Crosslinking Allosteric Sites on the Nucleosome Core Particle

Designing compounds that bind to the acidic patch of the nucleosome core particle (NCP) is an emerging therapeutic approach that can inhibit the binding of key chromatin factors. Pretreatment of human ovarian carcinoma, A2780, cells with RAPTA-T, an antimetastic agent which also binds to the acidic patch, sensitises the cells to auranofin, an antiarthritic agent, resulting in a three-fold increase in gold(I)-chromatin adducts. This synergism is accompanied by an allosteric relationship between RAPTA-T and auranofin. The binding of RAPTA-T to the carboxylate E61/E64 residues on the H2A protein (RU1) and imidazole H106 and carboxylate E102 residues of the H2B dimer (RU2) triggers a series of allosteric modifications that opens the previously inaccessible symmetry-related imidazole sites, H3 H113 (AU1) and H113’ (AU1’), on the H3-H4 tetramer. This series of systematic alterations in the inter-helical orientations of the H2A and H3/H3’ α-helices connecting the RU1/RU2 and AU1/AU1’ binding sites result in more compact AU1/AU1’ sites that favours auranofin binding. Based on this allosteric effect, multi-nuclear homo- and heterometallic drugs were designed and synthesised with the aim of targeting the RAPTA-T and auranofin sites on the NCP. Chapter 1 is an introduction into crosslinking anticancer agents inspired by cisplatin, RAPTA-T and auranofin. Both homo- and heterometallic complexes possessing the ability to form DNA-DNA, protein-protein and DNA-protein crosslinks are discussed. Chapter 2 follows an investigation into the impact of linker length on the cytotoxicity of homobimetallic ruthenium(II) and gold(I) complexes inspired by RAPTA-C and auranofin. As the lipophilicity of polyethylene glycol linkers plateaus between PEG6 and PEG10, the impact of linker length on the cytotoxicity of the resulting dinuclear complexes, independent of increasing lipophilicity, can be observed. Chapter 3 discusses the design, synthesis and in vitro cytotoxicity of a series of hetero-bimetallic ruthenium(II)-gold(I) complexes linked via a bis-phosphine ligand bearing different lengths of polyethylene glycol chains. The affinity of the complexes for histidine residues was assessed on ι-histidine and the 1-16 sequence of the amyloid β protein using mass spectrometry, demonstrating that the complexes have the potential bind to the RU2, AU1 and AU1’sites on the NCP. Chapter 4 describes the synthesis of a heterobimetallic ruthenium(II)-gold(I) complex linked via the arene of the RAPTA-moiety with a long polyethylene glycol linker. The interactions of the complex on the NCP were studied using X-ray crystallography and molecular dynamic simulations revealing that the complex can crosslink the RU1 and AU1 binding sites. A trinuclear diruthenium(II)-gold(I) complex, that has the potential to crosslink the RU1, RU2 and AU1 binding sites, is also described. Chapter 5 explores the possibility of igniting a similar allosteric effect as RAPTA-T using alternative molecules. Residues 1-23 Kaposi’s sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) and residues 535-551 prototype foamy virus (PFV) structural protein (GAG) bind to the acidic patch of the NCP. Gold(I)-peptide conjugates containing these peptide sequences were prepared to assess if the binding of the peptide can cause a structural alteration in the NCP that allows the gold(I) centre to bind to the AU1or AU1’ binding site.