Antiviral compounds via in situ RNA-click chemistry – ClickEnARN

After a thorough study, some aminoglycosides were chosen as a starting point for chemical modifications in order to adjust them to the DIS structure. Apart from classical chemical synthesis, with successive steps in «flask«, we wanted to develop a novel approach using the RNA sequence itself as « reactor «. In fact, by interacting with the RNA, molecules such as aminoglycosides are facing each other in the RNA (one aminoglycoside per RNA), they may, then, react together, forced by the RNA. Moreover, from a collection of molecules, only the best partners will interact with the RNA and would be transformed, thus selecting the best and most effective candidate. This strategy based on in situ click chemistry, the ultimate tool in drug discovery, was never applied to RNA ligands. Furthermore, innovative techniques must be developed to realize this first. Four teams have pooled their expertise in chemical synthesis, mass spectroscopy, biophysical and biology.candidate. This strategy based on in situ click chemistry, the ultimate tool in drug discovery, was never applied to RNA ligands. Furthermore, innovative techniques must be developed to realize this first. Four teams have pooled their expertise in chemical synthesis, mass spectroscopy, biophysical and biology.

This project dovetails very strongly 4 different laboratories. The results of one team strongly influence all project partners. Thus, poor affinity of certain compounds, forced us to change the synthesis program. Therefore, we developed new reaction sequences for the development of new triazoles linked to either neamine or neomycin. The biophysical properties (affinity for the DIS or A site) of these new families of aminoglycosides and their precursor, propargylic or azide precursors, will allow us to engage the latter in the «in situ click reactions« (with the initiation dimerization site of HIV-1 but also to A site), and, of course, to determine their antibiotic property. A new collaboration should come soon.

Preliminary studies of affinity of these compounds with the dimerization initiation site of HIV will begin: microcalorimetry, crystallization, exchange RNA in the presence of the molecules.

The ANR support allowed new collaborations that aims to study either ligand/structure interactions (van Delf, Micura, Micouin), either to understand the mechanisms leading the loop-loop complex of the initiation dimerization site to the extended duplex (Rueda).
With the aim of mimicking the side chain of butirosin, we have developed a simple method for the synthesis of azetidine. Finally, in all our attempts to functionalize alkynes, we discovered a green protocol for carbon monoxide generation.
Regard possible therapeutic implications term, a patent is not excluded. A first patent (PCT. Int. Appl 2007, WO 2007125423) had also been deposited at the origin of this project, and a second was initiated at the beginning of this project. This fact and the various problems encountered in this work have somewhat limited the traditional scientific production.

All this work led to 6 publications and 7 conferences.

Recent years have witnessed the emergence of a “RNA world” in biology. RNA is no longer considered as a passive carrier of DNA's sequence information. Indeed, various forms of RNA have been and are currently discovered, each one having huge implications in various biological domains, from protein synthesis to gene expression control.
Therefore, RNA molecules are also emerging as potent therapeutic targets. However, and despite advances in unveiling and understanding RNA roles in biology, relatively few works have so far been achieved on targeting RNA with small molecules. Interestingly, an increasing number of RNA structures are currently solved at atomic level and it thus become possible to design compounds, which can interfere with RNA, potentially leading to new drugs.

With the development of therapeutics or probes of cellular function in mind, we would like to study in situ click chemistry using RNA as template. This so far unprecedented approach will be applied to target special RNA sequences, and especially the RNA dimerisation initiation site of HIV.

To achieve this original and unique goal, and based on structural data, we will prepare a large panel of aminoglycoside derivatives, in which the N1 amino group has been either converted to an azide or grafted with an alkynyl chain. With libraries of azido and alkynyl aminoglycoside building blocks in hand, we will focus on in situ click chemistry, using RNA as template. The challenge is to find appropriate conditions for performing in situ click chemistry in the presence of RNA and for detecting click adducts in biological medium. Mass spectra analysis will extensively be used for this aspect.

This study will be performed in a collaborative work, with organic chemists well aware of synthesis especially with carbohydrates, with a team deeply involved in MS analysis in the field of proteomics analysis and of non-covalent complexes study in chemistry and biology, and with teams worldwide specialists in RNA handling and characterization, including crystallization, and involved in the discovery and study of the dimerisation initiation site of HIV.