New approaches to target RNA binding proteins

Abstract

RNA binding proteins (RBPs) are a large and diverse class of proteins that regulate all aspects of RNA biology. As RBP dysregulation has been implicated in a number of human disorders, including cancers and neurodegenerative disease, small molecule chemical probes that target individual RBPs represent useful tools for deciphering RBP function and guiding the production of new therapeutics. While RBPs are often thought of as tough-to-drug, the discovery of a number of small molecules that target RBPs has spurred considerable recent interest in new strategies for RBP chemical probe discovery. Here we review current and emerging technologies for high throughput RBP-small molecule screening that we expect will help unlock the full therapeutic potential of this exciting protein class.

Keywords: Chemical probes; Chemoproteomics; High throughput screening (HTS); RNA binding proteins (RBPs).

Figure 1. Fluorescence-based assays for RPI inhibitor screening.

(a) Förster Resonance Energy Transfer (FRET). Both RNA and RBP are appended to fluorophores with overlapping spectra, such that FRET emission is detected upon their interaction and abolished upon inhibition of their interaction. (b) Fluorescence polarization (FP). The RNA (or RBP) is appended to a fluorophore that is excited by plane-polarized light. If RBP binding is inhibited, the RNA will tumble faster in solution, increasing the detection of perpendicularly polarized light. (c) AlphaScreen®. RBP and RNA are appended to donor and acceptor beads, respectively. Excitation of donor beads leads to conversion of oxygen to singlet oxygen, which reacts to generate a chemiluminescent signal with the acceptor bead that then excites a fluorophore within the same bead, resulting in a fluorescent emission. No emission is detected upon small-molecule inhibition. (d) Enhanced fluorescence. The RNA is appended to a fluorophore whose fluorescence is enhanced upon RBP binding. Weak fluorescence indicates inhibition of interaction

Figure 2. Bioorthogonal chemistry-based assays for RPI inhibitor screening.

(a) Catalytic enzyme-linked click chemistry assay (cat-ELCCA). Inhibition of Dicer-mediated RNA processing is reported by CuAAC conjugation of alkyne-functionalized RNA to azide-HRP. The use of inverse electron demand Diels–Alder chemistry (IEDDA) can further increase the sensitivity and reproducibility of cat-ELCCA. (b) Nanoluciferase reporter assay. The combination of TCO-functionalized RNA with mTet-functionalized SmBiT affords a SmBiT-RNA fusion. Binding to a LgBiT-RBP fusion produces a detectable signal by luciferase formation. Inhibition of RNA-RBP binding abolishes such signal

Figure 3. Detection of druggable RBPs through chemoproteomics.

A reactive ligand featuring a bioorthogonal handle is introduced to cells or cell lysates to react either covalently (top scheme) or reversibly (bottom scheme) with protein targets. Reversible probes harbor a photocrosslinkable moiety for covalent linkage to protein targets upon UV irradiation. Probe-labeled targets are enriched by conjugation to biotin via CuAAC and then identified by LC-MS/MS.