title: Discovery and structural optimization of 1-phenyl-3-(1-phenylethyl)urea derivatives as novel inhibitors of CRAC channel.
abstract: OBJECTIVE
  Ca(2+)-release-activated Ca(2+) (CRAC) channel, a subfamily of store-operated channels, is formed by calcium release-activated calcium modulator 1 (ORAI1), and gated by stromal interaction molecule 1 (STIM1). CRAC channel may be a novel target for the treatment of immune disorders and allergy. The aim of this study was to identify novel small molecule CRAC channel inhibitors.
  
  
  METHODS
  HEK293 cells stably co-expressing both ORAI1 and STIM1 were used for high-throughput screening. A hit, 1-phenyl-3-(1-phenylethyl)urea, was identified that inhibited CRAC channels by targeting ORAI1. Five series of its derivatives were designed and synthesized, and their primary structure-activity relationships (SARs) were analyzed. All derivatives were assessed for their effects on Ca(2+) influx through CRAC channels on HEK293 cells, cytotoxicity in Jurkat cells, and IL-2 production in Jurkat cells expressing ORAI1-SS-eGFP.
  
  
  RESULTS
  A total of 19 hits were discovered in libraries containing 32 000 compounds using the high-throughput screening. 1-Phenyl-3-(1-phenylethyl)urea inhibited Ca(2+) influx with IC50 of 3.25±0.17 μmol/L. SAR study on its derivatives showed that the alkyl substituent on the α-position of the left-side benzylic amine (R1) was essential for Ca(2+) influx inhibition and that the S-configuration was better than the R-configuration. The derivatives in which the right-side R3 was substituted by an electron-donating group showed more potent inhibitory activity than those that were substituted by electron-withdrawing groups. Furthermore, the free N-H of urea was not necessary to maintain the high potency of Ca(2+) influx inhibition. The N,N'-disubstituted or N'-substituted derivatives showed relatively low cytotoxicity but maintained the ability to inhibit IL-2 production. Among them, compound 5b showed an improved inhibition of IL-2 production and low cytotoxicity.
  
  
  CONCLUSIONS
  1-Phenyl-3-(1-phenylethyl)urea is a novel CRAC channel inhibitor that specifically targets ORAI1. This study provides a new chemical scaffold for design and development of CRAC channel inhibitors with improved Ca(2+) influx inhibition, immune inhibition and low cytotoxicity.
authors.affiliation: State Key Laboratory of Drug Research, The National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Hai-zhen
authors.initials: HZ
authors.lastname: Zhang
authors.affiliation: National Key Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Xiao-lan
authors.initials: XL
authors.lastname: Xu
authors.affiliation: State Key Laboratory of Drug Research, The National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Hua-yan
authors.initials: HY
authors.lastname: Chen
authors.affiliation: National Key Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Sher
authors.initials: S
authors.lastname: Ali
authors.affiliation: National Key Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Wang
authors.affiliation: National Key Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Jun-wei
authors.initials: JW
authors.lastname: Yu
authors.affiliation: National Key Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Xu
authors.affiliation: State Key Laboratory of Drug Research, The National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Fa-jun
authors.initials: FJ
authors.lastname: Nan
mesh_terms: D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000737:chemistry* / Q000494:pharmacology*; D015220:Calcium Channels / Q000378:metabolism*; D015195:Drug Design; D057809:HEK293 Cells; D006801:Humans; D019169:Jurkat Cells; D008565:Membrane Proteins / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism; D000071740:ORAI1 Protein; D000071737:Stromal Interaction Molecule 1; D013329:Structure-Activity Relationship; D014508:Urea / Q000031:analogs & derivatives* / Q000494:pharmacology*
chemical_list: D002121:Calcium Channel Blockers; D015220:Calcium Channels; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; C103530:STIM1 protein, human; D000071737:Stromal Interaction Molecule 1; D014508:Urea; D002118:Calcium