New dsDNA binding unnatural oligopeptides with pyrimidine selectivity.
Solid phase peptide library screening followed by extension of a lead recognition element for binding to a dsDNA sequence (NF binding site of IL6) using solution phase screening, delivered a new DNA binding peptide, Ac-Arg-Ual-Sar-Chi-Chi-Tal-Arg-CONH2. In the present research, the contribution of the different amino acid side chains to the binding strength of the peptide to dsDNA was investigated using an ethidium bromide displacement test. Based on these results, the lead structure was optimized by deconvolution. Eight new unnatural amino acids were evaluated at two positions of the heptapeptide replacing the Ual-Sar fragment. The strongest dsDNA binding was observed using ([(3-chlorophenyl)methyl]amino)acetic acid (Cbg) and beta-cyclohexyl-l-alanine (Cha) respectively, at those two positions. A 10-fold increase in affinity compared to the Ual-Sar sequence was obtained. Further enhancement of dsDNA binding was obtained with hybrid molecules linking the newly developed peptide fragment to an acridine derivative with a flexible spacer. This resulted in ligands with affinities in the microM range for the dsDNA target (K(d) of 2.1 x 10(-6) M). DNase I footprinting with the newly developed oligopeptide motifs showed the presence of a pronounced pyrimidine specificity, while conjugation to an intercalator seems to redirect the interaction to mixed sequences. This way, new unnatural oligopeptide motifs and hybrid molecules have been developed endowed with different sequence selectivities. The results demonstrate that the unnatural peptide library approach combined with subsequent modification of selected amino acid positions, is very suited for the discovery of novel sequence-specific dsDNA binding ligands.
Zhang
Zhenyu
Z
Laboratory of Medicinal Chemistry, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, Leuven, Belgium.
Chaltin
Patrick
P
Van Aerschot
Arthur
A
Lacey
Jeff
J
Rozenski
Jef
J
Busson
Roger
R
Herdewijn
Piet
P
D000596:Amino Acids / Q000737:chemistry; D001665:Binding Sites / Q000187:drug effects; D001667:Binding, Competitive; D004247:DNA / Q000187:drug effects / Q000378:metabolism*; D018983:DNA Footprinting; D003850:Deoxyribonuclease I / Q000737:chemistry; D024202:Electrophoretic Mobility Shift Assay; D004996:Ethidium / Q000378:metabolism; D007202:Indicators and Reagents; D007364:Intercalating Agents; D015850:Interleukin-6 / Q000378:metabolism; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism*; D019151:Peptide Library; D011485:Protein Binding; D011743:Pyrimidines / Q000378:metabolism*; D013329:Structure-Activity Relationship
D000596:Amino Acids; D007202:Indicators and Reagents; D007364:Intercalating Agents; D015850:Interleukin-6; D008024:Ligands; D009842:Oligopeptides; D019151:Peptide Library; D011743:Pyrimidines; D004247:DNA; D003850:Deoxyribonuclease I; D004996:Ethidium