Virtual Screening-Based Discovery and Mechanistic Characterization of the Acylthiourea MRT-10 Family as Smoothened Antagonists

Abstract

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. 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.

Identification and Mechanism of Action of the Acylguanidine MRT-83, a Novel Potent Smoothened Antagonist

“…SAG was synthesized as described previously (Martínez et al, 2006 Pharmacophoric Model of MRT-83. We constructed a pharmacophoric model of MRT-83 based on chemical structures of Smo inhibitors as described previously (Manetti et al, 2010).…”

“…The plasmids pRK5 and pRK5-SP-myc-Smo encoding the mouse Smo sequence have been described previously (Manetti et al, 2010) and are referred to in the text as pRK5 and mouse Smo, respectively. The Wnt reporter plasmid M50 Super8xTOPFlash containing seven Tcf/Lef binding sites in the firefly luciferase promoter, the pLNCWnt-3aHA, and the control pRL-TK Renilla reniformis luciferase were obtained from Addgene (Cambridge, MA).…”

“…Zeocin, Geneticin (G418), penicillin-streptomycin, and all cell culture media or products were from Invitrogen (Cergy Pontoise, France) except as stated otherwise. HEK293 (American Type Culture Collection, Manassas, VA) and C3H10T1/2 (American Type Culture Collection) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (FCS) as described previously (Manetti et al, 2010). The Shh-light2 cells (from Pr.…”

“…For Tcf/Lef-dependent luciferase reporter assay, HEK293 were transfected by FuGENE (Roche Diagnostics, Mannheim, Germany) with 0.1 g of Tcf/Lef reporter, 0.25 g of R. reniformis luciferase and, as indicated in the legend to Fig. 3 (Manetti et al, 2010), using the restriction sites MluI and SalI to give pRK5-SPmyc-hSmo plasmid. The nucleotide sequence of hSmo construct was verified by DNA sequencing.…”

“…C3H10T1/2 cells were incubated for 6 days in the presence of SAG (0.1 M) and the studied compounds. The cell-based bioassay was performed as described previously (Manetti et al, 2010).…”