Characterization and initial demonstration of in vivo efficacy of a novel heat-activated metalloenediyne anti-cancer agent.

BACKGROUND: Enediynes are anti-cancer agents that are highly cytotoxic due to their propensity for low thermal activation of radical generation. The diradical intermediate produced from Bergman cyclization of the enediyne moiety may induce DNA damage and cell lethality. The cytotoxicity of enediynes and difficulties in controlling their thermal cyclization has limited their clinical use. We recently showed that enediyne toxicity at 37 °C can be mitigated by metallation, but cytotoxic effects of 'metalloenediynes' on cultured tumor cells are potentiated by hyperthermia. Reduction of cytotoxicity at normothermia suggests metalloenediynes will have a large therapeutic margin, with cell death occurring primarily in the heated tumor. Based on our previous in vitro findings, FeSO4-PyED, an Fe co-factor complex of (Z)-N,N'-bis[1-pyridin-2-yl-meth-(E)-ylidene]oct-4-ene-2,6-diyne-1,8-diamine, was prioritized for further in vitro and in vivo testing in normal human melanocytes and melanoma cells. METHODS: Clonogenic survival, apopotosis and DNA binding assays were used to determine mechanisms of enhancement of FeSO4-PyED cytotoxicity by hyperthermia. A murine human melanoma xenograft model was used to assess in vivo efficacy of FeSO4-PyED at 37 or 42.5 °C. RESULTS: FeSO4-PyED is a DNA-binding compound. Enhancement of FeSO4-PyED cytotoxicity by hyperthermia in melanoma cells was due to Bergman cyclization, diradical formation, and increased apoptosis. Thermal enhancement, however, was not observed in melanocytes. FeSO4-PyED inhibited tumor growth when melanomas were heated during drug treatment, without inducing normal tissue damage. CONCLUSION: By leveraging the unique thermal activation properties of metalloenediynes, we propose that localized moderate hyperthermia can be used to confine the cytotoxicity of these compounds to tumors, while sparing normal tissue.

Enhancement of Cytotoxicity of Enediyne Compounds by Hyperthermia: Effects of Various Metal Complexes on Tumor Cells.

Enediyne natural products are a class of compounds that were recognized for their potential as chemotherapeutic agents many years ago, but found to be highly cytotoxic due to their propensity for low thermal activation. Bergman cyclization of the enediyne moiety produces a diradical intermediate, and may subsequently induce DNA damage and account for the extreme cytotoxicity. While difficulties in controlling the thermal cyclization reaction have limited the clinical use of cyclic enediynes, we have previously shown that enediyne activity, and thus toxicity at physiological temperatures can be modulated by metallation of acyclic enediynes. Furthermore, the cytotoxicity of "metalloenediynes" can be potentiated by hyperthermia. In this study, we characterized a suite of novel metallated enediyne motifs that usually induced little or no cytotoxicity when two different human cancer cell lines were treated with the compounds at 37°C, but showed a significant enhancement of cytotoxicity after cells were exposed to moderate hyperthermia during drug treatment. Cultured U-1 melanoma or MDA-231 breast cancer cells were treated with various concentrations of Cu, Fe and Zn complexes of the enediyne (Z)-N,N'-bis[1-pyridyl-2-yl-meth-(E)-ylidene]octa-4-ene-2,6-diyne-1,8-diamine (PyED) and clonogenic survival was assessed to determine the effects of the drugs at 37°C and 42.5°C. Toxicity at 37°C varied for each compound, but hyperthermia potentiated the cytotoxicity of each compound in both cell lines. Cytotoxicity was concentration-, time- and temperature-dependent. Heating cells during drug treatment resulted in enhanced apoptosis, but the role of cell cycle perturbation in the response of the cells to the drugs was less clear. Lastly, we showed that hyperthermia enhanced the number of DNA double-strand breaks (DSBs) induced by the compounds, and inhibited their repair after drug treatment. Thus, thermal enhancement of cytotoxicity may be due, at least in part, to the propensity of the enediyne moiety to induce DSBs, and/or a reduction in DSB repair efficiency. We propose that "tuning" of metalloenediyne toxicity through better-controlled reactivity could have potential clinical utility, since we envision that such compounds could be administered systemically as relatively non-toxic agents, but cytotoxicity could be enhanced in, and confined to a tumor volume when subjected to localized heating.

Metal-mediated diradical tuning for DNA replication arrest via template strand scission.

A series of M(PyED)·X (X = 2Cl-, SO42-) pyridine-metalloenediyne complexes [M = Cu(II), Fe(II), or Zn(II)] and their independently synthesized, cyclized analogs have been prepared to investigate their potential as radical-generating DNA-damaging agents. All complexes possess a 1:1 metal-to-ligand stoichiometry as determined by electronic absorption spectroscopy and X-ray diffraction. Solution structural analysis reveals a pπ Cl [Formula: see text] Cu(II) LMCT (22,026 cm-1) for Cu(PyED)·2Cl, indicating three nitrogens and a chloride in the psuedo-equatorial plane with the remaining pyridine nitrogen and solvent in axial positions. EPR spectra of the Cu(II) complexes exhibit an axially elongated octahedron. This spectroscopic evidence, together with density functional theory computed geometries, suggest six-coordinate structures for Cu(II) and Fe(II) complexes and a five-coordinate environment for Zn(II) analogs. Bergman cyclization via thermal activation of these constructs yields benzannulated product indicative of diradical generation in all complexes within 3 h at 37 °C. A significant metal dependence on the rate of the reaction is observed [Cu(II) > Fe(II) > Zn(II)], which is mirrored in in vitro DNA-damaging outcomes. Whereas in situ chelation of PyED leads to considerable degradation in the presence of all metals within 1 h under hyperthermia conditions, Cu(II) activation produces >50% compromised DNA within 5 min. Additionally, Cu(II) chelated PyED outcompetes DNA polymerase I to successfully inhibit template strand extension. Exposure of HeLa cells to Cu(PyBD)·SO4 (IC50 = 10 µM) results in a G2/M arrest compared with untreated samples, indicating significant DNA damage. These results demonstrate metal-controlled radical generation for degradation of biopolymers under physiologically relevant temperatures on short timescales.