Synthesis, characterization, photochemistry and anticancer activity of novel photoactivatable platinum(IV) diazidodihydroxido complexes

PtIV-diazidodihydroxido complexes are inert in the dark, but can be selectively activated by irradiation with light and become potently cytotoxic towards cancer cells. By site-specific irradiation to tumour tissue, the side-effects to healthy tissue associated with conventional chemotherapeutics, such as cisplatin, can be circumvented. This thesis aims to develop design photoactivatable platinum(IV) diazidodihydroxido complexes to achieve higher photocytotoxicity, lower crossresistance and longer wavelength of activation. A series of PtIV diazidodihydroxido complexes with trans azido, trans hydryxido groups and mixed trans aliphatic/aromatic amines, was designed, synthesized and characterized. Trans, trans, trans-[Pt(N3)2(OH)2(MA)(Py)] (5) and trans, trans, trans-[Pt(N3)2(OH)2(MA)(Tz)] (8) are potently cytotoxic towards A2780, OE19 and HaCaT cell lines upon irradiation with UVA. Remarkably, they also showed potent cytotoxic effects towards A2780cis (cisplatin-resistant ovarian cancer cell subline). Also, the photocytotoxicity towards the A2780, A2780cis, OE19 and HaCaT cell lines upon irradiation with blue light (λmax = 420 nm) is still potent compared to that upon irradiation with UVA. These complexes are highly inert in the absence of light and have almost no dark toxicity. Upon irradiation with UVA/blue light, the complex 5 was observed to release free azide anions N3−, azidyl radicals N3•, nitrogen gas N2 and form nitrene intermediates. It was of importance to discover that singlet oxygen (1O2) is generated from photoreactions in the absence of an exogenous source of oxygen, whereas hydrogen peroxide (H2O2) and hydroxyl radical intermediates did not appear to be formed. Mono-functional and bi-functional Pt adducts were captured from the photoinduced binding of complexes 5 and 8 to 5'-GMP and a DNA oligonucleotide. It was discovered for the first time that the oxidation of 5'-GMP can occur during the photoreaction of complex 5 upon irradiation with UVA. Singlet oxygen and nitrene intermediate generated from this photoreaction are likely to be the cause of the oxidative damage to guanine. 4-Nitropyridine, 2,2'-bipyridine, and terpyridines were used as ligands in novel photoactivable PtIV (di)azido complexes and two were activated by green light. A new two-photon-activatable PtII complex, cis-[PtCl2(MOPEP)2](42), was also designed, synthesized and characterized. It was observed that this complex was sensitive to one-photon excitation below 500 nm and the ligand MOPEP underwent rapid solvent (acetonitrile) substitution upon irradiation. The same photoreaction was also triggered by two-photon excitation with fs-pulses laser light between 600 – 700 nm

Protein flexibility is key to cisplatin crosslinking in calmodulin

Chemical crosslinking in combination with Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) has significant potential for studying protein structures and proteinprotein interactions. Previously, cisplatin has been shown to be a crosslinker and crosslinks multiple methionine (Met) residues in apo-calmodulin (apo-CaM). However, the inter-residue distances obtained from nuclear magnetic resonance structures are inconsistent with the measured distance constraints by crosslinking. Met residues lie too far apart to be crosslinked by cisplatin. Here, by combining FTICR MS with a novel computational flexibility analysis, the flexible nature of the CaM structure is found to be key to cisplatin crosslinking in CaM. It is found that the side chains of Met residues can be brought together by flexible motions in both apo-CaM and calcium-bound CaM (Ca4-CaM). The possibility of cisplatin crosslinking Ca4-CaM is then confirmed by MS data. Therefore, flexibility analysis as a fast and low-cost computational method can be a useful tool for predicting crosslinking pairs in protein crosslinking analysis and facilitating MS data analysis. Finally, flexibility analysis also indicates that the crosslinking of platinum to pairs of Met residues will effectively close the nonpolar groove and thus will likely interfere with the binding of CaM to its protein targets, as was proved by comparing assays for cisplatin-modified/unmodified CaM binding to melittin. Collectively, these results suggest that cisplatin crosslinking of apo-CaM or Ca4-CaM can inhibit the ability of CaM to recognize its target proteins, which may have important implications for understanding the mechanism of tumor resistance to platinum anticancer drugs

Use of top-down and bottom-up fourier transform ion cyclotron resonance mass spectrometry for mapping calmodulin sites modified by platinum anticancer drugs

Calmodulin (CaM) is a highly conserved, ubiquitous, calcium-binding protein; it binds to and regulates many different protein targets, thereby functioning as a calcium sensor and signal transducer. CaM contains 9 methionine (Met), 1 histidine (His), 17 aspartic acid (Asp), and 23 glutamine acid (Glu) residues, all of which can potentially react with platinum compounds; thus, one-third of the CaM sequence is a possible binding target of platinum anticancer drugs, which represents a major challenge for identification of specific platinum modification sites. Here, top-down electron capture dissociation (ECD) was used to elucidate the transition metal–platinum(II) modification sites. By using a combination of top-down and bottom-up mass spectrometric (MS) approaches, 10 specific binding sites for mononuclear complexes, cisplatin and [Pt(dien)Cl]Cl, and dinuclear complex [{cis-PtCl2(NH3)}2(μ-NH2(CH2)4NH2)] on CaM were identified. High resolution MS of cisplatin-modified CaM revealed that cisplatin mainly targets Met residues in solution at low molar ratios of cisplatin–CaM (2:1), by cross-linking Met residues. At a high molar ratio of cisplatin:CaM (8:1), up to 10 platinum(II) bind to Met, Asp, and Glu residues. [{cis-PtCl2(NH3)}2(μ-NH2(CH2)4NH2)] forms mononuclear adducts with CaM. The alkanediamine linker between the two platinum centers dissociates due to a trans-labilization effect. [Pt(dien)Cl]Cl forms {Pt(dien)}2+ adducts with CaM, and the preferential binding sites were identified as Met51, Met71, Met72, His107, Met109, Met124, Met144, Met145, Glu45 or Glu47, and Asp122 or Glu123. The binding of these complexes to CaM, particularly when binding involves loss of all four original ligands, is largely irreversible which could result in their failure to reach the target DNA or be responsible for unwanted side-effects during chemotherapy. Additionally, the cross-linking of cisplatin to CaM might lead to the loss of the biological function of CaM or CaM–Ca2+ due to limiting the flexibility of the CaM or CaM–Ca2+ complex to recognize target proteins or blocking the binding region of target proteins to CaM

De novo generation of singlet oxygen and ammine ligands by photoactivation of a platinum anticancer complex

Worth the excitement: Highly reactive oxygen and nitrogen species are generated by photoactivation of the anticancer platinum(IV) complex trans,trans,trans-[Pt(N3)2(OH)2(MA)(Py)] (MA=methylamine, Py=pyridine). Singlet oxygen is formed from the hydroxido ligands and not from dissolved oxygen, and ammine ligands are products from the conversion of azido ligands to nitrenes. Both processes can induce oxidation of guanine