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Reactions That Translate mRNA into Drug-like Molecules

Periodic Reporting for period 4 - TRIGGDRUG (Reactions That Translate mRNA into Drug-like Molecules)

Reporting period: 2020-07-01 to 2020-12-31

The increased life span achieved in the most societies correlates with an increased probability of an individual to be diagnosed cancer. The aging of the society leads to an increase of cancer prevalence which concomitantly increases the number of cancer deaths. Despite the impressive improvements of cancer treatments, we do require new concepts for personalized therapies with little side effects.
In cancer, gene expression is deregulated due to amplification, mutation and translocation of genes. Next generation RNA sequencing provides us with the opportunity to identify the number and identity of the gene products aberrantly expressed in a patient. We propose a method that could take advantage of the personalized sequence data. The idea is to use the RNA molecules produced in cancer cells as instructors for the chemical synthesis of drug-like molecules that cure the disease. Accordingly, drug-like molecules would only be formed in those cells that express the disease-specific RNA molecules. Such a personalized molecular therapy would eliminate side effects caused by unwanted perturbation of healthy cells.
The idea to use cellular RNA molecules as triggers for drug synthesis requires chemical methods that couple recognition of the “cancer RNA” with a change of chemical reactivity. Reactive molecules must be able to “read” and “translate” the sequence of a RNA molecule into a drug-like output. We will develop mRNA-triggered reactions that i) proceed with turnover in template to cope with low mRNA copy numbers and ii) allow the single-step synthesis of highly active drug-like molecules to address deregulated protein targets inside cancer cells. To achieve this aim, we will advance chemical acyl transfer and alkylidene transfer as well as photocatalytic cleavage reactions. The reactions will form peptides, peptidomimetics or small molecules which will bind and inhibit those proteins that allow the cancer cell to survive. Since the products will able to target both RNA (by virtue of the “read” step) and deregulated proteins (by virtue pf the “translate” step) synergy between the nucleic acid and protein worlds will be harnessed.
Conclusion: Over the 5 years on this project, we developed four different chemical reaction systems to put the formation of cancer toxic peptides and small molecule drugs under the control of RNA. We succeeded in demonstrating that conjugates comprised of a nucleic acid component and a peptide/small molecule can have enhanced potencly compared to the components alone. While we succeeded in demonstrating cellular delivery of the molecules we did not succeed in verifying that endogeneous RNA can instruct the intracellular synthesis of drug-like compounds. However, the reaction systems are promising for applications in DNA-encoded libraries which are frequently applied in drug screening. Our work on TRIGGDRUG led to the serindipitous discovery of a new reaction paradigm for nucleic acid templated reaction. We introduced reactions that induce cleavage within the main chain of nucleic acid molecules. These truly catalytic reaction systems enable the design of signal amplifying detection chemistries that may compete with the enzymatic methods used for example for the detection of viral RNA.
The research project comprises four subprojects.
Subproject I. We developped reactions that are triggered by RNA and produce peptides that antagonize a protein (Bcl-XL) which normally allows the cancer cells to grow and escape programmed cell death. We found a reaction that puts the synthesis of a 16 amino acid long peptide under the control of a specific RNA molecule. We have shown that the product of this reaction binds cell death inhibiting protein (Bcl-XL), kills cancer cells and we have also identified the conditions that allow the delivery of the reagents into the cell. We expanded the approach to the RNA-instructed synthesis of D-peptides, which bind Bcl-XL and cannot be degraded by proteases. This reaction system performed well. However, we found that peptides targeting Bcl-XL are cell toxic when conjugated to nucleic acids.
In subproject II, we developed chemical syntheses and a RNA-triggered reaction system leading to dual activity agents. One part of the product molecule was designed to antagonize a road block installed by the cancer cell to prevent the intrinsic pathway of programmed cell death. The other part restored the blocked extrinsic pathway. We demonstrated that none of the parts has sufficient power to affect difficult to kill cancer cell lines. Rather, the two parts of the molecules are required to effectively kill cancer cells presumably by simultaneously targeting two different cancer Achilles heels.
In subproject III, we chemically modified small-molecules that inhibit cell growth signaling cancer proteins (receptor tyrosine kinases, RTKs). We demonstrated their sufficient bioactivity. We developed the chemical synthesis of reactive conjugates that can bind RNA targets. We performed RNA templated reactions in test tubes and demonstrated that the formation of the RTK inhibitors can be put under the control of RNA.
Subproject 4 deals with a new photocatalytic cleavage reaction that renders a RNA-templated reaction to produce many product molecules per RNA molecule. The approach relied on the development of cleavage linkers which were incorporated into nucleic acid probes. We developped the conjugation chemistry required for introduction of the cleavaga linkers and performed in depth studies on turnover frequenvcies (that is how many molecules can be converted under the control of a nucleic acid target per second).
We have established four different acyl transfer reactions which are driven/controlled by synthetic RNA. The reaction systems lead to compounds that contain i) a peptide nucleic acid (PNA) part (to bind and “read” cancer-specific mRNA) and ii) a peptide, phosphopeptide, peptidomimetic or small molecule compound . We showed that the starting materials can be delivered into live cells. However, we did not succeed in demonstrating that the drug-forming chemical reactions were triggered by intracellular RNA. We showed that peptides targeting a key player of the intrinsic pathway of programmed cell death (XIAP) can enhance the killing of cancer cells by antisense oligonucleotides. This synergetic activity was not obtained for peptides targeting the proapoptotic protein Bcl-xL. We described a reaction system that enables the synthesis of anticancer drugs in water. This reaction system may prove useful for fragment-based drug discovery and in DNA-encoded libraries. Furthermore, we discovered a new reaction paradigm for nucleic acid-templated chemistry. The method will enable chemical nucleic acid detection to proceed with sensitivities known from enzymatic reactions.