05 April 2021

A general fragment-based approach to… targeting RNA?

This is taken from the title of a recent open-access paper by Matthew Disney and collaborators at Scripps Research Institute Jupiter and Florida Atlantic University in Proc. Nat. Acad. Sci. USA. RNA has long been a target of FBLD: Practical Fragments first blogged about it in 2009, and a 2002 paper reported using fragment linking to obtain a low micromolar binder. So how general is the new approach?
The researchers describe chemical cross-linking and isolation by pull-down fragment mapping (Chem-CLIP-Frag-Map). This involves using photoaffinity probes that can crosslink to biomolecules such as RNA. The probes also have an alkyne tag that can be used to isolate bound molecules using click chemistry. We’ve written previously about such “fully functionalized fragments” (FFFs).
Earlier work had resulted in the identification of compound 1, which binds to a specific site on pre-miR-21, the precursor to a non-coding microRNA linked to cancer. An FFF version of compound 1 was shown to crosslink to pre-miR-21 after irradiation with UV light, and the site of modification could be mapped using a reverse-transcriptase-mediated primer extension, which stalled at the modified bases.
Next, the researchers screened 460 FFFs at 100 µM and found 21 that crosslinked to pre-miR-21. They were ultimately looking to link fragments with compound 1, and thus competition studies were used to eliminate fragments that bound at the same site. This left three fragments, and primer extension studies confirmed that these bound near but not at the binding site of compound 1.
Next, the researchers attached these three fragments to compound 1, with or without various linkers. Some of the resulting molecules had improved affinity, and compound 9 showed the tightest binding according to microscale thermophoresis (MST). Mutational and competition studies confirmed that the molecule binds to the expected site. Importantly, compound 9 not only bound to pre-miR-21, it also blocked processing by the enzyme Dicer. Moreover, it showed activity in cell models consistent with inhibition of pre-miR-21.

This is a nice paper, but there are several limitations. First, compound 9 is still a fairly modest binder with lackluster ligand efficiency. Indeed, while potency can be overrated, I would love to see a fully synthetic low nanomolar RNA binder. Second, while the approach may be general, it is not necessarily easy, and it requires specialized fragments. And as we noted last year, there is no relation between crosslinking efficiency and affinity. I wish the researchers had tried linking some of the non-selected fragments to see whether these were false negatives. Indeed, given the complexity of the approach, I wonder if the researchers would have been better off simply making and testing an anchor library around compound 1, in a similar fashion as described here.
But whether or not Chem-CLIP-Frag-Map turns out to be the solution to targeting RNA, I wholeheartedly agree with the conclusion: “It may be time to describe biomolecules that are perceived to be challenging small molecule targets as ‘not yet drugged’ rather than ‘undruggable.’”

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