title: Combining combinatorial chemistry and affinity chromatography: highly selective inhibitors of human betaine: homocysteine S-methyltransferase.
abstract: A new method to find novel protein targets for ligands of interest is proposed. The principle of this approach is based on affinity chromatography and combinatorial chemistry. The proteins within a crude rat liver homogenate were allowed to interact with a combinatorial library of phosphinic pseudopeptides immobilized on affinity columns. Betaine: homocysteine S-methyltransferase (BHMT) was one of the proteins that was retained and subsequently eluted from these supports. The phosphinic pseudopeptides, which served as immobilized ligands for the isolation of rat BHMT, were then tested for their ability to inhibit human recombinant BHMT in solution. The most potent inhibitor also behaved as a selective ligand for the affinity purification of BHMT from a complex media. Further optimization uncovered Val-Phe-psi[PO(2-)-CH(2)]-Leu-His-NH(2) as a potent BHMT inhibitor that has an IC(50) of about 1 microM.
authors.affiliation: Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, 16610 Praha 6, Czech Republic.
authors.forename: Michaela
authors.initials: M
authors.lastname: Collinsová
authors.forename: Carmen
authors.initials: C
authors.lastname: Castro
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Garrow
authors.forename: Athanasios
authors.initials: A
authors.lastname: Yiotakis
authors.forename: Vincent
authors.initials: V
authors.lastname: Dive
authors.forename: Jirí
authors.initials: J
authors.lastname: Jirácek
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D050940:Betaine-Homocysteine S-Methyltransferase; D015153:Blotting, Western; D002846:Chromatography, Affinity; D020650:Combinatorial Chemistry Techniques; D004591:Electrophoresis, Polyacrylamide Gel; D004746:Enoyl-CoA Hydratase / Q000037:antagonists & inhibitors; D004791:Enzyme Inhibitors / Q000138:chemical synthesis* / Q000494:pharmacology*; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors; D006801:Humans; D066298:In Vitro Techniques; D008024:Ligands; D008099:Liver / Q000378:metabolism; D008297:Male; D008780:Methyltransferases / Q000037:antagonists & inhibitors*; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification; D010721:Phosphinic Acids / Q000138:chemical synthesis; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000138:chemical synthesis / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D008024:Ligands; D019151:Peptide Library; D010455:Peptides; D010721:Phosphinic Acids; D011994:Recombinant Proteins; D008780:Methyltransferases; C494548:BHMT protein, human; D050940:Betaine-Homocysteine S-Methyltransferase; C494550:Bhmt protein, rat; D005982:Glutathione Transferase; D004746:Enoyl-CoA Hydratase