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It has been suggested that the alanine-based peptide with sequence Ac-XX-[A]7-OO-NH2, termed XAO where X denotes diaminobutyric acid and O denotes ornithine, exists in a predominantly polyproline-helix (PII) conformation in aqueous solution. In our recent work, we demonstrated that this “polyproline conformation” should be regarded as a set of local conformational states rather than as the overall conformation of the molecule. In this work, we present further evidence to support this statement. Differential scanning calorimetry measurements showed only a very small peak in the heat capacity of an aqueous solution of XAO at 57°C, whereas the suggested transition to the PII structure should occur at ∼30°C. We also demonstrate that the temperature dependence of the 3JHNHα coupling constants of the alanine residues can be explained qualitatively in terms of Boltzmann averaging over all local conformational states; therefore, this temperature dependence proves that a conformational transition does not occur. Canonical MD simulations with the solvent represented by the generalized Born model, and with time-averaged NMR-derived restraints, demonstrate the presence of an ensemble of structures with a substantial amount of local PII conformational states but not with an overall PII conformation. Fil: Makowska, Joanna. Uniwersytet Gdanski; Polonia. Cornell University; Estados Unidos Fil: Rodziewicz-Motowidlo, Sylwia. Uniwersytet Gdanski; Polonia. Cornell University; Estados Unidos Fil: Baginska, Katarzyna. Uniwersytet Gdanski; Polonia Fil: Makowski, Mariusz. Cornell University; Estados Unidos. Uniwersytet Gdanski; Polonia Fil: Vila, Jorge Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Matemática Aplicada de San Luis "Prof. Ezio Marchi". Universidad Nacional de San Luis. Facultad de Ciencias Físico, Matemáticas y Naturales. Instituto de Matemática Aplicada de San Luis "Prof. Ezio Marchi"; Argentina. Cornell University; Estados Unidos Fil: Liwo, Adam. Cornell University; Estados Unidos Fil: Chmurzynski, Lech. Uniwersytet Gdanski; Polonia Fil: Scheraga, Harold A.. Cornell University; Estados Unidos

2-methoxyestradiol (2-ME) is a physiological anticancer compound, metabolite of 17β-estradiol. Previously, our group evidenced that from mechanistic point of view one of anticancer mechanisms of action of 2-ME is specific induction and nuclear hijacking of neuronal nitric oxide synthase (nNOS), resulting in local generation of nitro-oxidative stress and finally, cancer cell death. The current study aims to establish the substantial mechanism of generation of reactive nitrogen species by 2-ME. We further achieved to identify the specific reactive nitrogen species involved in DNA-damaging mechanism of 2-ME. The study was performed using metastatic osteosarcoma 143B cells. We detected the release of biologically active (free) nitric oxide (•NO) with concurrent measurements of peroxynitrite (ONOO−) in real time in a single cell of 143B cell line by using •NO/ONOO− sensitive microsensors after stimulation with calcium ionophore. Detection of nitrogen dioxide (•NO2) and determination of chemical rate constants were carried out by a stopped-flow technique. The affinity of reactive nitrogen species toward the guanine base of DNA was evaluated by density functional theory calculations. Expression and localization of nuclear factor NF-kB was determined using imaging cytometry, while cell viability assay was evaluated by MTT assay. Herein, we presented that 2-ME triggers pro-apoptotic signalling cascade by increasing cellular reactive nitrogen species overproduction – a result of enzymatic uncoupling of increased nNOS protein levels. In particular, we proved that ONOO− and •NO2 directly formed from peroxynitrous acid (ONOOH) and/or by auto-oxidation of •NO, are inducers of DNA damage in anticancer mechanism of 2-ME. Specifically, the affinity of reactive nitrogen species toward the guanine base of DNA, evaluated by density functional theory calculations, decreased in the order: ONOOH > ONOO− > •NO2 > •NO. Therefore, we propose to consider the specific inducers of nNOS as an effective tool in the field of chemotherapy.