title: Immobilization of malarial (Plasmodium falciparum) dihydrofolate reductase for the selection of tight-binding inhibitors from combinatorial library.
abstract: A simple procedure for selection of tight-binding inhibitors of mutant dihydrofolate reductases from Plasmodium falciparum (PfDHFRs) based on preferential binding to the enzyme immobilized on a Sepharose column has been described. PfDHFRs with a cysteine residue at the C-terminal have been prepared in order to immobilize to a thiopropyl-Sepharose gel via S-S linkage. The amount of immobilized DHFRs was estimated to be 4-5 mg/g of dried gel, and the activities of bound DHFRs were comparable to that of free enzymes. The prepared immobilized enzyme has been used for the selection of tight-binding inhibitors from combinatorial libraries, based on the affinities of each ligand with the enzyme. Free ligands were then identified and analyzed quantitatively by high-performance liquid chromatography-mass spectrometry, and the components with high binding affinity of the library could thus be realized. Results could be confirmed by quantitative analysis of the bound ligands released from the enzyme by guanidine hydrochloride treatment.
authors.affiliation: National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Klong 1, Klongluang, Pathumtani 12120, Thailand. chawanee@biotec.or.th
authors.forename: Chawanee
authors.initials: C
authors.lastname: Thongpanchang
authors.forename: Supannee
authors.initials: S
authors.lastname: Taweechai
authors.forename: Sumalee
authors.initials: S
authors.lastname: Kamchonwongpaisan
authors.forename: Yongyuth
authors.initials: Y
authors.lastname: Yuthavong
authors.forename: Yodhathai
authors.initials: Y
authors.lastname: Thebtaranonth
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D020650:Combinatorial Chemistry Techniques; D003545:Cysteine / Q000737:chemistry; D004800:Enzymes, Immobilized; D005493:Folic Acid Antagonists / Q000302:isolation & purification*; D019791:Guanidine / Q000494:pharmacology; D008024:Ligands; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D019151:Peptide Library; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012685:Sepharose / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013440:Sulfides / Q000737:chemistry; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D004800:Enzymes, Immobilized; D005493:Folic Acid Antagonists; D008024:Ligands; D019151:Peptide Library; D013438:Sulfhydryl Compounds; D013440:Sulfides; D012685:Sepharose; D013762:Tetrahydrofolate Dehydrogenase; D019791:Guanidine; D003545:Cysteine