Fluorinated Fragments vs G-quadruplexes

Recently we highlighted an example of fragment-based ligand discovery against a riboswitch. Of course, RNA can form all kinds of interesting structures, and in a new paper in ACS Chem. Biol. Ramón Campos-Olivas (Spanish National Cancer Research Centre) and Carlos González (CSIC, Madrid) and their collaborators describe finding fragments that bind G-quadruplexes.

G-quadruplexes, as their name suggests, consist of groups of four guanine residues hydrogen bonding to one another in a planar arrangement. These individual tetrads then stack on top of one another. They can form in guanine-rich regions of RNA or DNA. Most famously, G-quadruplexes are found in telomeres at the ends of chromosomes. However, they are also found in telomeric repeat-containing RNA (TERRA), and are required for cancer cells to proliferate indefinitely.

The researchers used ¹⁹F-NMR screening to identify fragments that bound to an RNA containing 16 (UUAGGG) repeats (TERRA₁₆). ¹⁹F-NMR is a technique about which Teddy waxes rhapsodic, and in this incarnation involves examining the NMR spectra of fragments in the presence or absence of TERRA₁₆. Fragments that bind to the RNA show changes in ¹⁹F spin relaxation, resulting in broader, lower intensity signals. The library consisted of 355 compounds from a variety of sources, and although most of them were fragment-sized, a couple dozen had molecular weights above 350 Da.

The initial screen produced a fairly high hit rate (20 fragments), of which seven were studied in detail. Standard proton-based STD NMR confirmed the ¹⁹F-NMR results. The researchers then turned to a shorter RNA containing only two repeats (TERRA₂); this RNA sequence dimerizes to form a G-quadruplex. All seven fragments stabilized this complex against thermal denaturation, consistent with binding. Six of the fragments also induced changes to the ¹H NMR spectrum of TERRA₂, though one also caused general line broadening that could indicate aggregation. For the well-behaved fragments, dissociation constants (K_D) were determined by measuring changes in chemical shifts with increasing concentrations of ligand. K_D values ranged from 120 to 1900 micromolar, with modest ligand efficiencies ranging from 0.17-0.28 kcal/mol/atom.

Of course, selectivity against other nucleic acid structures is a major concern, so the researchers used ¹H and ¹⁹F NMR to assess compound binding to a tRNA, a DNA duplex, and a DNA analog of TERRA₂ also able to form a G-quadruplex. Aside from the putative aggregator, none of the seven compounds bound tRNA, and only two (including the aggregator) bound duplex DNA. However, all the compounds bound to the DNA G-quadruplex. Interestingly though, the DNA sequence used can form two types of G-quadruplexes in solution (parallel or antiparallel), whereas the equivalent RNA can only form a parallel dimer. In all cases the small molecules appeared to shift the equilibrium of the DNA to the parallel conformation, consistent with their initial identification as RNA binders.

Last year we highlighted another paper in which fragments were identified that may bind to a different DNA G-quadruplex. It would be interesting to functionally compare these two sets of hits. For example, do the hits identified initially against the DNA G-quadruplex also bind RNA G-quadruplexes? Of course, as with the riboswitch effort, there is a long way to go. It should be an interesting journey.