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.

Upon Request

Dan and I blog here because we love it; we don't get paid, it takes a lot of time, and has very little reward.  I love it when I meet someone new and they say, "Oh, I read your blog."  However, this allows us to have freedom to review what we want, when we want, and how we want.  We don't sell advertising, we don't generate revenue, and so on.  Sometimes people agree with us, sometimes they don't.  These posts are our opinions and like bellybuttons, everyone has one.  Sometimes, we get pinged by somebody who just published a paper and would really like to see us blog about it.  Sometimes we do, sometimes we already have and they missed it, and sometimes we don't.

I received a polite email recently, pointing out this paper.  It was already on my radar to blog about, so I bumped it up in the queue.  This paper caught my attention because it is a fragment screen against a DNA-target, specifically the G-quadruplex from c-MYC.  G-quadruplexes are found in the promoters to many oncogenes and the supposition is that by stabilizing them you can reduce their transcription.  It is an intriguing idea which has already been investigated with a number of compounds to date.  These authors decided to use fragments against the G-quadruplex without knowing if fragments would bind to a nucleic acid target with sufficient affinity and selectivity.  Their primary screen was an Intercalator Displacement Assay (IDA) which has been used previously to find G-quadruplex binding ligands.  A 1377 fragment library (@5mM) (previously used against riboswitches) was used and it obeyed the Voldemort Rule, had >95% purity, and 1mM aqueous solubility. The top 10 hits from this screen could be placed in three groups.

Then, in order to confirm their biochemical assay results they decided to dock them these top 10 fragments.  WHHAAAAT you say?  That was my initial reaction.  Why oh why doth they vex me so?  They then go into EXCRUCIATING detail about the docking results, even concluding from the results some SAR hypotheses.  I kid you not.  They also evaluated these top 10 fragments in a cellular assay (125um and 250uM) using a Western blot readout.  These concentrations were chosen in order to not show short or long-term toxicity, but Mirabile dictu, Data Not Shown.  All fragments, except two (7A3 and 2G5), showed significant changes in c-Myc expression levels. Interestingly, "no significant changes" still gives a 20% reduction in c-Myc levels. 

Four fragments were able to reproduce this effect, of which 11D6 was the best.  The four best were then run pair-wise to and every combination induced a significant reduction of c-Myc.  

So what does this tell us?  Well, I think they have found fragments which bind to the c-MYC promoter G-quadruplex.  It may be exhibiting this binding in the cells.  There are a few experiments that I would like to see (and would have asked for if I had reviewed this paper): a binding assay (SPR, ITC, NMR, whatevs) being he primary one.  We also continue to know that docking really does not add anything to the discovery process.