Final Activity Report Summary - EXPLORNA (Study of RNA Components by the Synthesis of Small Molecules) Our research objective was to exploit RNA as a pharmaceutical target and, thus, contribute to the development of novel and more efficient antibiotics. The work initially focused on substrates that bind specifically to the ribonucleic acid (RNA) components of the bacterial ribosome, namely the aminoglycoside antibiotics. During the first stage of our endeavour, interactions that increase the binding affinity of these small molecules for the ribosomal decoding RNA site (A-site) along with their required specific structural motifs was identified, based on existing three-dimensional structures of domains and subunits of the bacterial ribosome and antibiotic complexes thereof. These data were utilized for the rational design of small-molecule libraries derived from the neomycin- and kanamycin-classes of natural antibiotics. It was early on identified that the structures of most natural aminoglycosides embody a highly conserved diaminocyclohexitol, 2-deoxystreptamine (2-DOS), whose essential role for binding to RNA involves not only direct hydrogen bonding interactions but also the precise orientation of its peripheral functionalities for increased binding affinity. Consequently, we successfully accomplished and optimized specific methodologies for the synthesis of asymmetric 2-DOS analogues, exploiting its asymmetric substitution pattern in the natural products. Appropriately functionalized advanced intermediates from that effort served as the basis for the design and synthesis of new rigid scaffolds, locked in the ribosome-bound "bioactive" conformation. Thus several series of 5,6-, 6,6- and 7,6-spirocyclic analogues along with fused oxazolidinone-aminoglycoside hybrid structures were prepared. In order to further explore the chemical space that can be accessed with the aforementioned paradigm, cyclic 5, 6-spiroethers, flanked by a triazole moiety, were readily synthesized, as a result of the successful use of the "dynamic libraries" concept and the advantages of "click chemistry". Additionally, neamine-biotine conjugates have been synthesized to rationalize binding motifs as well as to identify additional RNA-targets of pharmaceutical significance. The small molecule libraries were tested for specific binding to the ribosomal target sites. Incorporating in our research current scientific discoveries illustrating the importance of aminoglycosides in viral infections (HIV, HCV) and genetic disorders, fluorescent based assays were developed in our laboratories. Specifically, binding was analysed by in vitro screening utilizing isolated RNA target domains for bacterial as well as eukaryotic systems. Furthermore, in vitro transcription/translation studies produced information regarding the ability of the synthetic molecules to interfere with protein production processes. RNA-complexes of ligands displaying the highest binding affinities in the screening assays were separately prepared in larger quantities for quantitative binding studies and further biochemical and biophysical characterization, including co-crystallization to identify the exact binding mode (orientation and site) of the analogues. Strategic collaborations were established for evaluating the therapeutic potential of selected molecules. The majority of the new entities proved capable of mimicking the interactions of the natural products for the bacterial decoding centre producing very good values for the ribosomal A-site construct utilized in our fluorescent studies. The variety observed in the correlation of binding affinity and biological effect is expected to significantly contribute in the understanding of the principals governing RNA recognition and more specifically the dynamic interplay of the small molecule attributes with the adaptable structural and energetic landscape of the ribosomal A-site. Additionally, it paves the way for the discovery not only of novel antibiotic agents but also of new antivirals with enormous therapeutic significance.
