Insights into the development of chemical probes for RNA

Morgan, Brittany S; Forte, Jordan E; Hargrove, Amanda E

A Screening Approaches ties prior to screening. While successful in protein-targeted drug discovery (41), only one report identified a bioactive ligand from a fragment-based library (commercial library) (42) . Once optimized, the scaffold yielded four additional molecules that targeted the Influenza A RNA promoter with IC 50 values ranging from 34 to 44 M in a cell-based luciferase assay (43) . Similarly, only two reports yielded bioactive small molecules from natural productbased libraries (synthetic library and academic library) (19, 44) . Both of these screens contained fewer than 150 small molecules, yet identified ligands that bind and modulate G-quadruplex structures located in the 5 -UTR of two distinct mRNAs. It is promising that small molecules were discovered from a variety of HTS libraries, suggesting that biologically active, RNA-binding ligands can be found in a subset of current small molecule chemical space (22) . Further validation and exploration of this space could lead to greater efficiency and success in identifying bioactive leads as well as RNA-privileged chemotypes. As expected, FcS used smaller libraries, typically containing fewer than 150 small molecules [ Table 1 ]. The largest FcS library (n = 320, academic library) was designed through a chemical similarity search of the bis-benzimidazole and similar cores, which have shown preferences for 1 × 1 nucleotide internal loops (45) . In addition, this library was filtered for favorable medicinal chemistry properties, and the screen resulted in three leads that: (i) bound r(CUG) exp in vitro; (ii) led to a statistically significant decrease in cTNT mini-gene exon inclusion in cells and (iii) were selective for a mini-gene with 960 r(CUG) repeats compared to a mini-gene without the repeats. FcS also encompassed RNA structure-guided design, which included two studies utilizing molecular modeling to identify ligands structurally similar to guanine for the xpt-pbuX riboswitch (46, 47) . In another structure-guided approach, a small library of p-terphenylene-based ligands was designed to mimic an ␣-helix of Rev, a protein-binding partner of the HIV-1 Rev Response Element (RRE) (48) . Leads were selected by docking, with one ligand disrupting the Rev-RRE interaction in vitro (IC 50 = 6.8 M), inhibiting HIV-1 replication (IC 50 values of 3.4-5.9 M), and exhibiting on-target effects via a RRE-luciferase reporter assay (IC 50 values of 10-21 M). Further, we did not identify successful reports of biologically active ligands from small molecule libraries biased to general RNAbinding. Recently, chemical companies have designed such focused libraries (ChemDiv, Nucleic Acid Ligands: http://www.chemdiv.com/nucleic-acid-ligands/; Otava Chemicals, RNA Targeted Library: http: //www.otavachemicals.com/products/targeted-librariesand-focused-libraries/rna-binding); however, the success of these libraries is yet to be reported. To date, successful FcS strategies have utilized knowledge of the RNA structure and/or a small molecule binder(s), neither of which is known for many therapeutically-relevant RNAs.