Many chemotherapeutic drugs interact with DNA to induce cytotoxicity. Mass spectrometry has become an essential technique in the investigation and identification of anticancer DNA adducts. Traditionally, identification of therapeutic DNA adducts was conducted by P1 enzymatic digestion followed by separation via gel electrophoresis or high performance liquid chromatography (HPLC). Structural information about binding was identified via NMR and x-ray crystallography. These methods are arduous and require significant sample consumption. Mass spectrometry is a high-through put methodology that requires a minimal amount of sample consumption to produce site specific binding information. Anticancer agents may bind directly to DNA via formation of a covalent bond creating a monoadduct, a single covalent bond at one site of double stranded DNA, or a crosslink, two covalent bonds on each strand of duplex DNA. Cytotoxicity of covalent anticancer agents is achieved by effectively blocking replication of DNA, thus preventing proliferation of cancerous cells. Much effort has been directed in the search for new chemotherapies to increase binding specificity for cancerous cells. In designing new drugs it is essential to understand DNA interactive properties--such as differences in the cellular conditions, the number of nucleobases within the binding site, and the tendency to form a monoadduct or a crosslink. These factors can then be exploited to design more selective anti-cancer drugs. Several covalent bond-forming anti-cancer DNA adducts have been investigated using mass spectrometry. These include mitomycin C, nitrogen mustards, cisplatin, psoralen derivatives, a bioreductive prodrug (RH1), and an enediyene. Mitomycin C is an anticancer antibiotic that forms a DNA crosslink at the 2-amino group of guanine. The DNA/mitomycin C adduct was evaluated by tandem mass spectrometry and the results demonstrated that the mitomycin C adduct formed isomeric tetramer nucleotides upon activation for dissociation.. Nitrogen mustards can form DNA crosslinks and monoadducts. The extent of DNA alkylation with a sulfur acridine mustard derivative was evaluated by tandem mass spectrometry using infrared multiphoton dissociation. . Cisplatin is an anticancer therapeutic that crosslinks DNA at N7 guanine residues. The fragmentation pattern of cisplatin/DNA adducts investigated by tandem mass spectrometry confirmed the formation of a crosslink in the platinated diagnostic fragment ions detected. Psoralens, used for centuries to treat psoriasis, form crosslinks preferentially at thymine nucleobases by the sequential absorption of two photons. The results of tandem mass spectrometry were used to identify the sequence selectivity of psoralen derivatives. Recently, a study of a bioreductive prodrug, 2,5-diaziridinyl-3-[hydroxymethyl]-6-methyl-1,4-benzoquinone (RH1), was shown to form crosslinks with DNA at N7 guanine residues and the fragment ions produced via tandem mass spectrometry confirmed the site of the crosslink. Lastly, enediynes are of therapeutic interest because they exhibit a high cytotoxicity when the drug moiety forms a DNA crosslink through a biradical intermediate across opposing cytosine nucleobases. The tandem mass spectrometry results indicated that the enediyne moiety binds in a somewhat nonselective manner, as it associated with thymine as well as cytosine, and the formation of a covalent crosslink was confirmed by the retention of the enediyne by diagnostic fragment ions. . Other anticancer agents associate with DNA through noncovalent interactions like minor groove binding or intercalation. In both cases, the electrostatic interactions between the chemotherapeutic agent and the DNA double helix interfere with DNA transcription leading to incomplete proteins synthesis and ultimately cell death. Noncovalent anticancer moieties historically suffer from a lack of specificity, as most drug moieties have only two to four base pair binding sites. Thus, current research focuses on increasing the specificity of these small molecules. A novel tetraintercalator, 1,4,5,8-tetracarboxylic naphthalene diimide units connect by peptides (TET), has four intercalation units and a 14 base pair binding site allowing for dramatically greater sequence selectivity. The novel tetraintercalator shows the highest specificity amongst known intercalating moieties. An investigation into the sequence selectivity and binding site affinity compared to well characterized small molecule intercalators, actinomycin D and echinomycin, was assessed by mass spectrometry. The results show that TET preferentially binds to sequences that contain the unmodified binding site and also shows a slight preference to adenine and thymine rich sequences, indicating the peptide linkers play an important role in DNA interactions. Tandem mass spectrometry results demonstrated that TET binds with high affinity to its binding site compared to small molecule intercalators. Upon collision induced dissociation (CID) the predominant species in the mass spectrum was the DNA/TET – G ion peak. Intercalating adducts generally dissociate by strand scission with either strand retaining the drug moiety or by ejection of the drug, as seen with both actinomycin D and echinomycin in this study. Therefore, TET shows promise as a new development toward an anticancer therapeutic with high sequence selectivity and binding affinity with DNA. This workfocuses on reviewing the advancements of covalent bond forming DNA interactive anticancer therapeutics that have been studied by mass spectrometry, and presents a study of the interactions of a novel intercalation drug with DNA explored by mass spectrometry. / text

Cis-diamminedichloroplatinum (II), or cisplatin, is a widely prescribed anticancer compound, currently one of only three platinum (II) complexes FDA approved for cancer treatment. Despite its widespread use, we lack a comprehensive picture of global drug targets, which would lend valuable insights into the molecular mechanisms of action and resistance in different tissues. Drug binding to genomic DNA is an accepted cause of downstream apoptotic signaling, but less than 10% of Pt (in the case of cisplatin) accumulates within genomic DNA. Non-genomic contributions to cisplatin's therapeutic action are also under active investigation. In particular, cisplatin treatment can disrupt RNA-based processes such as splicing and translation. Pt(II) targeting of non-DNA species such as RNA may contribute to or sensitize a cell to the downstream effects of this drug, including the induction of apoptosis. Chapter I summarizes the activity profile of Pt(II) therapeutics, describing cellular uptake, cellular localization, incidences of Pt(II) accumulation within RNA, and RNA processes affected following drug treatment. Chapter II reports our thorough investigation of the distribution of Pt species throughout messenger and ribosomal RNA, with the discovery that Saccharomyces cerevisiae ribosomes act as a de facto cellular Pt sponge. In Chapter III, we report the synthesis of an azide-functionalized platinum (II) species, picazoplatin, for post-treatment click labeling and isolation of drug targets in vivo. Picazoplatin was designed to circumvent mislocalization and misprocessing of Pt typically encountered when trying to track small molecules tethered to large, charged fluorophores. This chapter contains several proof-of-principle studies validating the use of this class of reagents for future purification and sequencing of Pt-bound nucleic acids. Chapter IV describes the first application of the click-capable Pt reagent technology: the demonstration of significant in-gel fluorescent detection of Pt-bound ribosomal RNA and transfer RNA extracted from picazoplatin-treated S. cerevisiae and the first evidence that cellular tRNA is a platinum substrate. Chapter V summarizes these data, which suggest a potential ribotoxic mechanism for cisplatin cytotoxicity and broadly describe a convenient click chemistry methodology that can be applied to identify other metal or covalent modification-based drug targets. This dissertation includes previously published and unpublished co-authored material.

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Yes / The integrins are a family of 24 heterodimeric transmembrane cell surface receptors. Involvement in cell attachment to the extracellular matrix, motility, and proliferation identifies integrins as therapeutic targets in cancer and associated conditions; thrombosis, angiogenesis and osteoporosis. The most reported strategy for drug development is synthesis of an agent that is highly selective for a single integrin receptor. However, the ability of cancer cells to change their integrin repertoire in response to drug treatment renders this approach vulnerable to the development of resistance and paradoxical promotion of tumor growth. Here, we review progress towards development of antagonists targeting two or more members of the RGD-binding integrins, notably αvβ3, αvβ5, αvβ6, αvβ8, α5β1, and αIIbβ3, as anticancer therapeutics.

Cyclobutanes represent an underutilised structural feature in medicinal chemistry, partially due to difficulties in forming them in an easy and controlled manner. Herein is described their application to a drug discovery project and development of the enamine [2+2] cyclisation; a straightforward synthesis of functionalised cyclobutanes. A library of 30 cyclobutane based integrin antagonists have been designed and synthesised to explore the SAR around the hit dual β3 integrin antagonist ICT9055. Several of which were shown to be highly potent antagonists inhibiting cancer cell adhesion, migration and invasion while remaining non-toxic. ICT9072 had comparable β3 activity to hit compound ICT9055 but also had activity against αvβ5 and therefore showed greater inhibition of migration of DLD-1 cells. This showed the ability to modify this scaffold for multi integrin antagonism and potential benefit of this. Synthetic studies towards the marine natural product providencin has led to the development of a previously unknown intramolecular enamine [2+2] cyclisation which has been shown to proceed in a diastereoselective manner. This reaction has been applied to the synthesis of a highly functionalised enatiopure cyclobutene suitable for inclusion into the total synthesis. A model furyl cyclobutane has also been synthesised to exemplify the route from the enantiopure cyclobutene through to the furyl cyclobutane fragment of providencin. / Yorkshire Cancer Research

Cyclobutanes represent an underutilised structural feature in medicinal chemistry, partially due to difficulties in forming them in an easy and controlled manner. Herein is described their application to a drug discovery project and development of the enamine [2+2] cyclisation; a straightforward synthesis of functionalised cyclobutanes. A library of 30 cyclobutane based integrin antagonists have been designed and synthesised to explore the SAR around the hit dual β3 integrin antagonist ICT9055. Several of which were shown to be highly potent antagonists inhibiting cancer cell adhesion, migration and invasion while remaining non-toxic. ICT9072 had comparable β3 activity to hit compound ICT9055 but also had activity against αvβ5 and therefore showed greater inhibition of migration of DLD-1 cells. This showed the ability to modify this scaffold for multi integrin antagonism and potential benefit of this. Synthetic studies towards the marine natural product providencin has led to the development of a previously unknown intramolecular enamine [2+2] cyclisation which has been shown to proceed in a diastereoselective manner. This reaction has been applied to the synthesis of a highly functionalised enatiopure cyclobutene suitable for inclusion into the total synthesis. A model furyl cyclobutane has also been synthesised to exemplify the route from the enantiopure cyclobutene through to the furyl cyclobutane fragment of providencin.

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