A click chemistry assay to identify natural product ligands for pre-microRNAs

Abstract

Despite the great diversity of structure and function and relevance to human health, RNA remains an underexploited area of drug discovery. A major bottleneck toward this goal has been the identification of probes and drug leads that are specific for select RNAs and methods that will facilitate such discovery efforts. Our laboratory has recently developed an innovative approach for assaying RNA-small molecule interactions, catalytic enzyme-linked click chemistry assay or cat-ELCCA, which is a functional assay that takes advantage of the power of catalytic signal amplification combined with the selectivity and bioorthogonality of click chemistry. Importantly, through application of this platform assay technology to the challenging problem of identifying selective inhibitors of pre-microRNA maturation, we identified natural products as a potential source of such compounds. Herein we describe this methodology in addition to the downstream pipeline toward the discovery of natural product ligands for premicroRNAs. Through cat-ELCCA, our goal is to discover novel ligands to facilitate our investigation of RNA recognition by small molecules.

Discovery of Surfactins as Inhibitors of MicroRNA Processing Using Cat-ELCCA

“…We first measured IC 50 values for each of the surfactins 1 – 6 using cat-ELCCA (Figure ). , To determine if the purified compounds retained any selectivity for pre-miR-21, we also performed the assay with pre-let-7d as a structurally similar control pre-miRNA . As shown in Figure , while metabolites 1 and 2 were inactive in the assay, analogues 3 – 6 showed dose-dependent inhibition of Dicer processing of both pre-miRNA substrates, albeit with only a modest favoring toward pre-miR-21 inhibition.…”

“…Encouraged by these results, we next set out to determine the mechanism of inhibition. To determine if the isolated surfactins had pre-miRNA binding capacity, we measured the binding affinity ( K d ) of surfactins 3 – 6 for pre-miR-21 using surface plasmon resonance spectroscopy (SPR). , To our delight, all surfactins bound to the RNA with K d values of 380–800 nM (Figure C; see Figure S95 for sensorgrams). We then profiled binding to pre-let-7d.…”