Fragment-based lead discovery against RNA has been a theme on Practical Fragments for well over a decade. Unfortunately, most of the resulting hits are either weak or non-druglike. A paper published late last year in J. Am Chem. Soc. by Matthew Disney and collaborators at the Scripps Research Institute in Florida provides a nice counter example.
The researchers started by building a new fragment library based on known RNA-binding molecules – a venerable approach we first described back in 2009. The most common scaffolds differ somewhat from the most common scaffolds found in drugs (see here), with pyrimidines, triazoles, furans, and benzimidazoles over-represented. A set of 2500 fragments mostly conforming to the rule of three was purchased from ChemDiv, and the structures of all of them are helpfully provided in the supporting information.
Having identified specific RNA sequences bound by compound 3, the researchers searched human microRNAs (miRNAs) and found that the pre-miR-372 contains a bulge predicted to bind. When this RNA is processed it produces miR-372, which represses translation of the tumor suppressor LATS2, thereby increasing cellular proliferation. Perhaps the binding of compound 3 to the pre-miRNA would impede its processing.
The affinity of compound 3 for pre-miR-372 was measured to be 300 nM, giving it an impressively high ligand efficiency. In vitro experiments showed that the compound blocked processing by the enzyme Dicer. The researchers conducted a series of cellular experiments showing that compound 3 decreased levels of miR-372 and increased pre-miR-372m while having no effect on 379 other miRNAs. Encouragingly, compound 3 also increased levels of LATS2 mRNA and protein and decreased cell proliferation. Additional experiments with siRNA, mutated versions of pre-miR-372, and cell lines with lower levels of miR-372 all support the on-target mechanism.
This is a lovely paper, and compound 3 appears to be a good starting point for further optimization. However, the work also suggests why finding lead-like RNA binders may be so difficult. In the library-vs-library approach described, the researchers studied 12,800,000 different molecular interactions and came up with just three (somewhat) specific binders. The ligandability of RNA – or at least the small internal loops studied here – appears to be low. To prospectively find hits against specific RNAs may require much larger fragment libraries than are typically used. Perhaps this could be an application for DNA-encoded fragment libraries, which we wrote about here.