateral ventricle of adult mice of MRT-83 but not of a structurally related compound inactive at Smo abolished up-regulation of Patched transcription induced by Sonic Hedgehog in the neighboring subventricular zone. These data demonstrate that MRT-83 efficiently antagonizes Hh signaling in vivo. All together, these molecular, functional and biochemical studies provide evidence that MRT-83 interacts with Smo. Thus, this novel Smo antagonist will be useful for manipulating Hh signaling and may help develop new therapies against Hh-pathway related diseases.

Virtual screening-based discovery and mechanistic characterization of the acylthiourea MRT-10 family as smoothened antagonists.

The seven-transmembrane receptor Smoothened (Smo) is the major component involved in signal transduction of the Hedgehog (Hh) morphogens. Smo inhibitors represent a promising alternative for the treatment of several types of cancers linked to abnormal Hh signaling. Here, on the basis of experimental data, we generated and validated a pharmacophoric model for Smo inhibitors constituted by three hydrogen bond acceptor groups and three hydrophobic regions. We used this model for the virtual screening of a library of commercially available compounds. Visual and structural criteria allowed the selection of 20 top scoring ligands, and an acylthiourea, N-(3-benzamidophenylcarbamothioyl)-3,4,5-trimethoxybenzamide (MRT-10), was identified and characterized as a Smo antagonist. The corresponding acylurea, N-(3-benzamidophenylcarbamoyl)-3,4,5-trimethoxybenzamide (MRT-14), was synthesized and shown to display, in various Hh assays, an inhibitory potency comparable to or greater than that of reference Smo antagonists cyclopamine and N-((3S,5S)-1-(benzo[d][1,3]dioxol-5-ylmethyl)-5-(piperazine-1-carbonyl)pyrrolidin-3-yl)-N-(3-methoxybenzyl)-3,3-dimethylbutanamide (Cur61414). Focused virtual screening of the same library further identified five additional related antagonists. MRT-10 and MRT-14 constitute the first members of novel families of Smo antagonists. The described virtual screening approach is aimed at identifying novel modulators of Smo and of other G-protein coupled receptors.

Acylthiourea, acylurea, and acylguanidine derivatives with potent hedgehog inhibiting activity.

The Smoothened (Smo) receptor is the major transducer of the Hedgehog (Hh) signaling pathway. On the basis of the structure of the acylthiourea Smo antagonist (MRT-10), a number of different series of analogous compounds were prepared by ligand-based structural optimization. The acylthioureas, originally identified as actives, were converted into the corresponding acylureas or acylguanidines. In each series, similar structural trends delivered potent compounds with IC(50) values in the nanomolar range with respect to the inhibition of the Hh signaling pathway in various cell-based assays and of BODIPY-cyclopamine binding to human Smo. The similarity of their biological activities, in spite of discrete structural differences, may reveal the existence of hydrogen-bonding interactions between the ligands and the receptor pocket. Biological potency of compounds 61, 72, and 86 (MRT-83) were comparable to those of the clinical candidate GDC-0449. These findings suggest that these original molecules will help delineate Smo and Hh functions and can be developed as potential anticancer agents.