title: High throughput screening identified a substituted imidazole as a novel RANK pathway-selective osteoclastogenesis inhibitor.
abstract: Receptor activator of nuclear factor-kappaB (NF-kappaB) (RANK) plays a key role in the differentiation, activation, and survival of osteoclasts. Upon activation of RANK with RANK ligand (RANKL), osteoclast precursor cells differentiate into tartrate-resistant acid phosphatase (TRAP)-positive, multinucleated osteoclasts. To identify compounds that block osteoclastogenesis, a cell-based assay was developed using RAW264.7 cells stably transfected with a TRAP promoter-dependent reporter gene as a surrogate readout for differentiation. Described herein is the strategy for high throughput screening and subsequent secondary biological assays for hit triage, which resulted in the identification of compound 1, a 4-nitroimidazole derivative, that specifically inhibited RANKL-induced TRAP gene and protein expression. Compound 1 did not affect the tumor necrosis factor-alpha- or lipopolysaccharide-induced TRAP-luciferase response, suggesting selective inhibition of the RANKL-induced pathway. Reverse transcription polymerase chain reaction analysis confirmed the inhibition of expression of osteoclast marker genes, such as TRAP, cathepsin K, and carbonic anhydrase type II. Compound 1 did not inhibit the RANKL-induced activation of a NF-kappaB reporter gene, or p38 kinase activity, suggesting a mechanism of action downstream of NF-kappaB. Together, these results suggest that we have identified a RANK pathway-specific inhibitor able to block the RANKL-induced osteoclast differentiation process. The hit identification strategy described here can be applied to other cell-based assays using an indirect surrogate readout to improve success rates.
authors.affiliation: Pharmaceutical Research Institute, Bristol-Myers Squibb Company, Wallingford, CT, USA. taosheng.chen@stjude.org
authors.forename: Taosheng
authors.initials: T
authors.lastname: Chen
authors.forename: Anita C
authors.initials: AC
authors.lastname: Knapp
authors.forename: Yang
authors.initials: Y
authors.lastname: Wu
authors.forename: Jinwen
authors.initials: J
authors.lastname: Huang
authors.forename: Jean S
authors.initials: JS
authors.lastname: Lynch
authors.forename: John K
authors.initials: JK
authors.lastname: Dickson
authors.forename: R Michael
authors.initials: RM
authors.lastname: Lawrence
authors.forename: Jean H M
authors.initials: JH
authors.lastname: Feyen
authors.forename: Michele L
authors.initials: ML
authors.lastname: Agler
mesh_terms: D000135:Acid Phosphatase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007093:Imidazoles / Q000737:chemistry / Q000494:pharmacology; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D009593:Nitroimidazoles / Q000737:chemistry / Q000494:pharmacology*; D010010:Osteoclasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D053244:Osteoprotegerin / Q000737:chemistry / Q000494:pharmacology*; D011725:Pyridines / Q000737:chemistry / Q000494:pharmacology; D053245:RANK Ligand / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects*; D000071681:Tartrate-Resistant Acid Phosphatase; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D007093:Imidazoles; D007527:Isoenzymes; D008070:Lipopolysaccharides; D009593:Nitroimidazoles; D053244:Osteoprotegerin; D011725:Pyridines; D053245:RANK Ligand; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D008156:Luciferases; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase; C093642:SB 203580; C052587:4-nitroimidazole; D004121:Dimethyl Sulfoxide