Targeting RNA with Small Molecules To Capture Opportunities at the Intersection of Chemistry, Biology, and Medicine

Abstract

The biology of healthy and disease-affected cells is often mediated by RNA structures, desirable targets for small molecule chemical probes and lead medicines. Although structured regions are found throughout the transcriptome, some even with demonstrated functionality, human RNAs are considered recalcitrant to small molecule targeting. However, targeting structured regions with small molecules provides an important alternative to oligonucleotides that target sequence. In this Perspective, we describe challenges and progress in developing small molecules interacting with RNA (SMIRNAs) to capture their significant opportunities at the intersection of chemistry, biology, and medicine. Key to establishing a new paradigm in chemical biology and medicine is the development of methods to obtain, preferably by design, bioactive compounds that modulate RNA targets and companion methods that validate their direct effects in cells and pre-clinical models. While difficult, demonstration of direct target engagement in the complex cellular milieu, along with methods to establish modes of action, is required to push this field forward. We also describe frameworks for accelerated advancements in this burgeoning area, their implications, key new technologies for development of SMIRNAs, and milestones that have led to broader acceptance of RNA as a small molecule druggable target.

Figure 1.

RNA structural hierarchy and examples of the 3D structures of small molecules bound to RNA. (Top) Sequence, secondary structure, and three-dimensional structure of tRNA. (Bottom) Left: structure of the bacterial ribosome (Protein Data Bank (PDB) ID 4V52) with protein in blue, RNA in dark gray, and neomycin B in green. Middle: binding of neomycin B to the A-site of the bacterial ribosome extracted from the structure on the left. Right: structure of a cyclic peptide bound to the HIV TAR RNA that was developed via structure-based design (PDB ID 2KDQ).

Figure 2.

Identifying and drugging targetable RNA structures in the human transcriptome. Diverse RNA sequences in the human transcriptome, or the composite of RNAs made from an organism, fold into defined three-dimensional structures. Indeed, human RNAs have hubs of defined structure, some of which are evolutionarily conserved and likely functional. These regions are ideal targets for small molecules (SMIRNAs), complementary to unstructured regions targeted with oligonucleotide-based modalities. We describe the design of bioactive SMIRNAs from sequence and how these SMIRNAs have enabled target validation tools.

Figure 3.

A library-versus-library screen, dubbed two-dimensional combinatorial screening (2DCS), defines interactions between three-dimensionally folded RNA structures and small molecules. (Top) 2DCS is completed with a library of RNA motifs that are embedded in a unimolecular, and thus amplifiable, hairpin structure. The cassette is general, as internal loop (3×3 and 4×3), bulge (3×2), hairpin (5-mer and 6-mer) and other RNA fold libraries can be studied using this approach. RNA libraries are labeled and screened for binding to small molecules in the presence of a large excess of RNAs that mimic the constant regions in the library (C1 and C2) and DNA oligonucleotides (C3 and C4). (Bottom) A microarray with an agarose surface provides a medium to spatially array and encode small molecules that can be studied for binding to RNA folds, for example by incubation with labeled 3×3 ILL in the presence of C1–C4. The three-dimensional RNA folds that bind small molecules are excised from the array and sequenced. Bioinformatic analysis is used to score the selected interactions. Briefly, by sequencing the starting library to define sequencing biases and comparing the RNA fold frequencies to those of the RNA folds selected to bind a small molecule, binding landscapes are quickly defined (via Z_obs). Those binders are then assigned a fitness based on the highest affinity interaction identified. These 2DCS-defined SMIRNA partners are then used to design small molecules targeting RNA and have a variety of validated activities. Atomic coordinates for the 4×3 IL, 3×3 IL, and 3×2 IL were obtained from RCSB PDB IDs 1JO7, 1HWQ and 2LU0, respectively. Atomic coordinates for the 6-nt HP and 5-nt HP were obtained from RCSB PDB IDs 1HWQ and 2B7G, respectively.

Figure 4.

Non-coding microRNAs play pervasive roles in biology to repress the amount of protein translated from an mRNA. Like other cellular RNAs, microRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNA) that undergo various processing steps. Indeed, pri-miRNAs are cleaved by the nuclease Drosha to liberate a precursor miRNA (pre-miRNA) that is translocated to the cytoplasm and further processed by Dicer to generate mature (functional) miRNAs. Mature miRNAs bind via base-pairing to the 3′ UTR of mRNAs with sequence complementarity and decrease the amount of protein synthesized. SMIRNAs that target nuclease processing sites inhibit biogenesis, reduce mature miRNA levels, and increase protein production of downstream targets. Many cancers aberrantly express miRNAs to repress the synthesis of pro-apoptotic proteins, and SMIRNAs have been developed against them as targeted lead medicines in several cancer indications.

Figure 5.

Factors affecting selectivity of small molecules targeting RNA. (A) Compounds can bind to RNAs but not affect biology. Targeting functional sites (i.e., nuclease processing sites) can affect RNA biology by inhibiting key processes. (B) Protein binding can enhance the selectivity of SMIRNAs. For example, sub-optimal RNA folds that bind a small molecule may be not occupied due to insufficient affinity to compete with protein binding. (C) Approaches for targeted destruction of an RNA are selective because of the inherent selectivity of the compound and the presence of a nuclease cleavage site(s) near the binding site. That is, selectivity is due to ligand binding and proper positioning of the cleaving entity. (D) Compound binding sites may not be accessible due to additional folding interactions.

Figure 6.

Target validation and profiling tools enabled by SMIRNAs. SMIRNAs have been developed that cross-link with their cellular targets (Chem-CLIP), cleave their cellular targets (RiboSNAP), change target RNA sequences, or compete with ASOs for binding (ASO-Bind-Map). The methods are complementary, and the ideal method to employ will depend on the RNA target and the inherent sequence specificity of the cross-linking, cleavage, or reactive species generated. (A) Chem-CLIP, a cross-linking approach in which small molecules bind to RNA targets and undergo a proximity-based reaction at the binding site, tagging the RNA with a purification tag. (B) Ribo-SNAP, a cleavage-based approach in which small molecules bind to RNA targets in cells and undergo a proximity-based cleavage reaction at the binding site, allowing transcriptomewide assessment of target engagement. (C) Changing RNA sequence with a small molecule. A small molecule that targets an RNA is appended with ruthenium bipyridine. Irradiation of cells and animals with light produces reactive oxygen species that convert G to *-oxo-G. Recognition of 8-oxo-G lesions by antibodies allows immunoprecipitation of bound RNAs, which can then be analyzed. (D) ASO-Bind-Map is a competition-based experiment between ASOs and small molecules. The binding of small molecules thermodynamically stabilizes a region of defined structure and inhibits ASO binding. Inhibition of ASO cleavage indicates SMIRNA binding of the targeted mRNA. (E) On-site drug synthesis can be used to study RNA target engagement. Briefly, two pro-drugs harboring a complementary donor or acceptor bind adjacent structures in an RNA target, triggering a proximity-based click reaction (RNA is the catalyst) and producing a FRET signal. This allows imaging target engagement and also tracking of the target upon binding to the drug.

Figure 7.

Ribonuclease-targeted chimeras (RIBOTACs) as an approach to cleave RNAs with endogenous nucleases. (Top) Targeted recruitment of RNase L with a small molecule. This approach has been shown to selectively and potently cleave a targeted RNA in a catalytic and sub-stoichiometric manner. (Bottom) RIBOTACs can be extended to other RNA-modifying enzymes by changing the recruiter module. Enzymes have defined substrate specificity and expression levels. As recruiters are developed for more enzymes, these factors can be used to control selectivity. Other factors that control selectivity include the linker between RNA-binding modules and the recruiter, which also influences cellular/tissue uptake and localization. Incorporation of enzyme-recruiting small molecules into Inforna allows a streamlined and designer way to affect RNA biology via cleavage, akin to antisense.

Figure 8.

Experimental workflow for developing validated small molecules interacting with RNA (SMIRNA) as lead chemical probes or medicines. The design and validation of small molecules that target RNA can be challenging. Illustrated is a scheme for their comprehensive evaluation, including in vitro and cellular studies, as well as other factors that should be considered. Notably, cellular studies should comprise an assessment of the small molecule’s effect on transcript and protein levels, its selectivity, direct target engagement, and its ability to reverse phenotype.

Figure 9.

Chemically diverse RNA-binding small molecules and the RNA folds that interact with them have been defined by 2DCS. Novel compounds, including derivatives of nucleic acid binders to ablate inherent substrate specificity, as well as both known and experimental drugs have been studied. Analysis of RNA binders from 2DCS has shown that various fragments, both present and absent in known drugs, bind RNA avidly. The observation that known drugs bind RNA, especially kinase and topoisomerase inhibitors, suggests that RNA should be considered as both an on- and off-target of experimental and known drugs. The canonical targets of known drugs that target RNA are listed under the compound’s structure.