A metal-free DNA nuclease based on a cyclic peptide scaffold.
The ability to cleave DNA with the aid of chemical nucleases has been a challenge in the scientific community, particularly in the absence of a redox active metal ion. Inspired by structural characterization of the active site found in Staphylococcal nuclease, we have designed a series of organic molecule comprising cyclic pentapeptides conjugated to a DNA intercalator (e.g., anthraquinone). The cyclic peptide is designed to cleave the phosphodiester backbone, whereas the intercalator is expected to improve binding affinity to the substrate (DNA). Our lead compound (1-AQ), composed of the cyclic peptide cyc-d-Lys-Gly-Arg-Ser-Arg conjugated to anthraquinone, degrades DNA into small fragments at physiologically relevant conditions (i.e., 37 degrees C, pH = 7.4). We find that 1-AQ is highly effective in degrading duplex DNA at micromolar concentrations as corroborated by agarose and polyacrylamide gel electrophoresis. Changing the DNA intercalator to acridine (1-Ac) renders the compound comparable in nuclease activity to 1-AQ. In comparison to control compounds (Lin-1 and 1) that lack either the cyclic scaffold or the DNA intercalator, our lead compound (1-AQ) is found to be significantly more active as a DNA chemical nuclease. We have studied the importance of the triad (Arg-Ser-Arg) as the designed module for DNA cleavage. Changing l-Ser to l-Glu (cyc-d-Lys-Gly-Arg-Glu-Arg, Glu-AQ) results in an inactive compound, whereas the cyclic peptide Gly-AQ (cyc-d-Lys-Gly-Arg-Gly-Arg, where glycine replaces l-serine) has similar DNA nuclease activity to 1-AQ. In addition, changing the stereochemistry from d-lysine to l-lysine results in a cyclic peptide (1-L-AQ) exerting weak DNA nuclease activity, highlighting the importance of the cyclic backbone conformation for efficient DNA nuclease activity. The addition of ROS scavengers does not reduce DNA nuclease activity; an observation that supports a hydrolytic cleavage mechanism. Finally, we have estimated the kinetics of DNA cleavage of a 15-mer duplex DNA substrate by compound 1-AQ. By monitoring DNA duplex degradation by following the change in absorbance (hyperchromicity) at various 1-AQ concentrations, we report a maximal k(obs) value (as an underestimation of k(max)) of 1.62 h(-1) at a 7.5-fold of 1-AQ. We have also compared the other two active peptide conjugates, namely, 1-Ac and Gly-AQ to that of 1-AQ. Both compounds exert similar nuclease activity to that of 1-AQ. To the best of our knowledge, this is the most active metal-free DNA nuclease reported to date that exerts its DNA nuclease activity at biologically relevant conditions.
Alkhader
Shadad
S
The Institute for Drug Research, The School of Pharmacy, The Hebrew University of Jerusalem, Hadassah Ein-Karem, Jerusalem, Israel.
Ezra
Aviva
A
Kasparkova
Jana
J
Brabec
Viktor
V
Yavin
Eylon
E
D000880:Anthraquinones / Q000737:chemistry; D004247:DNA / Q000138:chemical synthesis / Q000737:chemistry* / Q000187:drug effects; D053837:DNA Cleavage / Q000187:drug effects; D003851:Deoxyribonucleases / Q000737:chemistry* / Q000378:metabolism; D015394:Molecular Structure; D010456:Peptides, Cyclic / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D013237:Stereoisomerism
D000880:Anthraquinones; D010456:Peptides, Cyclic; D004247:DNA; D003851:Deoxyribonucleases