title: A benzamide-linked small molecule HS-Cf inhibits TNF-α-induced interferon regulatory factor-1 in porcine chondrocytes: a potential disease-modifying drug for osteoarthritis therapeutics.
abstract: BACKGROUND
  Using tumor necrosis factor-alpha (TNF-α)-activated porcine chondrocytes as a screening tool, we aim to synthesize and identify small-molecule inhibitors preserving immunomodulatory effects as therapeutics for osteoarthritis (OA).
  
  
  METHODS
  Chondrocytes were isolated from pig joints. A minilibrary of 300 benzamide-linked small molecules was established. The levels of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) were measured by Western blot and Griess reaction, respectively. Proteoglycan degradation in cartilage explants was determined by histochemistry analysis. The activation of transcription factors and protein kinases was determined by electrophoretic mobility shift assays or Western blots. Zymography and real-time reverse transcriptase-polymerase chain reaction were used to determine enzyme activity and expression of matrix metalloproteinases (MMPs) and aggrecanases, respectively.
  
  
  RESULTS
  Bioassay screening of benzamide-linked small molecules revealed that 2-hydroxy-N-[3-(trifluoromethyl)phenyl]benzamide (HS-Cf) was a potent inhibitor of NO production and iNOS expression in TNF-α-stimulated porcine chondrocytes. HS-Cf suppressed TNF-α-induced activity of MMP-13 and expressions of several aggrecanases and prevented TNF-α-mediated reduction of collagen II. Histochemistry analysis confirmed that HS-Cf could prevent TNF-α-induced degradation and release of proteoglycan/aggrecan in cartilage explants. Such effects by HS-Cf were likely through suppressing TNF-α-induced interferon regulatory factor-1 (IRF-1) but not nuclear factor-kappaB signaling. The significance of IRF-1 was further confirmed by short hairpin knockdown studies.
  
  
  CONCLUSIONS
  In a minilibrary containing 300 small molecules, we identified a benzamide-linked small molecule, HS-Cf, that through down-regulating TNF-α-induced IRF-1 activity suppressed chondrocyte activation and prevented cartilage destruction. HS-Cf might be a potential disease-modifying drug for OA therapeutics.
authors.affiliation: Graduate Institute of Medical Science, National Defense Medical Center, Neihu 114, Taipei, Taiwan, Republic of China.
authors.forename: Feng-Cheng
authors.initials: FC
authors.lastname: Liu
authors.forename: Hsu-Shan
authors.initials: HS
authors.lastname: Huang
authors.forename: Chuan-Yueh
authors.initials: CY
authors.lastname: Huang
authors.forename: Ro
authors.initials: R
authors.lastname: Yang
authors.forename: Deh-Ming
authors.initials: DM
authors.lastname: Chang
authors.forename: Jenn-Haung
authors.initials: JH
authors.lastname: Lai
authors.forename: Ling-Jun
authors.initials: LJ
authors.lastname: Ho
mesh_terms: D000818:Animals; D001549:Benzamides / Q000737:chemistry / Q000494:pharmacology*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D024043:Collagen Type II / Q000235:genetics / Q000378:metabolism; D004195:Disease Models, Animal; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects / Q000276:immunology; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D050856:Interferon Regulatory Factor-1 / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D007596:Joints / Q000473:pathology*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism; D010003:Osteoarthritis / Q000188:drug therapy* / Q000276:immunology / Q000473:pathology; D015398:Signal Transduction / Q000187:drug effects; D054852:Small Molecule Libraries; D013552:Swine; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology / Q000378:metabolism
chemical_list: C571233:2-hydroxy-N-(3-(trifluoromethyl)phenyl)benzamide; D001549:Benzamides; D024043:Collagen Type II; D007166:Immunosuppressive Agents; D050856:Interferon Regulatory Factor-1; D054852:Small Molecule Libraries; D014409:Tumor Necrosis Factor-alpha; D052247:Nitric Oxide Synthase Type II; D010450:Endopeptidases; C083641:aggrecanase