The naturally occurring polyamines, spermidine and spermine, have been shown to be critical for cell growth and carcinogenesis. Their N-bisethyl synthetic analogs and homologs were found to be strongly inhibitory of cell proliferation in many human tumor cell lines, some of which were chemoresistant to other therapeutic agents. Even though the cell killing effects of several of the bisethyl derivatives is so efficient that some of them have reached the stage of clinical trials, the molecular rationale behind these effects is still unknown. This proposal focuses on the clarification of this problem. It is known that polyamines govern the extent and fidelity of the translation of the genetic code by binding to tRNA, but the ways in which this interaction proceeds are still unexplained. We have recently shown, using 15N NMR, that the natural polyamines and the synthetic N-bisethyl analogs with antiproliferative effects bind to tRNA through hydrogen bonds; to elucidate how the nature of these interactions and to pinpoint the domains of tRNA which bind to the polyamines we propose to use additional novel NMR techniques. This will help fill a basic scientific gap arising from the fact that currently available X-ray and solution NMR data afford conflicting results concerning these systems. Our analyses on polyamine/tRNA will use 2D NOSEY and ROESY solution NMR techniques to locate the sites of binding as a function of increasing relative concentrations of polyamines; these studies will involve yeast and E. coli tRNAPhe, as well as thirteen natural and therapeutically-related polyamines. The 9.5-15 ppm region of the 1H NMR spectrum (imino protons) of E. coli and yeast tRNAPhe will be used to monitor the sites of binding, by measuring Overhauser cross peaks between the 1H imino resonances from the macromolecule and the polyamine protons. Since all the 1H NMR studies that have been so far carried out on tRNA revealed significant changes in the spectral parameters of at least some imino protons upon addition of polyamines, our spectroscopic approach will provide highly reliable information about the location of the binding sites. The Overhauser experiments will use polyamines enriched with 13C in the carbons bonded to the amino groups. This will allow us to acquire 13C-half-filtered 2D NMR spectra where only protons bound to 13C heteroatoms appear along one of the frequency dimensions. We expect to characterize from these measurements a binding interaction that reflects the X-ray diffraction results as low polyamine/tRNA rations, while explaining the NMR behaviour of base-paired imino protons as relative polyamine concentrations increase. If our hypotheses are confirmed, the NMR analyses will detect the interaction of therapeutically-relevant polyamines with specific sites of the tRNA backbone. These results will be a decisive contribution toward the development of new synthetic polyamines to control cell protein synthesis.
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