Nucleic Acids as Drugs and Drug Targets With Focus on Anticancer Active Platinum Complexes and Small Interfering RNAs

Detta är en avhandling från Department of Chemistry, Lund University

Sammanfattning: To ensure that patients receive a safe and effective treatment, it is important to understand how drugs interact with and affect molecules within a cell. Here, evaluation of platinum based drugs and their ability to interfere with nucleic acid functions is presented. The main focus of this thesis has been to evaluate whether siRNA efficiency can be modified by two clinically used anticancer active platinum complexes; cisplatin and oxaliplatin. To evaluate the potency of platinum based drugs with respect to direct modulation of protein synthesis, siRNA methodology and protein silencing capacity in a luciferse based system was used. In addition to cellular based assays, platinated nucleic acids were characterized by thermal meting studies, HPLC, MALDI-MS, UPLC-ESI-MS and enzymatic and chemical probing resolved by PAGE. siRNA guide and/or passenger strands were pre-platinated and gel-purified prior to experimental evaluation. Platination of the siRNA passenger strand was found not to perturb the protein down regulation significantly. Platination of the siRNA guide strand on the other hand was found to decreases the protein down regulation capacity up to seven fold, when the platinum modification was located in non-seed regions. The effect was more pronounced for oxaliplatin. The thermal stability of RNA duplexes is reduced after platination. To evaluate if the observed platinum induced destabilization in the RNA environment was transferable to DNA, a thermal melting study was conducted. Analysis of the contributions from enthalpy and entropy to the free energy of duplex dissociation showed a larger variation in RNA. A study was also conducted to evaluate the DNA binding properties of novel platinum complexes/peptide chimeras. The platinum complexes contained a nonapeptide with either basic (lysine) or aliphatic (glycine, alanine) amino acids. The bulky peptide retarded the platinum binding to DNA, an effect that was counteracted by the electrostatic attraction of the positively charged lysine residues. Evaluation of the binding preference of cis-diamminedichloridoplatinum(II) (cisplatin) and dichloride N,O- (L)-ornitineplatinum(II) (O-platin) to rRNA was performed by incubation of rRNA with the two complexes followed by enzymatic degradation and evaluation by HPLC and mass spectrometry. A guanosine preference over adenosine was confirmed for cisplatin and the opposite binding preference was observed for O-platin. We conclude that platinum drugs affect RNA function and can reduce siRNA efficiency.

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