The prospect of RNA targeting using small molecules

December 13, 2017
David Chenoweth, PhD, Associate Professor, Department of Chemistry University of Pennsylvania

Small molecules that bind to macromolecular nucleic acids are well known. Before the 1960s, dyes such as aminoacridines, were utilized by histologists and biologists to image specific sub-nuclear structures.1 This was perhaps the earliest recognition that small molecules can bind macromolecular nucleic acids. In 1961, the first nucleic acid-small molecule binding hypothesis (the “intercalator hypothesis”) was formulated by Leonard Lerman, and thus, the first of the canonical small molecule-nucleic acid binding modes was born.2 As with many new ideas, the scientific community remained skeptical of Lerman’s intercalation hypothesis for many years. But his discovery was a pivotal turning point that would eventually usher in a new age of biophysical studies that would reveal molecular-level details of many nucleic acid-binding drugs, some of which are still used as first-line chemotherapeutics today.

The minor groove of DNA represents another binding target for small molecules with the earliest examples of DNA minor groove binders represented by the natural products netropsin and distamycin. Despite their discovery in the early 1950s and 1960s, their mode of binding would not be elucidated until 1980’s. Landmark studies using X-ray, NMR, and affinity cleavage experiments would for the first time demonstrate the plasticity of the DNA double helix through its ability to accommodate small molecule minor groove binding ligands in 1:1 and 2:1 binding modes. Molecular design efforts through the next three decades would elegantly demonstrate that the minor groove of DNA can be targeted with structure and sequence specificity by rationally designed high affinity small molecule ligands. This approach has been beautifully exemplified in the large body of work from the Dervan laboratory along with many others.3a-g

RNA is structurally more diverse and more complicated than most DNA and thus refractory to the kind of modular ligand design efforts that have worked for B-form duplex DNA.4 Double helical RNA proves refractory to targeting with small molecule groove binders since the minor groove is nearly non-existent in the A-form helix, unlike the deep and narrow minor groove of B-form DNA. The classic solution to this problem involves targeting linear RNA using complementary oligonucleotides to enable a rational modular pharmacophore design. Advances in chemical synthesis of oligonucleotides through the 1980s and 1990s and unnatural oligonucleotide modifications were critical to the development of the antisense oligonucleotide (ASO) approach.

Chemical modifications dating back to the mid 1960s, such as 2’-fluoro5, phosphorothioate6, and 2’-O-methyl modifications7 helped to address stability and delivery problems that plague the field. Alternate backbone recognition motifs, such as PNAs8 and LNAs9, appeared through the 1990s along with triple helix recognition10 and aptamer11 breakthroughs. These approaches have been broadly successful, yielding multiple clinical candidates including both ASOs and RNAi. Ignoring the delivery challenges inherent in this class of compounds, beyond common target organs such as the liver and eye, ASOs serve as an important proof of principle that RNA can be targeted for therapeutic benefit.

Another broad class of RNA-binding ligands that has been studied for many years includes antibiotics that bind to ribosomal RNA and block various critical steps in bacterial translation.12 These ligands have been primarily discovered by screening efforts and are over-represented with natural products. Although many of these agents may not be considered “drug-like,” as they are polyamines with poor selectivity and tissue distribution, they are nevertheless in widespread clinical use. Moreover, many (e.g., clindamycin) are quite drug-like and some are completely synthetic (e.g., linezolid). For many years, it was not entirely appreciated that these agents directly bound rRNA rather than the associated ribosomal proteins. As X-ray structures of ribosomes become available, the fascinating molecular mechanisms of action of these agents have been elucidated.

So, what’s next? With the example of drug-like SMs binding to rRNA, perhaps other RNAs can be targeted with small molecules.13 A particularly attractive target class includes riboswitches.14 In a brilliant series of papers from Ron Breaker’s group, it has become apparent that lower organisms often employ dynamic RNA folds in the 5’ UTRs to bind endogenous small molecules and thereby regulate transcription or translation of the downstream RNA.15 The discovery of drug-like analogs of these endogenous ligands could produce interesting biology – a path that is very familiar to scientists that design ligands against proteins. In fact, a team at Merck recently found drug-like ligands (i.e., ribocil), binding to the FMN riboswitch that regulates FMN biosynthesis.16

Are there other targets beyond riboswitches and rRNA that are amenable to small molecule binding? The broad, undiscovered domain of the transcriptome demands that we answer this question. The fact that we can take a pretty arbitrary small molecule and use SELEX methods to identify high-affinity aptamers indicates that there is nothing intrinsically averse about tight and selective small molecule-RNA interactions. In an effort to identify nucleic acid structures that model endogenous RNA structures, we made and studied simple multi-helical nucleic acid junctions and assessed the binding behavior of small molecules based on a new triptycene scaffold that were complementary to the cavity at the three-way junction interface.17 We were able to discover molecules that once complexed with the RNA, conferred substantial stabilization of the folded structure.

The lingering question remains, are there endogenous structures with similar pockets that lend themselves to small molecule targeting? Sigma32 RNA, responsible for regulating the bacterial heat-shock response, provides one unique example.18 Thermal destabilization of this RNA leads to increased translation and initiation of the heat-shock response in E. coli. Therefore, small molecules that stabilize this RNA in cells should suppress the heat-shock response program. We demonstrated that shape-complementary small-molecule ligands (i.e., triptycenes) suppressed the heat-shock response in a dose-dependent fashion.

We see these findings as a promising beginning, offering the prospect of discovery of ligands that bind folded RNAs and impact the biological functions of these RNAs.


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