Parallel Discovery Strategies Provide a Basis for Riboswitch Ligand Design

Abstract

Highlights d Searched for synthetic ligands to the ZTP riboswitch using two approaches d Identified hits that replace the natural ligand's ribose moiety with a pyridine d Lead compound binds to and activates ZTP riboswitches in vitro and in vivo d Co-crystal structures suggest affinity is achieved via p-p stacking interactions Authors

Small molecule targeting of biologically relevant RNA tertiary and quaternary structures

“…Both methods yielded medium-and high-affinity compounds, but the only ones that resulted in in vivo activity were synthetic analogues of the cognate ligand ZMP. Notably, the analogue found to be 3-fold more potent than the cognate ligand in vivo reported a 2-fold lower affinity than ZMP to the riboswitch in vitro, thereby highlighting once again the importance of not relying on affinity to rank compounds fitness when targeting riboswitches (Tran et al, 2020). Despite their impressive cross-species sequence/function conservation, recent successes have proved that the differences in 3D conformations of riboswitches can be harnessed to develop ligands with high specificity for a single species-specific topology.…”

“…A recent joint effort by the Ferre ´-D'Amare ´and Schneekloth laboratories allowed for a direct comparison between synthetic and high-throughput approaches (Tran et al, 2020). In the study, the authors aimed at targeting a purine-related riboswitch, the ZTP riboswitch.…”

Targeting RNA structures with small molecules

“…Small-molecule microarrays (SMMs), created by delivery of minute amounts of compounds to glass slides in a spatial array, were initially used to interrogate protein binding [91][92][93][94] and later extended to study the binding of aminoglycosides to the rRNA A-site 95 and how binding is affected by aminoglycoside modification by resistance enzymes 96 . SMMs have now been used to screen a wide variety of compounds and RNA targets [97][98][99][100][101][102][103] (fIg. 2c).…”

Subsite Ligand Recognition and Cooperativity in the TPP Riboswitch: Implications for Fragment-Linking in RNA Ligand Discovery

“…Cooperativity and induced fit are critical in molecular recognition and biological function. − Ligand cooperativity has been extensively studied in protein–small molecule ligand systems, ,, and principles derived from these studies have been used to guide fragment-based ligand design for multiple protein targets. ,− The field of RNA-targeted drug discovery is undergoing a notable shift to emphasize low-molecular-weight, drug-like molecules − and fragment-based approaches represent a promising strategy for discovering molecules with favorable properties that bind RNA. − RNA molecules clearly experience significant cooperativity at the level of their global folding and interactions with divalent ions, , in binding with large oligonucleotide ligands, , and for interactions between dimeric and multivalent ligands with duplex RNAs . However, structure–function relationships that underlie cooperativity at individual RNA–ligand interaction sites are poorly understood.…”