Validating Fragment-Based Drug Discovery for Biological RNAs: Lead Fragments Bind and Remodel the TPP Riboswitch Specifically

Thiamine pyrophosphate (TPP) riboswitches regulate essential genes in bacteria by changing conformation upon binding intracellular TPP. Previous studies using fragment-based approaches identified small molecule fragments that bind this gene-regulatory mRNA domain. Crystallographic studies now show that, despite having micromolar K(d)s, four different fragments bind the TPP riboswitch site-specifically, occupying the pocket that recognizes the aminopyrimidine of TPP. Unexpectedly, the unoccupied site that would recognize the pyrophosphate of TPP rearranges into a structure distinct from that of the cognate complex. This idiosyncratic fragment-induced conformation, also characterized by small-angle X-ray scattering and chemical probing, represents a possible mechanism for adventitious ligand discrimination by the riboswitch, and suggests that off-pathway conformations of RNAs can be targeted for drug development. Our structures, together with previous screening studies, demonstrate the feasibility of fragment-based drug discovery against RNA targets.