title: Properties influencing the relative binding affinity of pteroate derivatives and drug conjugates thereof to the folate receptor.
abstract: OBJECTIVE
  Using in vitro competition assays, determine salient chemical features of pteroates and pteroate-drug conjugates which afford high affinity to the folate receptor.
  
  
  METHODS
  Both folate binding protein-coated polystyrene plates and adherent human cell-based assays were used to evaluate the effects of assay temperature and buffer composition on pteroate/pteroate-drug conjugate binding affinity. Following assay selection and optimization, the relative binding affinities of ten vitamers and derivatives as well as seven pteroate-drug conjugates were evaluated.
  
  
  RESULTS
  Compared to polystyrene plates containing immobilized folate binding protein, adherent KB cells were determined to be an equally effective, more desirable source of folate receptor for such analyses. Using the latter method, we discovered that a charged group positioned in close proximity to the pteroate's aryl moiety is critical for retaining high binding affinity. We also found that a diverse set of bioactive small molecule agents can be attached to folic acid in a manner that does not appreciably disturb this vitamin's intrinsic high affinity for the folate receptor. However, conjugation of lipophilic, high protein-binding agents to folate was sometimes found to dramatically reduce affinity, which is a finding that best exemplifies the need for having a reliable in vitro assay for determining a compound's RA.
  
  
  CONCLUSIONS
  Molecules which bind best to the human folate receptor are those that contain hydrophilic regions distal to the ligand's aryl group, and for drug conjugates, an extended hydrophilic spacer placed in-between the pteroate and drug cargo moieties.
authors.affiliation: Endocyte, Inc., 3000 Kent Ave., West Lafayette, IN 47906, USA. Chrisleamon@endocyte.com
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Leamon
authors.forename: Fei
authors.initials: F
authors.lastname: You
authors.forename: Hari Krishna
authors.initials: HK
authors.lastname: Santhapuram
authors.forename: Mingjin
authors.initials: M
authors.lastname: Fan
authors.forename: Iontcho R
authors.initials: IR
authors.lastname: Vlahov
mesh_terms: D001667:Binding, Competitive; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008024:Ligands; D015394:Molecular Structure; D009305:Nasopharynx / Q000166:cytology; D004364:Pharmaceutical Preparations / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011622:Pterins / Q000737:chemistry* / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013696:Temperature
chemical_list: D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D008024:Ligands; D004364:Pharmaceutical Preparations; D011622:Pterins; D011956:Receptors, Cell Surface; D005492:Folic Acid