Properties Influencing the Relative Binding Affinity of Pteroate Derivatives and Drug Conjugates Thereof to the Folate Receptor

• Published in: Pharmaceutical research 2009-06, Vol.26 (6), p.1315-1323
• Main Authors: Leamon, Christopher P, You, Fei, Santhapuram, Hari Krishna, Fan, Mingjin, Vlahov, Iontcho R
• Format: Article
• Language: English
• Subjects: Binding sites; Binding, Competitive; Biochemistry; Biological and medical sciences; Biomedical and Life Sciences; Biomedical Engineering and Bioengineering; Biomedicine; Carrier Proteins - metabolism; Cell Line; conjugate; folate receptor; Folate Receptors, GPI-Anchored; Folic Acid - chemistry; Folic Acid - metabolism; General pharmacology; Humans; ligand affinity; Ligands; Medical Law; Medical sciences; Molecular biology; Molecular Structure; Nasopharynx - cytology; neoplasms; Pharmaceutical Preparations - chemistry; Pharmaceutical Preparations - metabolism; Pharmaceutical sciences; Pharmaceutical technology. Pharmaceutical industry; Pharmacology. Drug treatments; Pharmacology/Toxicology; Pharmacy; Protein Binding; Proteins; Pterins - chemistry; Pterins - metabolism; Receptors, Cell Surface - metabolism; Research Paper; Structure-Activity Relationship; Temperature; Vitamin B
• ISSN: 0724-8741; 1573-904X
• DOI: 10.1007/s11095-009-9840-3

Purpose Using in vitro competition assays, determine salient chemical features of pteroates and pteroate-drug conjugates which afford high affinity to the folate receptor. Materials and 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. Conclusion 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.