Structural and functional studies of platinum antitumor agent binding to DNA and the nucleosome

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2010.Vita. Cataloged from PDF version of thesis.Includes bibliographical references.Cisplatin, carboplatin, and oxaliplatin are three FDA-approved members of the platinum anticancer drug family. These compounds induce apoptosis in tumor cells by binding to nuclear DNA, forming a variety of adducts, and triggering cellular responses, one of which is the inhibition of transcription. The focus of this thesis is on studying the structure of these adducts, and correlating these effects with inhibition of transcription. Chapter 1 presents (i) a detailed review of the structural investigations of various Pt-DNA adducts and the effects of these lesions on global DNA geometry; (ii) research detailing inhibition of cellular transcription by Pt-DNA adducts; and (iii) a mechanistic analysis of how DNA structural distortions induced by platinum damage may inhibit RNA synthesis in vivo. These hypotheses will be explored in subsequent chapters of the thesis. In Chapter 2, features of the 2.17 A resolution X-ray crystal structure of cisdiammine( pyridine)chloroplatinum(II) (cDPCP) bound in a monofunctional manner to deoxyguanosine in a DNA duplex are discussed and compared to those of a cisplatin-1,2- d(GpG) intrastrand cross-link in double-stranded DNA. The global geometry of cDPCPdamaged DNA is quite different from that of DNA containing a cisplatin 1,2-d(GpG) cross-link. The latter platinated duplex is bent by ~40* toward the major groove at the site of the adduct; however, the monofunctional Pt-dG lesion causes no significant bending of the double helix. Like the cisplatin intrastrand adduct, however, the cDPCP moiety creates a distorted base pair step to the 5' side of the platinum site that may be correlated to its ability to destroy cancer cells. Structural features of monofunctional platinum adducts are analyzed, the results of which suggest that such adducts may provide a new platform for the design and synthesis of Pt anticancer drug candidates. The role of carbonate in the binding of cis-diamminedichloroplatinum(II) to DNA was investigated in Chapter 3 in order to understand the potential involvement of carbonato-cisplatin species in the mechanism of action of platinum anticancer agents. Cisplatin was allowed to react with single-stranded DNA in carbonate, phosphate, and HEPES buffers, and the products were analyzed by enzymatic digestion/mass spectrometry. The data from these experiments demonstrate (1) that carbonate, like other biological nucleophiles, forms relatively inert complexes with platinum that inactivate cisplatin, and (2) that the major cisplatin-DNA adduct formed is a bifunctional cross-link. These results are in accord with previous studies of cisplatin- DNA binding and reveal that the presence of carbonate has no consequence on the nature of the resulting adducts. The 1.77-A resolution X-ray crystal structure of a dodecamer DNA duplex with the sequence 5'-CCTCTGGTCTCC-3' that has been modified to contain a single engineered 1,2-cis- {Pt(NH 3)2}2+-d(GpG) cross-link, the major DNA adduct of cisplatin, is reported in Chapter 4. These data represent a significant improvement in resolution over the previously published 2.6-A structure. The ammine ligands in this structure are clearly resolved, leading to improved visualization of the cross-link geometry with respect to both the platinum center and to the nucleobases, which adopt a higher energy conformation. Also better resolved are the deoxyribose sugar puckers, which allow us to re-examine the global structure of platinum-modified DNA. Another new feature of this model is the location of four octahedral [Mg(H 20)6]2+ ion associated with bases in the DNA major groove and the identification of 124 ordered water molecules that participate in hydrogen bonding interactions with either the nucleic acid or the diammineplatinum(II) moiety. Chapter 5 discusses structural investigations of nucleosomal DNA modified by sitespecific platinum adducts. Nucleosome core particles containing a single 1,3-cis-{Pt(NH 3)2}2+_ d(GpTpG) intrastrand cross-link were synthesized and crystallized, and the X-ray structure was determined at 3.2 A resolution. The cisplatin adduct adopts a conformation facing toward the octamer core, in agreement with previous experiments. DNA in the vicinity of the Pt adduct has a similar helical bend as that observed in the NMR solution structure of free DNA containing the same cross-link, indicating that the rotational positioning power of cisplatin intrastrand crosslinks stems from the propensity to align the bent Pt-DNA structure with the DNA curvature arising from the nucleosome superhelix. Functional consequences of cisplatin binding to nucleosomal DNA are explored in Chapter 6. The effect of a single engineered platinum intrastrand cross-link on ATPindependent nucleosome mobility was investigated in vitro. Both 1,2-d(GpG) and 1,3-d(GpTpG) adducts of cisplatin inhibit translocation of DNA along the histone octamer, with the former Pt lesion providing a larger barrier. In vitro transcription assays with T7 RNA polymerase were conducted to determine whether cisplatin-DNA cross-links inhibit RNA synthesis by preventing access to nucleosomal DNA. Immobilized transcription templates containing a T7 RNAP promoter site, a single engineered cisplatin 1,2-d(GpG) or 1,3-d(GpTpG) intrastrand cross-link, and a phased nucleosome core particle were prepared. Analysis of resulting RNA transcript length revealed that the T7 RNAP elongation complex can overcome the energy barrier to nucleosome sliding caused by platinum intrastrand cross-links, but stalls when it reaches a Pt- DNA adduct placed on the DNA template strand. These results provide further evidence that intrastrand cross-links of cisplatin inhibit transcription by creating a physical barrier that the polymerase cannot pass. Appendices A and B summarize incomplete work that may be of use to future researchers working in this area. Appendix A describes attempts to isolate isomerically pure Pt- DNA probes containing a photoreactive benzophenone moiety, for use in cross-linking experiments that identify proteins that recognize and interact with cisplatin-DNA damage. In Appendix B efforts to obtain an X-ray crystal structure of an 1 Imer duplex DNA containing the 1,3- cis-{Pt(NH 3)2} 2 -d(GpTpG) intrastrand cross-link are reported. Appendix C details HPLC and mass spectrometric methods for purification and analysis of platinated oligonucleotides that were developed in the course of this research.by Ryan Christopher Todd.Ph.D