Chartering chemical space of riboswitch ligands - towards future antibiotics

There is an urgent need for new antibiotics to prevent a looming antibiotic crisis. The grim reality is that the antibiotics of today are losing effectiveness faster than they are being replaced by new treatment options. Infections caused by multiple drug resistant bacteria which are difficult or impossible to treat are tremendously increasing. Accordingly, new antibiotics, preferable with new chemical structures to avoid evolved resistance mechanisms are in high demand. Potential targets for antibiotics are riboswitches. Riboswitches are part of certain ribonucleic acids (RNAs) and they function as genetic switches in bacteria. This means that they can shut off the expression of essential genes and thus kill bacteria. This makes riboswitches promising targets for antibiotics which act differently as known antibiotics. In this project, we want to develop new ligands for two specific riboswitches, namely the flavin mononucleotide (FMN) and S-adenosyl methionine (SAM)-I riboswitch. This will allow us to explore if they are really as good targets for new antibiotics as suggested. If this is the case, the compounds will also serve as starting points for the development of the next generation of antibiotics. As a basis for this project, we have established a robust pipeline to transcribe the FMN and SAM-I riboswitches in vitro and to label the riboswitches which is required for subsequent binding assays. A lot of time has been invested in developing a robust and sensitive binding assay and this process proves to be much more tedious than expected. Therefore, we are now in the process of establishing an in vitro transcription / translation assay as an alternative way to determine compound binding to the riboswitch. We have also collected diffraction data for a new FMN riboswitch construct and data processing is ongoing. Further, synthetic routes for six different compound series for the FMN riboswitch have been developed and a number of analogues has been synthesized.

In terms of drug discovery, the RNA field is much less advanced than the protein field and tools and assays are much less established. We therefore expect that the developed robust SPR assay will be highly beneficial to push drug discovery for RNA forward, not only for the FMN riboswitch but also for other targets. Likewise, very little is known about SAR of RNA ligands. The insights gained from dissecting ribocil (once the final compounds have been characterized) will there very valuable to increase our understanding about what drives affinity for RNA ligands. This will help to design FMN riboswitch ligands with high affinity, but also more general for RNA binders.

There is an urgent need for new antibiotics. Potential targets for antibiotics are riboswitches. Riboswitches are noncoding RNAs that function as genetic switches in bacteria. It has been demonstrated that some compounds with antibiotic activity act by riboswitch binding. This makes riboswitches promising targets for antibiotics with a new mechanism of action. However, rigorous exploration of the chemical space of riboswitch ligands and chemical validation are urgently needed to drive this field forward and to open it up for drug discovery. Therefore, we will use structure-based methods such as virtual and fragment screening to derive new ligands for two riboswitches that were found in pathogenic bacteria and either shown or predicted to regulate the expression of essential genes, namely the flavin mononucleotide (FMN) and S-adenosyl methionine (SAM)-I riboswitches. Discovered hit compounds will be thoroughly validated. We will follow up with structure-based ligand optimization and in vitro and in vivo mode of action studies with the goal to explore the chemical space of riboswitch ligands and to chemically validate riboswitches as drug targets. If riboswitches meet the high expectations, this project will also deliver starting points for the development of antibiotics with a new mode of action which are highly desired to prevent a looming antibiotic crisis.