Interactions of anticancer therapeutics with DNA investigated via mass spectrometry

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.