Fragments vs SARS-CoV-2 RNA

The first mention of SARS-CoV-2 on Practical Fragments in early March of last year highlighted a crystallographic fragment screen against the main viral protease. As discussed last week this effort has now led to compounds with nanomolar activity in cells. We’ve also highlighted a separate crystallographic screen against this target as well as a screen against the Nsp3 macrodomain. But proteins are not the only potential viral targets.

 

A recent (open access) paper in Angew. Chem. Int. Ed. by Harald Schwalbe and a large group of collaborators mostly at Johann Wolfgang Goethe-University focuses not on proteins but on RNA. Harald also presented this work at Discovery on Target last week, where he noted that the effort is part of the COVID19-NMR project, a collaboration of 240 people in 18 countries.

 

The researchers investigated 15 RNA regulatory elements that are conserved between SARS-CoV-2 and SARS-CoV, ranging from 29-90 nucleotides, as well as 5 larger multielement RNAs (118-472 nucleotides). These were screened against the DSI-poised library (discussed here): 768 fragments designed for rapid follow-up chemistry.

 

Three different ligand-detected NMR methods were used for screening: chemical shift perturbation (CSP) or line-broadening, waterLOGSY, and T2-relaxation. Fragments were screened at 200 µM in pools of 12 against 10 µM RNA. Compounds that hit in at least two assays were investigated individually.

 

In total 40 fragments bound to one or more of the 15 shorter RNAs, and an additional 29 fragments bound to the five longer RNAs. Between 5 and 49 hits were found for all but two of the RNAs. Selectivity varied: some fragments bound to just one RNA while one fragment bound to 18 of 20.

 

Given the negatively-charged phosphate backbone of RNA, it is not surprising that many of the fragment hits are positively charged at physiological pH. Nearly one-third of the 40 hits against the shorter RNAs contain a basic amine; pyrimidine and benzimidazole moieties are enriched, and not one of the hits contain a carboxylic acid. All the hits have at least one aromatic ring and most have two or three, perhaps suggesting intercalation. Moreover, as seen in a previous ambitious RNA screen from the same group, hits tend to have fewer sp³-carbons than non-hits.

 

The highest affinity fragment had a dissociation constant of just 64 µM but an impressive ligand efficiency of 0.38 kcal/mol/atom. A search of commercial analogs yielded a compound with low micromolar affinity against two RNA targets. In his presentation Harald noted that this series has since been optimized to a 200 nM binder.

 

This paper is a tour de force, but as I have noted, there remains a dearth of high-affinity specific RNA binders. The researchers also note another potential problem: viral RNA accounts for roughly two-thirds of total RNA in cells infected with SARS-CoV-2. Would this necessitate high concentrations of drug for effective antiviral activity?

 

Whether or not the work leads to drugs, it should further basic research. Laudably, structures of all the hits and non-hits are provided in the paper, and the extensive supporting information provides more details. Hopefully we will soon see whether fragments poised for ready elaboration really will enable rapid progress against RNA.