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Metal Anti-Tumor Agents

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The discovery of the anticancer drug cisplatin over 40 years ago spawned a new era of modern metal-based pharmaceuticals. Among the recognized non-platinum containing antitumor agents are dinuclear carboxylate species of Rh, Re and Ru but early investigations that commenced in the 1970’s steadily declined, in part, due to the fact that the compounds did not surpass the anticancer activity of cisplatin. The revival of this research area in our laboratories, and, indeed by others working in the field of metals in medicine over the past decade, has led to renewed interest in the cellular metabolism and biological targets of metal antitumor active compounds.

We are investigating the interactions ofmetal-metal bonded systems vis-à-vis their interactions with DNA, the primary target of platinum and platinum group metal anticancer agents. Our work has unearthed new concepts regarding viable substitution pathways and binding modes of dimetal units with DNA1 the results of which are being applied to the design of new and improved dirhodium anticancer active candidates.

The primary targets under investigation in our laboratories are dirhodium paddlewheel complexes of the type Rh2(O2CR)4L2 (Figure 1A) or [Rh2(O2CCH3)2(N-N)2L2]2+ (Figure 1B; N-N = chelating ligands such as bpy, dppz).2 Their adducts with DNA fragments are studied by 1D and 2D NMR spectroscopies, mass spectrometry, as well as molecular modeling. Our unprecedented findings indicate that, contrary to conventional wisdom, the equatorial sites in the dirhodium complexes are favorably poised to bind DNA purine bases (e.g., guanine, adenine) and DNA fragments (e.g., d(GpG), d(pGpG), d(ApA), d(GpA), d(ApG));3-6 these interactions lead to the formation of rare nucleobase tautomers implicated in DNA mutations. Superposition of the low energy Rh2(O2CCH3)2{d(pGpG)} conformer, generated by from 2D NMR data and simulated annealing calculations, with the crystal structure of cis-[Pt(NH3)2{d(pGpG)}], reveals remarkable similarities between the adducts; the two octahedral rhodium atoms are capable of engaging in cis binding to GG intrastrand sites by establishing N7/O6 bridges that span the Rh–Rh bond (Figure 2).4  Furthermore, detailed 2D NMR data analyses performed on the double-stranded DNA 12mer d(CTCTC5*A6*ACTTCC)·d(GGAAGTTGAGAG) revealed that the duplex forms an intrastrand adduct with the dirhodium unit binding to residues C5 and A6 (Figure 3), which significantly destabilizes the duplex.7 Coordinative binding, to the dirhodium unit, of A6 takes place via N7 whereas binding of the C5 base takes place via the exocyclic N4 site resulting in the anti cytosine rotamer in its metal-stabilized rare iminooxo form.7 Formation and stabilization of rare base forms and mispairs can be one of the factors that contribute to the antitumor activity of dirhodium compounds.


Figure 1. Structures of (A) Rh2(O2CR)4L2 and (B) [Rh2(O2CCH3)2(N-N)2L2]2+.


Figure 2. Superposition of low energy Rh2(O2CCH3)2{d(pGpG)} conformer (colored model), generated from 2D NMR data and simulated annealing calculations, with the crystallographically determined cis-[Pt(NH3)2{d(pGpG)}] (light blue).


Figure 3. Model of the DNA duplex d(CTCTC*A*ACTTCC)·d(GGAAGTTGAGAG) cross-linked by the dirhodium unit cis-[Rh2(m-O2CCH3)2(h1-O2CCH3)]+at the cytosine-adenine step; DNA base colors: A: pink; C: light blue; G: green; T: yellow. Blue ribbon: phosphodiester backbone. Atom colors: Rh: green; O: red; C: grey; H: white.


Figure 4. Phase contrast and fluorescent image of HeLa cells treated with Rh2(O2CCH3)4. Left: Phase contrast. Center: SYTOX® Blue fluorescence emission. Right: Overlay of the phase contrast and SYTOX® Blue fluorescence emission (pseudo-colored blue) images.

In our laboratories, we also perform cytotoxicity studies of various cancer cell lines (e.g., Hela cells) as well as biochemical techniques (e.g., gel electrophoresis, PAGE, viscosity measurements) in the presence of dirhodium compounds to further probe their interactions with biological targets of interest such as key enzymes of the cellular cycle (e.g., polymerases, topoisomerases and endonucleases). To better understand their possible mechanism(s) of activity, the interactions of dirhodium complexes with DNA are studied in vitro and in cellulo. For the in vitro experiments we use different biophysical assays including viscosity measurements, determination of DNA binding constants and photocleavage experiments whereas for the in cellulo experiments we apply different techniques such as the comet assay, the annexin V assay, the glutathione modification assay and fluorescence dye techniques (Figure 4).

References
1. Chifotides, H. T.; Dunbar, K. R. Acc. Chem. Res., 2005, 38, 146-156.
2. H. T. Chifotides and K. R. Dunbar ‘Rhodium Compounds’, Chapter 12 In ‘Multiple Bonds Between Metal Atoms’, 3rd Edition, F. A. Cotton, C. Murillo and R.A. Walton, Eds., Springer-Science and Business Media, Inc.: New York, 2005, pp 465-589.
3. Chifotides, H. T.; Koshlap, K. M.; Pérez, L. M.; Dunbar, K. R. J. Am. Chem. Soc. 2003, 125, 10703-10713.
4. Chifotides, H. T.; Koshlap, K. M.; Pérez, L. M.; Dunbar, K. R. J. Am. Chem. Soc. 2003, 125, 10714-10724.
5. Chifotides, H. T.; Dunbar, K. R. J. Am. Chem. Soc. 2007, 129, 12480-12490.
6. Chifotides, H. T.; Dunbar, K. R. Chem. Eur. J. 2008, 14, 9902-9913.
7. Kang, M.; Chifotides, H. T.; Dunbar, K. R. Biochemistry, 2008, 47, 2265-2276.

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Department of Chemistry | Texas A&M University | State of Texas