RESUMO
Peptide libraries displayed by T7 phage, which contain random cDNA fragments insets, were screened for their ability to bind to a biotinylated derivative of clarithromycin. Phage particles bound to an immobilized derivative of the antibiotic were isolated and the inserted cDNA was amplified and sequenced. A common selected peptide sequence, composed of 19 amino acids, was obtained and a synthetic peptide with this sequence was produced. Surface plasmon resonance experiments showed that the synthetic peptide immobilized on a sensor chip bound to clarithromycin and the dissociation constant was determined to be 2.1 x 10(-3) M. The dissociation constants of other macrolide antibiotics, erythromycin, roxithromycin, azithromycin and josamycin were also determined to be 5.4 x 10(-3) M, 6.2 x 10(-5) M, 1.1 M and 3.4 x 10(-2) M, respectively. These results indicated that T7 phage display method might be useful to determine relatively weak interactions between small molecule drugs and the selected peptides which could represent a possible binding site conserved in binding proteins.
Assuntos
Antibacterianos/metabolismo , Bacteriófago T7/genética , Claritromicina/metabolismo , Biblioteca de Peptídeos , Peptídeos/metabolismo , Sequência de Aminoácidos , Biotinilação , Claritromicina/análogos & derivados , Biblioteca Gênica , Ligantes , Dados de Sequência Molecular , Estrutura Molecular , Peptídeos/química , Peptídeos/genética , Ligação Proteica , Ressonância de Plasmônio de SuperfícieRESUMO
During screening for mammalian DNA polymerase inhibitors, we found and succeeded in isolating a potent inhibitor from a higher plant, Taxus cuspidate. The compound was unexpectedly determined to be taxinine, an intermediate of paclitaxel (taxol) metabolism. Taxinine was found to selectively inhibit DNA polymerase alpha (pol.alpha) and beta (pol.beta). We therefore, tested taxol and other derivatives and found that taxol itself had no such inhibitory effect, and only taxinine could inhibit both pol.alpha and beta. The other compounds used, one derivative, cephalomannine, and five intermediates synthesized chemically inhibited only the pol.alpha activity in vitro. None of the compounds, including taxinine, influenced the activities of the other DNA polymerases, which are reportedly targeted by many pol.beta inhibitors. With both pol.alpha and beta, all of the compounds tested noncompetitively inhibited with respect to both the DNA template-primer and the dNTP substrate.
