Fragment linking on the bacterial TPP riboswitch
Last week’s post focused on fragment screening against RNA, and we continue the theme this week with a paper published in Proc. Nat. Acad. USA by Kevin Weeks and collaborators at University of North Carolina Chapel Hill, New York University, and Université de Sherbrooke.
The researchers developed a screening technology called SHAPE-MaP (Selective 2’-Hydroxyl Acylation analyzed by Primer Extension and Mutational Profiling). Essentially, RNA in the presence or absence of potential ligands is treated with an acylating agent that reacts with the 2’-hydroxyl group on ribose subunits. This addition requires the hydroxyl groups to be exposed, so ligands that bind in the vicinity may directly block or cause conformational changes to change the patterns of acylation. Conveniently, acylation causes mutations when the modified RNA is sequenced, making modified sites easy to detect. Moreover, by clever uses of “barcodes” in other regions of the RNA, multiple samples can be pooled and analyzed.
Because the approach uses sequencing to identify binding sites, long strands of RNA can be tested. In this case, the researchers built an RNA construct containing a ‘pseudoknot’ structure in the dengue virus genome as well as a thiamine pyrophosphate (TPP) riboswitch, which changes conformation when it binds to TPP. (We wrote about a different fragment screen against this riboswitch back in 2014). A set of 1500 rule-of-threecompliant fragments from Maybridge was screened, resulting in 41 hits. These were then rescreened in triplicate, which winnowed the field to just eight fragments, of which seven bound TPP and one appeared to be nonspecific. All eight were assessed by isothermal titration calorimetry (ITC), which produced measurable affinities for six.
Compound 2 was the most potent hit, and when the researchers tested 16 analogs they found some, such as compound 17, with improved affinity. With an eye towards fragment linking, they took a conceptually similar approach to SAR by NMR by rescreening the original 1500 fragments in the presence of compound 2 to look for ligands that would bind at a second site. This yielded five hits, including compound 28. ITC characterization of a more soluble analog, compound 31, revealed that it had weak but measurably improved affinity in the presence of compound 2. A handful of linked analogs were made, and while most of these had affinity similar or worse than the best initial fragment, compound Z1 bound with sub-micromolar affinity as assessed by ITC.
The natural ligand TPP binds to the riboswitch with a Kd of 110 nM, and in doing so blocks in vitro transcription of bound RNA. Despite having a similar affinity as TPP, compound Z1 was much less effective at blocking transcription. Unfortunately, although the researchers were able to obtain crystal structures of several molecules bound to the riboswitch, including compound 17, they were unable to obtain one with compound Z1.
This is a rare example of fragment linking on RNA, and although the linked molecule does not show fully additive affinity, it does have reasonable ligand efficiency. But like the example last week, this paper illustrates how difficult discovering RNA binders is likely to be. The confirmed hit rate is less than 0.5%, and this is for an RNA sequence that evolved specifically to bind low molecular weight ligands. As the researchers note, none of the fragments bound the dengue virus pseudoknot. Perhaps most RNA is truly undruggable, at least with small molecules.