title: Biochemical characterization of smoothened receptor antagonists by binding kinetics against drug-resistant mutant.
abstract: Hedgehog (Hh) signaling critical for development, differentiation, and cell growth is involved in several cancers, including medulloblastoma and basal cell carcinoma. Although antagonism of the smoothened receptor (SMO), which mediates Hh signaling, is an attractive therapeutic target, a drug-resistant mutation in SMO (SMO-D473H) was identified in a clinical trial of the approved drug vismodegib. TAK-441 potently inhibits SMO-D473H, unlike vismodegib and another SMO antagonist, cyclopamine, whereas the differences in binding modes between these antagonists remain unknown. Here we report the biochemical characterization of TAK-441, vismodegib, and cyclopamine by binding kinetics. The association (kon) and dissociation (koff) rates were determined by kinetic binding studies using [(3)H]TAK-441, and dissociation was confirmed by label-free affinity selection-mass spectrometry (AS-MS). In the [(3)H]TAK-441 competition assay, TAK-441 but not vismodegib and cyclopamine showed time-dependent inhibition. Quantitative kinetic binding analysis revealed that koff of TAK-441 was >10-fold smaller than those of vismodegib and cyclopamine. To further assess the binding mode of antagonists, kinetic binding analysis was performed against SMO-D473H. The D473H mutation affected koff of TAK-441 but not kon. In contrast, only kon was changed by the D473H mutation in the case of vismodegib and cyclopamine. These results suggest that the difference in antagonist efficacy against D473H is associated with the binding mode of antagonists. These findings provide a new insight into the drug action of SMO antagonists and help develop potential therapeutics for drug-resistant mutants.
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan. Electronic address: yuuji.shimizu@takeda.com.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Shimizu
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Ishii
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan.
authors.forename: Kazumasa
authors.initials: K
authors.lastname: Ogawa
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Sasaki
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Matsui
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan.
authors.forename: Masaharu
authors.initials: M
authors.lastname: Nakayama
mesh_terms: D000813:Anilides / Q000494:pharmacology*; D001667:Binding, Competitive; D002460:Cell Line; D019008:Drug Resistance, Neoplasm; D006801:Humans; D007700:Kinetics; D009154:Mutation; D011725:Pyridines / Q000494:pharmacology*; D011758:Pyrroles / Q000494:pharmacology*; D011869:Radioligand Assay; D043562:Receptors, G-Protein-Coupled / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000072086:Smoothened Receptor; D014704:Veratrum Alkaloids / Q000494:pharmacology*
chemical_list: D000813:Anilides; C538724:HhAntag691; D011725:Pyridines; D011758:Pyrroles; D043562:Receptors, G-Protein-Coupled; C491023:SMO protein, human; D000072086:Smoothened Receptor; C577980:TAK-441; D014704:Veratrum Alkaloids; C000541:cyclopamine
keywords: Binding kinetics; Cyclopamine; Cyclopamine (PubChem CID: 442972); D473H; Smoothened receptor; TAK-441; TAK-441 (PubChem CID: 44187367); Vismodegib; Vismodegib (PubChem CID: 24776445)