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Peptide Inhibition of Topoisomerase IB from Plasmodium falciparum

  • Roy A
  • D'Annessa I
  • Nielsen C
  • et al.
Molecular Biology International (2011) 2011 1-10
DOI: 10.4061/2011/854626
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Abstract

Control of diseases inflicted by protozoan parasites such as Leishmania, Trypanosoma , and Plasmodium , which pose a serious threat to human health worldwide, depends on a rather small number of antiparasite drugs, of which many are toxic and/or inefficient. Moreover, the increasing occurrence of drug-resistant parasites emphasizes the need for new and effective antiprotozoan drugs. In the current study, we describe a synthetic peptide, WRWYCRCK, with inhibitory effect on the essential enzyme topoisomerase I from the malaria-causing parasite Plasmodium falciparum. The peptide inhibits specifically the transition from noncovalent to covalent DNA binding of P. falciparum topoisomerase I, while it does not affect the ligation step of catalysis. A mechanistic explanation for this inhibition is provided by molecular docking analyses. Taken together the presented results suggest that synthetic peptides may represent a new class of potential antiprotozoan drugs.

Figures

  • Figure 1: Effect of peptides on pfTopoI-mediated DNA relaxation. The effect of peptides on relaxation was assayed by incubating 200 fmol supercoiled plasmid with enzyme and peptide WRWYCRCK, KCCRCK, or WYCRCK at the following concentrations: 1.3 μM, 2.5 μM, 5 μM, 7.5 μM, 12.5 μM, 25 μM, or 50 μM. (a) Representative gel picture showing the relaxation activity of pfTopoI in the presence of increasing concentrations of peptide WRWYCRCK. (b) Representative gel picture showing the relaxation activity of pfTopoI in the presence of increasing concentrations of peptide KCCRCK. (c) Representative gel picture showing the relaxation activity of pfTopoI in the presence of increasing concentrations of peptide WYCRCK. C: negative control lane without any enzyme added; 0: positive control lane with pfTopoI but no peptide added; SC: supercoiled plasmid; Rn: relaxed topoisomers; Cat: supercoiled catenated plasmid.
  • Figure 2: Effect of peptide WRWYCRCK on pfTopoI-mediated DNA cleavage. (a) Schematic depiction of the cleavage and religation reactions. The substrate (OL19/OL27) used for assaying cleavage allows covalent attachment of the enzyme to the 3′ end of the 5′- radiolabeled scissile strand (OL19) by cleaving off a trinucleotide. Ligation is prevented by diffusion of the trinucleotide. To initiate ligation, the ligator strand (OL36) is added to covalent cleavage complexes generated by incubating pfTopoI with radiolabeled OL19/OL27. (b) Gel picture showing the cleavage products obtained by incubating 5′-radiolabeled OL19/OL27 with pfTopoI (lane 1) or hTopoI (lane 2). (c) Graphical depiction of the cleavage activity of pfTopI plotted as a function of peptide WRWYCRCK concentration. The cleavage activity was calculated as described in Section 2. (d) Representative gel picture showing the ligation activity of pfTopoI in the presence of peptide WRWYCRCK at the following concentrations: 1.3 μM, 2.5 μM, 5 μM, 7.5 μM, or 12.5 μM. T: topoisomerase I; asterisk: 5′-radiolabel with [γ-32P]; filled circle: 5′-cold phosphorylation; S: substrate; Cl1: cleavage product resulting from cleavage at the black arrow in the schematic depiction; Cl2: cleavage product resulting from cleavage two nucleotides upstream of the black arrow in the schematic depiction; C: negative control lanes without any enzyme added; Cl: cleavage control lane without ligator strand added; 0: positive control lane with pfTopoI but no peptide added.
  • Figure 3: Effect of peptide WRWYCRCK on restriction digestion by restriction endonucleases. Representative gel picture showing the result of incubating pUC19 plasmid with EcoRI (lanes 4–7) or PvuII (lanes 8–11) in the presence of peptide WRWYCRCK at the following concentration: 12.5 μM, 25 μM, or 50 μM. The sizes, kbp, of the DNA marker (lane 1, labeled M) are shown to the left of the gel picture. 0: control lanes with no peptide added; +: control lane with 50 μM peptide added; asterisks indicate the gel electrophoretic mobility of the digested plasmid, for EcoRI, 2.7 kbp, and for PvuII, 0.3 kbp and 2.4 kbp.
  • Figure 4: Docking of peptides into the noncovalent pfTopoI-DNA complex. Side view of the ternary pfTopoI-DNA-peptide complex. The structure represents the best complex obtained from the docking experiment. The protein is shown in red cartoon, the DNA in grey ribbon and the peptide in licorice, with the atoms coloured with the following code: carbon: cyan, nitrogen: blue, oxygen: red, sulphur: yellow. (a) Octapeptide WRWYCRCR, (b) hexapeptide WYCRCR, and (c) hexapeptide KCCRCR.
  • Table 1: Contacts between the peptide WRWYCRCK, and the noncovalent pfTopoI-DNA complex calculated for the best docked complex.

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APA

Roy, A., D’Annessa, I., Nielsen, C. J. F., Tordrup, D., Laursen, R. R., Knudsen, B. R., … Andersen, F. F. (2011). Peptide Inhibition of Topoisomerase IB from Plasmodium falciparum. Molecular Biology International, 2011, 1–10. https://doi.org/10.4061/2011/854626

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