Small molecule targeting of biologically relevant RNA tertiary and quaternary structures

Abstract

Initial successes in developing small molecule ligands for non-coding RNAs have underscored their potential as therapeutic targets. More recently, these successes have been aided by advances in biophysical and structural techniques for identification and characterization of more complex RNA structures; these higher-level folds present protein-like binding pockets that offer opportunities to design small molecules that could achieve a degree of selectivity often hard to obtain at the primary and secondary structure level. More specifically, identification and small molecule targeting of RNA tertiary and quaternary structures have allowed researchers to probe several human diseases and have resulted in promising clinical candidates. In this review we highlight a selection of diverse and exciting successes and the experimental approaches that led to their discovery. These studies include examples of recent developments in RNA-centric assays and ligands that provide insight into the features responsible for the affinity and biological outcome of RNA-targeted chemical probes. This report highlights the potential and emerging opportunities to selectively target RNA tertiary and quaternary structures as a route to better understand and, ultimately, treat many diseases.

Keywords: G-quadruplex; RNA structure; high-throughput screening; pseudoknot; quaternary; riboswitch; small molecule; synthetic library; tertiary; triple helix.

Figure 1.

(A) MALAT1 triplex binder with highest affinity and selectivity (left), proposed mode of binding obtained through docking (center), and representative assay set up to test the effects of the small molecule on RNAse R-induced MALAT1 degradation (right) (Donlic et al., 2020). (B) Small molecule lead found through HTS (left), proposed mode of binding obtained through docking (middle), and effect of the small molecule on MALAT1 RNA levels and branching in organoid ex vivo models (right) (Abulwerdi et al., 2019). (C) Small molecule reported binder (left) of SARS-CoV pseudoknot element discovered through a high-throughput docking effort (middle), which resulted in a decrease in frameshifting upon binding (right) (Park et al., 2011).

Figure 2.

(A) NRAS mRNA G4-binder (left) reported to decrease the expression of NRAS (right) (Katsuda et al., 2016). (B) TERRA mRNA G4 stabilizer (left), which decreases increases the affinity of the structure for TRF2, thereby hijacking it from binding telomeric DNA (right) (Zhang et al., 2017). (C ) Small molecule (left) reported to destabilize ADAM10 and increase its expression in AD models (right) (Dai et al., 2015). (D) VEGF G4 binder (left) that results in the destabilization of the G4 structure thereby reducing expression of the angiogenic protein (right) (Wang et al., 2017b).

Figure 3

(A) Representative effect of a small molecule that stabilizes the inactive state of a riboswitch resulting in a decrease in transcription/translation adapted from Mulhbacher et al. (B) Schematic of a small molecule that favors the active conformation of a riboswitch increasing transcription/translation. (C) Small molecule lead (right) and its binding pocket (light blue) on PreQ1 riboswitch of T. tencongensis PDB: 3Q51 (Connelly et al., 2019). (D) Small molecule fragment (magenta) bound to E.coli thiM TPP riboswitch (PDB: 4NYB) (Warner et al., 2014). While not being the best fragment from Cressina et al. it was chosen for representation due to its detailed follow-up by Warner et.al. (E) Ribocil-C (magenta) binding-site on FMN riboswitch of F. nucleatum (magenta) PDB 5C45 (Vicens et al., 2018). (F) Modeled small molecule (magenta) bound to FMN riboswitch with improved cellular accumulation and activity against gramnegative bacteria PBD: 6BFB (Motika et al., 2020; Rizvi et al., 2018).

Figure 4.

(A) Representative effect of a small molecule that stabilizes the inactive state of a riboswitch resulting in a decrease in transcription/translation adapted from Mulhbacher et al. (B) Schematic of a small molecule that favors the active conformation of a riboswitch increasing transcription/translation. (C) Reported small molecule analogue binder of guanine riboswitch PDB: 1U8D (Mulhbacher et al., 2010). (D) Cyclic C -di-GMP analogue bound to V. choloeae riboswitch PDB: 3IRW (Furukawa et al., 2012).

Figure 5.

Group II intron binder (left) recently reported to prevent splicing in COX1-containing C. parapsilosis (Fedorova et al., 2018)

Figure 6.

(A) First small molecule reported binder (left) to promote full length expression of SMN1 protein in SMA patients (right) (Naryshkin et al., 2014) and its reported mode of binding (center) (Campagne et al., 2019). (B) SMN 5’-splice site binder(left) and its reported mode of binding (center) (Palacino et al., 2015).

Figure 7.

(A) small molecule binder (left) reported to decrease the affinity of HOTAIR essential binding element (center) for EZH2 and promote NLK expression (right) (Ren et al., 2019). (B) Small molecule (left) found to induce a conformational change (center) that exposes AUF1 binding site but not the hnRNP A1 site, thereby halting viral proliferation (right) (Davila-Calderon et al., 2020).