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Towards microRNA modulators by fragment-based drug discovery (FBDD) approaches

Final Report Summary - MICRORNA FBDD (Towards microRNA modulators by fragment-based drug discovery (FBDD) approaches)

MicroRNAs are short ribonucleic acid (RNA) molecules, on average only 22 nucleotides long. They have been identified as important endogenous regulators of gene function. Aberrant regulation of them has been linked to various human diseases. Intervention by new chemical entities has the potential to provide a new therapeutic strategy. Anti-sense agents (chemically engineered oligonucleotides) have been developed to inhibit microRNA function. However, due to their chemical structure anti-sense agents do not show the essential pharmacokinetic properties as necessary for therapeutic use. Thus, our main objective was the development of new molecular entities with better pharmacokinetic properties to target microRNA for therapy.

MicroRNA silencing processes are mediated by a specialized family of RNA-binding proteins named Argonaute. These proteins bind microRNA and silence complementary target mRNAs either by direct cleavage or by recruitment of additional silencing factors. Recently, it was shown that mRNA binding to the complex of target microRNA and Argonaute 2 protein occurs in a rate-dependent step to the solvent-exposed first eight nucleotides of microRNA's 5'-end, the so-called seed region. The blockage of microRNA's seed region inhibits totally microRNA binding to the target mRNA.

Due to the fact that microRNA functions are mediated by the Argonaute 2 protein we have developed a novel inhibitor class: microRNA-specific Argonaute 2 protein inhibitors. These kind of inhibitors bind via their short but specific nucleotide sequence to the solvent-exposed target microRNA's seed region and then with their small molecule moiety in the active site of the Argonaute 2 protein. As for the first binding step depends upon specific base pairing the inhibitor should not bind to the complex of AGO2 and non-target microRNA. By targeting a protein binding site in an analogous way to established drugs this provides a chance to develop microRNA inhibitors with better pharmacokinetic properties for therapeutic use than reported oligonucleotide inhibitors.

We developed a model for rational drug design enabling the identification of Argonaute 2 active site binders and their linkage with a peptide nucleic acid sequence (PNA), which is used to address the microRNA of interest. The designed inhibitors targeting microRNA-122, a hepatitis C virus drug target, had an IC50 of 100 nM, ten-fold more active than the simple PNA sequence (IC50 of 1 μM) giving evidence that the strategy has potential for inhibition of microRNA functions. We are now exploring the use of other potential oligonucleotide alternatives and are also focusing on further active site ligand optimization in the hope to obtain microRNA-specific Argonaute 2 protein inhibitors able for therapeutic use.

In summary, we developed new chemical entities with a great chance to have better pharmacokinetic properties than reported oligonucleotide inhibitors for microRNA inhibition. From there, this work has a socio-economic impact as it is highly relevant for the European pharmaceutical industry. As microRNAs are promising drug targets for diabetes or cardiovascular diseases our inhibitors have the potential for the development to future 'block busters', drugs with a sales revenue of more than 1 billion USD per year. With this outlook, our work is a significant contribution to maintain Europe as one of the most innovative places in the world.