Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 45 (18), 10861-10871

Producing Irreversible Topoisomerase II-mediated DNA Breaks by Site-Specific Pt(II)-methionine Coordination Chemistry

Affiliations

Producing Irreversible Topoisomerase II-mediated DNA Breaks by Site-Specific Pt(II)-methionine Coordination Chemistry

Ying-Ren Wang et al. Nucleic Acids Res.

Abstract

Human type II topoisomerase (Top2) isoforms, hTop2α and hTop2β, are targeted by some of the most successful anticancer drugs. These drugs induce Top2-mediated DNA cleavage to trigger cell-death pathways. The potency of these drugs correlates positively with their efficacy in stabilizing the enzyme-mediated DNA breaks. Structural analysis of hTop2α and hTop2β revealed the presence of methionine residues in the drug-binding pocket, we therefore tested whether a tighter Top2-drug association may be accomplished by introducing a methionine-reactive Pt2+ into a drug to further stabilize the DNA break. Herein, we synthesized an organoplatinum compound, etoplatin-N2β, by replacing the methionine-juxtaposing group of the drug etoposide with a cis-dichlorodiammineplatinum(II) moiety. Compared to etoposide, etoplatin-N2β more potently inhibits both human Top2s. While the DNA breaks arrested by etoposide can be rejoined, those captured by etoplatin-N2β are practically irreversible. Crystallographic analyses of hTop2β complexed with DNA and etoplatin-N2β demonstrate coordinate bond formation between Pt2+ and a flanking methionine. Notably, this stable coordinate tether can be loosened by disrupting the structural integrity of drug-binding pocket, suggesting that Pt2+ coordination chemistry may allow for the development of potent inhibitors with protein conformation-dependent reversibility. This approach may be exploited to achieve isoform-specific targeting of human Top2s.

Figures

Figure 1.
Figure 1.
Chemical structures of etoposide and the two etoplatins synthesized in this study. The polycyclic aglycone (rings A–D) and pendant ring (E-ring) of etoposide are labeled. A cis-dichlorodiammineplatinum(II) moiety was introduced via an amide linkage to the C4 position of the aglycone core in α and β configuration about the E-ring to produce etoplatin-N2α and N2β, respectively. Both etoplatins contain an additional chiral center (marked with asterisks) whose chirality was not specified during synthesis.
Figure 2.
Figure 2.
Etoplatin-N2β, but not the N2α epimer, more potently inhibits the supercoil relaxation activity of Top2 by inducing the formation of ethylenediaminetetraacetic acid (EDTA)-resistant DNA breaks. (A) Inhibition of the relaxation activity of hTop2 by etoposide and etoplatins. Each relaxation reaction contains 300 ng of supercoiled (SC) pRYG plasmid DNA. The enzyme-positive reactions contain 80 ng of hTop2β△CTD (designated as hTop2β). OC stands for open circle (full-relaxed product produced by Top2) DNA. (B) The DNA cleavage assay shows the production of EDTA-resistant, hTop2-mediated DNA breaks in the presence of etoplatin-N2β. Each cleavage reaction contains 250 ng of HindIII-linearized (L) pRYG plasmid DNA. A total of 1.2 μg of hTop2β△CTD (upper panel, designated as hTop2β) or hTop2α△CTD (lower panel, designated as hTop2α) was added to the respective enzyme-positive reactions. To stop the cleavage reaction, Sodium dodecyl sulphate (SDS) and EDTA were added in the indicated order and the denatured enzyme was removed by proteinase K digestion. Lanes labeled with -ProK indicates no proteinase K treatment after the reaction was stopped. The disappearance and reemergence of the linear substrate DNA (L) indicate the production and resealing of DNA breaks, respectively. Refer to Supplementary Figure S2 for the full-sized image of this gel.
Figure 3.
Figure 3.
Both etoplatin-N2β and -N2α bind to the DNA cleavage sites in the hTop2βcc crystal structure as etoposide but only etoplatin-N2β forms the irreversible Pt2+–thioether coordinate bond. (A) The electron density maps of the bound drugs in the crystal structures of hTop2βcore-derived cleavage complexes stabilized by etoplatin-N2β (upper panel) and etoplatin-N2α (lower panel). The final 2DFo–mFc maps (at 1.5 σ) of the respective drugs are shown as blue meshes. Continuous electron density can be observed between Pt2+ and the Sδ of M782, indicating the formation of a coordinate bond. (B and C) Stereo views of the drug-binding site in the etoplatin-N2β- and etoplatin-N2α-stabilized cleavage complexes derived from hTop2βcore, respectively. The Pt2+–thioether coordination in the etoplatin-N2β-stabilized structure in B is specified by the red arrow. DNA is shown in sticks representation (blue) and labeled with positive and negative numbers to designate nucleotides downstream and upstream, respectively, of the scissile phosphate. The bound drugs are shown in sticks representation (yellow) with the Pt2+ in gray and Cl in green spheres. The drug’s aromatic rings are labeled in red letters. The two protein chains are shown in a cartoon/stick representation and colored in magenta and cyan, respectively. The scissile phosphate-linked active site Y821 is labeled with a prime to specify that this residue is from the second protein chain.
Figure 4.
Figure 4.
The etoplatin-N2β-mediated Pt2+–thioether coordination relies on the integrity of the hTop2cc structure. (A) The release of etoplatin-N2β (as reflected by the amount of membrane permeable Pt2+) from structurally intact guanidine hydrochloride (-GdnHCl) and denatured (+GdnHCl) Top2cc derived from hTop2α△CTD was quantified by inductively coupled plasma mass spectrometry (ICP-MS). The membrane permeable Pt2+ level of the +GdnHCl group gradually increases with time and was significantly higher than the -GdnHCl group started from 16 h. (*: P < 0.05, **: P < 0.01) (B) Cartoon representations illustrating that the release of etoplatin-N2β from Top2cc upon GdnHCl treatment (in vitro route) may be achieved by targeting Top2 to the 26S proteasome for degradation (in vivo route). Specifically, the trapped Top2cc on genomic DNA in the cell will be ubiquitinated (Ub) and channeled to the 26S proteasome for degradation (46,47). Upon structural disruption of its binding site, etoplatin-N2β can no longer form stable coordination with the targeting methionine, allowing its release from the protein.
Figure 5.
Figure 5.
Like etoposide, etoplatins induced chromosomal DNA breaks and cancer cell death in a hTop2-dependent manner. (A) Levels of etoposide- and etoplatin-induced chromosomal DNA breaks are significantly reduced in the hTop2-deficient HL-60/MX2 cells compared to the parental HL-60 cells. It has been shown that HL-60/MX2, a mitoxantrone-resistant variant of HL-60 cell line, expresses much lower levels of both hTop2α and hTop2β (28). Cells were treated with 50 μM of etoposide or etoplatins for 1 h, comet assay were then carried out to measure chromosomal DNA breaks and quantified data were shown in (B). In the microscopic images, each green circular head or comet (head + tail) image represents one cell. The green dots are indicative of intact nucleoids containing SC loops of chromosomal DNA attached to the nuclear matrix. Upon drug treatments, damaged nuclear DNA becomes relaxed due to the presence of DNA breaks resulting from drug-stabilized Top2cc and a tail of damaged DNA migrates toward the anode during electrophoresis, leading to the comet-like appearance. Values as determined by the percentage of cells with DNA damage are presented as mean ± SD (n = 3). (C) Etoplatin-N2β exhibits clear hTop2-dependent cancer cell killing activity. HL-60 leukemia cells were treated with 100 μM of etoposide, etoplatin-N2β or etoplatin-N2α, and after 4 days of incubation, MTT assay were performed to determine cytotoxicity. N.S. (not significant, P > 0.05); *P < 0.05; **P < 0.01; ***P < 0.001.

Similar articles

See all similar articles

Cited by 4 articles

References

    1. Zarzycka B., Kuenemann M.A., Miteva M.A., Nicolaes G.A., Vriend G., Sperandio O. Stabilization of protein-protein interaction complexes through small molecules. Drug Discov. Today. 2016; 21:48–57. - PubMed
    1. Potashman M.H., Duggan M.E. Covalent modifiers: an orthogonal approach to drug design. J. Med. Chem. 2009; 52:1231–1246. - PubMed
    1. Singh J., Petter R.C., Baillie T.A., Whitty A. The resurgence of covalent drugs. Nat. Rev. Drug Discov. 2011; 10:307–317. - PubMed
    1. Lavergne S.N., Park B.K., Naisbitt D.J. The roles of drug metabolism in the pathogenesis of T-cell-mediated drug hypersensitivity. Curr. Opin. Allergy Clin. Immunol. 2008; 8:299–307. - PubMed
    1. Uetrecht J. Immune-mediated adverse drug reactions. Chem. Res. Toxicol. 2009; 22:24–34. - PubMed

MeSH terms

Feedback