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Targeting nucleic acid structures by site-directed ligand discovery

Final Report Summary - TNSBSLD (Targeting nucleic acid structures by site-directed ligand discovery)

Nucleic acids (deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)) are involved in many cellular functions and play an important role in many biological processes. Nucleic acids have the ability to fold under physiological conditions into complex tertiary structures comprising local motifs. Analogously to the protein binding pockets, these particular nucleic acid structures can be considered as molecular receptors that are attractive targets for drug design. The design of molecules that recognise and bind to DNA secondary structures in a sequence and / or structure specific manner has emerged as an important and challenging goal in chemical biology.

Clinically used agents in antitumor therapy are excellent examples for small molecules acting at the nucleic acid level in cancer cells. Temodar and Doxil are examples for such antitumor therapeutics acting by alkylation of DNA or by intercalation into the DNA, respectively. The aim of this research is to establish a novel method to exploit functional nucleic acid structures for the generation of new therapeutics.

A DNA structure that has generated significant scientific interest is the four stranded G-quadruplex. This structural motif has been shown to play an important role in a number of biological processes including cancer by acting as a regulatory element for controlling oncogene transcription and may therefore be considered as a therapeutic target. Induction and stabilization of such G-quadruplex structures by small molecules can modulate the expression level of these genes and is coupled to down-regulation of the c-Myc gene.

In this work, we achieved a new and effective method to find small molecules (also called fragments) in a short period of time, which are able to recognize and bind to such DNA structure and down regulate the oncoprotein production in cancer cell lines.

Summary of the major project achievements over the entire period

The information provided in this section will only be made available to ERCEA staff and to the evaluation panel members.

- Research and technological achievements and the impact and use of them

We introduced an extension of the classical tethering approach, a method that allows us to probe specific regions / microenvironments on a target surface by small molecules and for the first time applied this to nucleic acid structures. Unlike other currently used methods, this method provides a de novo design of small molecules for a given nucleic acid target and can distinguish between non-covalently bound and specific bound fragments by comparison of the chemical profile of the supernatant with the tethering sample.

It is noteworthy that this analytical process is far easier than the conventional DCC approach which is commonly used and which requires a more detailed analysis of all homo- and hetero-disulphide dimers present in equilibrium mixtures. In contrast to templated dynamical combinatorial chemistry, this approach does not require large amounts of the target. We have also improved the final analytical part and intend to use this approach as a fast screening method using large thiol libraries against different nucleic acids and nucleic acid- protein complexes by pooling the compounds into groups of 10 thiol fragments with unique high-performance liquid chromatography (HPLC) traces. Our findings also provide a specific guideline for the design of a new generation of nucleic acid specific ligands and provide lead structures for optimization by SAR-methods to improve weak binding.

- Novel and / or unconventional methodologies

In the second year we extended our method to the field of fragment-based (FB)-ligand discovery. FB methods have successfully been used to discover high affinity ligands for protein active sites, as well as target protein-protein-interfaces. For nucleic acids, only a few examples of a fragment based approach are available in the literature: most notably the recent efforts to target RNA riboswitches. To date, however, targeting DNA by an FB approach is still unexplored. We used the FB approach as a novel and unconventional method to target DNA. In order to select hit fragments from a fragment library which bind to the c-Myc G-Quadruplex target, a new assay was designed. For the screening an intercalator-displacement-assay (IDA) was used with thiazole orange (TO) as an intercalator. TO is highly fluorescent if bound to the target DNA and quenched after displacement (figure 1). The kd and binding stoichiometry of TO to C-Myc DNA was determined (kd = 3.5 µM ± 0.69). The IDA was applied in a high throughput fashion to screen the available fragments against the G-quadruplex target.

By including negative and positive controls, an excellent Z'-score was calculated (Z' = 0.89) indicating a statistically robust assay. A liquid handling robot was used to screen 1377 fragments. 15 hits were identified and subdivided in groups based on their chemical structure. From the 15 hits the best ten were retested in a dose dependent manner (figure 3). The 50 % displacement value (DP50 %) for each hit fragment was determined and converted to their corresponding kd, by using the prusoff-cheng equation. The ligand efficency (LE) of the best 10 hits were also determined. The ten best hits were ranked regarding their kd values and tested again in an independent method (surface plasmon resonance).

- Inter and cross disciplinary developments

Following the novel and unconventional FB method applied on DNA, we were able to identify several hit fragments, which were used in an interdisciplinary manner for in-cellular testing.

The best 5 fragments were investigated in cellular experiments using cancer cell lines. The tested fragments show a down regulation of the oncoprotein c-Myc in an In-cell-Western assay specific for c-Myc. As reported the c-Myc protein is involved in cancer development and is over expressed in over 80 % of all cancer cells.

- Knowledge and technology transfer

The knowledge gained by this research has been transferred through interdisciplinary collaborations started during the last two years. The initial results in the first year led to collaborations with the CRI (Cambridge Research Institute), with Prof. C. Abell's group (Cambridge) and with prof. S. Neidle (London). By using Prof. Abell's in-house fragment library we were able to screen more than 1 500 fragments against the c-Myc G-quadruplex DNA target. In addition prof. Neidle provided us with docking support in order to get structural insight into the fragment-target recognition motif.

- Enhancing the immediate research environment

The Balasubramanian laboratory and others around the globe are searching for novel small molecules that interfere with different cancer pathways, in this case particularly by binding to the DNA G-quadruplex DNA motif located in the promoter region of different oncogenes. The FB approach has been introduced for the first time in this field and showed high potential for discovery of small molecules for G-Quadruplex DNA targets. This approach improved and accelerated the lead-structure finding process in the early hit discovery phase.