EXPLORNA Science Publications Presentations Members

Marie Curie Excellence Grant

“Study of RNA components by the Synthesis of Small Molecules”

Contract No. MEXT-CT-2006-039149, Dr. Dionisios Vourloumis,

Budget : € 1.619.960, Duration : Feb 2007– Jan 2011

Scientific highlights

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. Thus, classification of 2-DOS as a “privileged” RNA-targeting chemical entity, for the development of novel antiviral and antimicrobial pharmaceuticals, is highly substantiated. We have successfully accomplished and optimized the synthesis of a chiral 2-DOS analogue from neomycin in just five steps, exploiting the asymmetric substitution pattern of 2-DOS in the natural products. Furthermore, we have used the specific compound for the construction of appropriately protected 2-DOS analogues, allowing us to synthesize molecules with the diversified substitution patterns found in natural aminoglycosides (manuscript in preparation). Other representatives are currently underway. Specifically for the case of oxazolidinone intermediates (figure), they represent an interesting case of hybrid structures, since they contain important structural elements of the aminoglycoside family. Selected analogues from that series have been synthesized and biologically evaluated, producing exceptional antibacterial activities with only moderate binding affinities (manuscript in preparation). The latter observation suggests altered binding sites and mechanisms of action and these compounds are currently further investigated.

The bacterial ribosome represents the confirmed biological target for many known antibiotics that interfere with bacterial protein synthesis. Aminoglycosides represent a lead paradigm in RNA molecular recognition and constitute ideal starting points for the design and synthesis of novel RNA binders. Previous rational design approaches of RNA-targeting small molecules have been mainly concentrating on direct functionalization of aminoglycosidic substructures. We successfully designed and synthesized rigid spirocyclic scaffolds locked in a predicted ribosome-bound “bioactive” conformation. These analogues are capable to mimic many of the interactions of the natural products for the A-site, as proven by their obtained binding affinities. The development of an optimized approach for their synthesis and their potential to inhibit protein production in vitro are established. Our results formulate a preliminary SAR study for the identification of important interactions, supporting the development of analogues with improved antibiotic profiles. Six-membered spirocyclic scaffold 2 represents the champion of the present effort, being able to maintain the desired activity with half the number of the notorious amino-moieties present in neamine. Also, direct comparison between the 6,6- and 7,6-analogues introduces the possibility of controlling the selectivity of action between bacteria and eukaryotic organisms through fine-tuning of structural elements. Additional examples, co-crystallographic studies and biological evaluations are currently underway. (ChemBioChem 2009, 10, 1969-1972 and ChemBioChem 2011, 12, 71-87).

Furthermore, the development of a new synthetic approach enabled the construction of novel 6,7-spiro bicyclic aminoglycoside mimics. Our improved synthetic protocol resolved previous issues for the introduction of an allyl group on a tertiary hydroxyl by utilizing Pd-chemistry performed under neutral conditions. Consequently, the retention of the protecting carbamates for the amines present enabled the differentiation of N1 for further functionalization, through the formation of a trans-oxazolidinone intermediate. Also, careful selection of the dihydroxylation conditions delivered our final analogues with complete stereocontrol. A significant improvement of the binding affinities for the bacterial ribosome’s decoding center was achieved, in comparison to the parent compounds. Also, we observed that a direct correlation between binding affinities and in vitro biological potency is not always possible (Bioorg. Med. Chem. Lett. 2010, 20, 7488-7492).

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 and assessed for their binding potential. The synthetic molecules produce very good EC₅₀ values for the ribosomal A-site construct utilized in our fluorescent studies. Although the exact binding mode (orientation and site) of the spiroether triazole analogues is uncertain, we expect them 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. As oppose to the oxazolidinone-aminoglycoside hybrids, the observed binding affinities do not correlate well with the obtained biological effects. Consequently, the specific compounds represent exceptional candidates as anti-HIV or anti-HCV agents, targeting the Dimerization Initiation Site (DIS) or the Internal Ribosome Entry Site (IRES) of the viruses respectively, without the toxic side effects associated with inhibiting protein production within a host organism. Strategic collaborations (Prof. Hermann, Dr. Ennifar and industrial collaborators) have been established to examine these possibilities (ChemBioChem 2011, in press).

The potential of aminoglycoside antibiotics to induce premature stop codon readthrough in eukaryotic systems has been recently reported, inspiring the evaluation of structural alterations within the Homo sapiens cytoplasmic decoding center upon ligand binding. As a result, we reported the employment of an affinity screen capable of monitoring conformational changes of adenines 1492 and 1493 in solution. Thus, changes induced by the presence of a ligand can be directly translated to binding affinities for the eukaryotic decoding center. Furthermore, a good correlation is obtained between the experimental binding affinities and the biological activity of the compounds examined. Additionally, illustrating the generality of the assay, unnatural rigid-aminoglycoside analogues of potential therapeutic significance were evaluated.

We have selected a Homo sapiens cytoplasmic decoding center construct, where the two adenines at positions 1492 or 1493 have been replaced by 2-aminopurine.

Signal intensity and dose response is indicative of binding affinity and protein production inhibition potency. Overall, the good correlation obtained between the experimental binding affinities and the biological activity of the compounds examined, associated with functionality differences of the chemical entities and the importance of A1492 within the decoding center, inspire additional studies towards the elucidation of the complex mechanism of protein production in eukaryotes (Anal. Biochem. 2011, in press).

Overall, many interesting biological observations have been made regarding the correlation of RNA recognition and the mechanism of action for the synthetic entities. For certain cases the specific binding values were exceptional, surpassing the affinities of the parent natural products. In many occasions binding was directly related to antibacterial activity suggesting identification of novel antibacterial agents with potential therapeutic significance. In other instances very good antibiotic activities were not associated with binding in the specific RNA constructs, indicating alternative mechanisms of action. Finally in some series, exceptional binding affinities produced minimal antibacterial effects, indicating alternate structural characteristics of the produced complex with the RNA. In spite of the intriguing potential to study the exact mechanism of protein production in prokaryotes, the lack of toxic effects of these compounds provides an enormous opportunity to examine their action for the treatment of viral infections and genetic disorders.