Periodic Reporting for period 1 - DrugSearchTool (Innovative FRET-based toolkit for screening of drugs to fight miRNA-related diseases and its use in the development of cancer treatment with miRNA-96 as a therapeutic target)
Okres sprawozdawczy: 2018-09-01 do 2020-08-31
The screening of drug candidates against so far undruggable disease states, such as various cancers and certain heart failures, is a vital procedure in the R&D development process in pharmaceutical industry. Efficient screening methods enable a fast and accurate way to find promising drug candidates among myriads of molecules usually available in the molecule libraries of pharmaceutical industry companies. The DrugSearchTool research project carried out by Dr. Anna Wypijewska del Nogal within MSCA-IF in the Wilhelmsson Lab at Chalmers University of Technology, Gothenburg, Sweden, in collaboration with University of Gothenburg (Grøtli Lab) and AstraZeneca (CVRM, BioPharmaceuticals R&D, Gothenburg) resulted in the development of a Förster resonance energy transfer (FRET)-based method to screen for drug candidates for several forms of cancer and heart failure.
The method employs light emissive mimics of cellular nucleobases (so-called fluorescent base analogues, FBAs) incorporated into microRNA precursor molecules (pre-miRNA) to report which ligands have a potential to efficiently inhibit miRNA-driven cancerogenesis or heart failure. This innovative approach enables accurate determination of the binding site of a drug candidate within pre-miRNAs, facilitating further optimization of drug structure.
The method has been implemented into evaluation of drug candidate libraries for pre-miRNA-21 (upregulated in brain, lung, liver, ovaries, breast, prostate and pancreas cancers) and pre-miRNA-377 (associated to the etiology of heart failure), contributing to the industrial R&D and to the benefit of the society by providing a new tool to help fighting life-threatening malfunctions. Moreover, the method is universal to any RNA/DNA system, which makes it interesting for several academic investigators, working in such fields as biochemistry, biophysics or medicinal chemistry. To date, results acquired during this project have been published in Nucleic Acids Research (2020) and submission of three more manuscripts to well-reputed peer-reviewed journals is envisaged. The project has also been presented in the RadioScience podcast: http://www.radioscience.se/tag/phd-students/(odnośnik otworzy się w nowym oknie).
The method employs light emissive mimics of cellular nucleobases (so-called fluorescent base analogues, FBAs) incorporated into microRNA precursor molecules (pre-miRNA) to report which ligands have a potential to efficiently inhibit miRNA-driven cancerogenesis or heart failure. This innovative approach enables accurate determination of the binding site of a drug candidate within pre-miRNAs, facilitating further optimization of drug structure.
The method has been implemented into evaluation of drug candidate libraries for pre-miRNA-21 (upregulated in brain, lung, liver, ovaries, breast, prostate and pancreas cancers) and pre-miRNA-377 (associated to the etiology of heart failure), contributing to the industrial R&D and to the benefit of the society by providing a new tool to help fighting life-threatening malfunctions. Moreover, the method is universal to any RNA/DNA system, which makes it interesting for several academic investigators, working in such fields as biochemistry, biophysics or medicinal chemistry. To date, results acquired during this project have been published in Nucleic Acids Research (2020) and submission of three more manuscripts to well-reputed peer-reviewed journals is envisaged. The project has also been presented in the RadioScience podcast: http://www.radioscience.se/tag/phd-students/(odnośnik otworzy się w nowym oknie).
To facilitate the use of fluorescent RNA base analogues in the FRET-based drug screening method developed in this project they needed to be carefully characterized inside RNA. To this end we incorporated the base analogues at various positions in the pre-miRNAs and investigated their influence on the native structure of the pre-miRNA as well as how fluorescence properties depend on their position in the RNA sequence. We found that the modified bases behave structurally as their native counterparts and obtained understanding that helped us in the design of the pre-miRNAs used for screening, i.e. where to place the light emissive reporter RNA base analogues in the pre-miRNAs.
With the knowledge on how to position the fluorescent base analogues in the pre-miRNA we set up an initial qualitative small-molecule screen in which we benchmarked our novel FRET method against existing techniques, such as ITC (calorimetry) and SPR (surface plasmon resonance). We found that our method can report on the binding process of the small molecule to pre-miRNA-21, which is upregulated in various cancers, and that the binding information obtained correlates correctly with currently used techniques.
As the next step, we further developed our method to enable screening of a larger number of molecules and to be quantitative. This enabled determination of the small molecule-pre-miRNA binding constant. Importantly, we were also able to determine the site of binding of a set of small molecules on the pre-miRNA-377 (associated with the etiology of heart failure), which is not obtainable with currently used high-throughput techniques. The knowledge of the nature and the position of the binding site is important in the development of improved drug candidates targeting a certain pre-miRNA. Finally, as a way to understand the details of conformation and dynamics of the pre-miRNA with and without the drug candidate bound to it, we have initialized single-molecule investigations using optical tweezers. Together with similar information obtained from the FRET-measurements of the pre-miRNA using the RNA base analogues and at similar conditions, we, thanks to our new method, help pharmaceutical R&D in the development of improved drug candidates targeting pre-miRNAs.
With the knowledge on how to position the fluorescent base analogues in the pre-miRNA we set up an initial qualitative small-molecule screen in which we benchmarked our novel FRET method against existing techniques, such as ITC (calorimetry) and SPR (surface plasmon resonance). We found that our method can report on the binding process of the small molecule to pre-miRNA-21, which is upregulated in various cancers, and that the binding information obtained correlates correctly with currently used techniques.
As the next step, we further developed our method to enable screening of a larger number of molecules and to be quantitative. This enabled determination of the small molecule-pre-miRNA binding constant. Importantly, we were also able to determine the site of binding of a set of small molecules on the pre-miRNA-377 (associated with the etiology of heart failure), which is not obtainable with currently used high-throughput techniques. The knowledge of the nature and the position of the binding site is important in the development of improved drug candidates targeting a certain pre-miRNA. Finally, as a way to understand the details of conformation and dynamics of the pre-miRNA with and without the drug candidate bound to it, we have initialized single-molecule investigations using optical tweezers. Together with similar information obtained from the FRET-measurements of the pre-miRNA using the RNA base analogues and at similar conditions, we, thanks to our new method, help pharmaceutical R&D in the development of improved drug candidates targeting pre-miRNAs.
Overall, the project has been able to deliver a new method for screening of small-molecule drugs that target pre-miRNAs. The method not only facilitates determination of quantitative binding data in a more straightforward way than current techniques but also provides binding site and pre-miRNA conformation information not obtainable with current high-throughput techniques. Hence, the interbase FRET-based approach to study pre-miRNA conformation and drug binding that has been developed herein is of high significance both for academia and industry, advancing the field of RNA and rational drug design in general and facilitating faster progress in the field of small molecules targeting pre-miRNAs in particular. This could simplify the development of drugs against currently undruggable diseases, for example various cancers and certain heart failures, and, consequently, increase life quality for human beings world-wide.