Fragments vs (lots of) RNA

RNA is hot. Hundreds of millions of dollars have gone to startups focused on finding small molecules that bind RNA, and plenty of large pharmaceutical companies are pursuing this class. As with all difficult targets, fragments have a role to play, as demonstrated by a paper just published (open access!) in ChemBioChem by Harald Schwalbe and collaborators at Johann Wolfgang Goethe-University Frankfurt and Saverna Therapeutics.

 

The researchers assembled a collection of 101 fluorine-containing fragments and pooled these into five sets of 20-21 compounds each. These were then screened (at 50 µM) against 14 different RNA targets, ranging from 14-nucleotide hairpins to a 127-nucleotide riboswitch. The primary screen was ligand-detected ¹⁹F NMR using CPMG, in which ligand binding causes a change in relaxation which is detected as a decreased signal. All hits from the mixtures were confirmed as single molecules. To assess selectivity, all the compounds were also screened against 5 DNA targets and 5 proteins.

 

The results are not entirely unexpected. Some of the fragments did not hit any targets, while others were rather promiscuous: a couple showed strong binding to 8 of the 14 RNA targets. That said, strong is a relative term; the highest affinity of any fragment measured was 375 µM.

 

The RNA targets spanned a variety of structures, but the most hit-rich were aptamers and riboswitches, which bind specific small molecules or ions. These had 7 to 26 hits each. In contrast, the other RNA targets tested all had six or fewer fragment hits. For the aptamers, competition experiments with the natural ligands suggested binding at the orthosteric sites in some cases but not others.

 

Hit rates against four of the five proteins were also high, with 16 to 55 hits each. The fifth protein, a challenging phosphatase, had only four hits. This was still better than the 24-nucleotide DNA duplex, with a single hit. The four G-quadruplex DNA targets had between 12 and 20 hits, consistent with prior research. Taken together, the results suggest that RNA riboswitches and aptamers may be reasonably ligandable, while RNA targets that do not normally bind small molecules may be more challenging.

 

The researchers also conducted cheminformatic analyses. Not surprisingly given the relatively small library, there were no strong correlations between molecular features and targets bound, though fragments that hit had a slight tendency towards more aromatic atoms and fewer sp³-hybridized carbons. This is consistent with a vigorously debated paper we highlighted in July.

 

Finding weak hits is one thing, but advancing them has been challenging for RNA targets. The researchers provide an example in which they link a fragment to an intercalator to generate a low micromolar binder, but intercalators are often nonspecific, and affinity would still need to be further improved. Practical Fragments first highlighted a fragment screen against RNA more than a decade ago, and earlier this year we noted a high nanomolar binder, but I have yet to see an attractive low nanomolar lead emerge.

 

Nonetheless, this paper provides a solid launching point for such an effort to succeed. In particular, the researchers laudably include structures of all the library members as well as the raw screening data of all the hits on all the targets in the Supporting Information. If you are feeling adventurous, you now have plenty of starting points to choose from.