RNA-Targeted Small Molecule Drug Development

Why Develop RNA-Targeted Drugs?

For small molecule therapies, the vast majority of targets are proteins. This strategy has led to a large number of new drugs and breakthrough discoveries over the past few decades, but there are a number of problems and bottlenecks with this route:

• Firstly, many proteins are not druggable, which means that it is difficult to develop small molecules with inhibitory properties.
• Secondly, proteins account for only a tiny fraction of the information in the genome. Only 1.5% of the human genome encodes proteins, and disease-related proteins account for only 10-15% of this total.

However, RNA is one such potential target that may be able to break through the blockages that exist in the development of protein-targeted drugs. In normal cells, RNA has an important physiological function, carrying genetic information from genes to direct protein synthesis. Non-coding RNAs, on the other hand, regulate gene expression. Developing drugs that target RNA has multiple benefits:

• As it is in the upstream of proteins, targeting RNA is expected to directly up-regulate or down-regulate the translation efficiency of proteins, solving the problem that proteins are not drug-ready.
• RNA is extremely abundant in the human genome, and the sequences that produce non-coding RNA account for 70% of the genome, which is much more abundant than the sequences that code for proteins.

Important Structural Regions of RNA

For different structural analyses and tests of RNA there are these methods: nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography, and cryo-electron microscopy (cryo-EM) can be used to determine the tertiary structure of RNA; programs such as RNAfold, Sfold, and CONTRAfold can be used to predict the secondary structure (base-pairing) of RNA, and programs such as FARFAR2, MC-Fold/MC-Sym, and iFoldRNA can be used to predict the tertiary structure of RNA from the RNA sequence. The important structural regions of common RNAs are as follows

• Iron Response Element (IRE): A highly conserved cis-acting stem-loop structure containing one or more unpaired adenine bases. Typically found in RNA molecules associated with iron metabolism, such as mRNA for transferrin receptors, ferritin, etc., Iron Response Proteins (IRP) can bind to IREs, thereby influencing their stability or translation processes, affecting the expression of iron metabolism-related proteins, and thus regulating the cellular metabolism of iron.
• Splicing Modulator: This includes splicing enhancers and silencers. They interact with splicing regulatory factors to induce the inclusion or exclusion of exons, guiding RNA splicing, and generating different mRNA isoforms, thereby affecting protein translation.
• RNA Repeat Sequences: Repetitive nucleotide sequences occurring in RNA molecules, which may play a role in RNA structure and function. Abnormal or excessively long repeat sequences, known as RNA repeat expansions, can cause abnormalities in RNA structure and function, leading to certain diseases (such as microsatellite diseases).
• Drosha and Dicer Processing Sites: These sites appear in precursor microRNA (miRNA) and are associated with miRNA maturation (pri-miRNA transcribed by RNA polymerase II is processed by the endonuclease Drosha into pre-miRNA, which is further processed by the endonuclease Dicer into mature miRNA). miRNA, along with the RNA-induced silencing complex (RISC), regulates mRNA degradation, etc.
• Other Sites: Such as Internal Ribosome Entry Site (IRES) in viral RNA, bacterial riboswitches, and regulatory structures in the 5' and 3' untranslated regions (UTRs).

Strategies Design for Targeting RNA

Most of the ligands targeting RNA are basic and positively charged in normal physiological environments. In addition, these small molecules have a flat structure and bind well to the bases of RNA. Briefly, these drugs can be categorized into three main groups based on the structure of the RNA targeted:

• multiple closely packed helices of RNA.
• irregular and usually bulge-containing secondary structures.
• triplet repeats.

The first of these classes of drugs is considered to be at the forefront of current advances in RNA-targeted drug development. Unlike proteins, RNA consists mainly of four classes of nucleotide elements, which carry a large charge and are also more hydrophilic than proteins.RNA, after all, has folded three-dimensional structures, and these complex structures are expected to result in sufficient drug-forming conformations to allow small-molecule drug binding and recognition. Here are some examples of mechanisms of action of small molecule drugs targeting RNA:

• Small molecules bind directly to functional sites in RNA, where simple binding is sufficient to produce biological effects that precisely and predictably disrupt downstream biological processes.
• Binding to viral RNAs, such as HIV TAR and RRE RNAs, and IRES in HCV mRNA. these small molecules are able to interfere with viral infection processes.
• Target human miRNAs and selectively inhibit the production of pathogenic miRNAs by binding to functional structures in miRNA precursors.
• Inhibition of proteins that are not easily treatable, such as by targeting coding RNA to interfere with the translation of alpha-synuclein, thereby affecting the expression of proteins associated with neurodegenerative diseases.
• Regulation of splicing by small molecules, e.g., by targeting splicing regulatory elements of the MAPT gene to generate specific protein isoforms.
• Directly targeting the RNA degradation process.

Methods for Identifying or Screening Small Molecule RNA Binding Molecules

• RNA targets were incubated with a small molecule library. Unbound ligands are removed by size exclusion chromatography, and then bound ligands are identified by LC-MS.
• The fluorescence-based assay relies on the change in fluorescence when the small molecule binds to the RNA target.
• Microarray-based screening where a set of small molecules are immobilized to the array surface and incubated with labeled RNA targets, then washed and imaged to identify target-bound compounds.
• RNA binders can be identified and binding sites mapped using Chem-CLIP (chemical crosslinking and put-down).
• RNA-binding small molecules are identified by DEL. It is possible to simultaneously screen for binding to a target of interest and an associated RNA that is not required for binding, which are labeled with different fluorophores and identified and isolated by flow cytometry.
• Inforna is a lead identification strategy that compares structures present in cellular RNAs with a database of experimentally determined RNA-small molecule interactions.
• Structure-based small molecule design. Dependent on structural models of RNA or RNA-ligand complexes, both of which can be used for docking studies, while the latter can be used to optimize RNA-small molecule interactions.

Optimization Strategies for RNA-targeted Small Molecules

• Ligand-based molecular optimization, including classical SAR optimization and discovery of new backbones through pharmacophore.
• Structure-based molecular optimization, which relies on virtual screening of the structure of the RNA target and design based on ligand-RNA interactions.
• Sequence-based molecular optimization, which explores the differences between target and off-target structures and modifies the hit based on differentiated structural features (i.e., modular assembly or dimerization).

In recent years, the emergence of RNA drugs, such as siRNA, ASO, and mRNA drugs, has overturned the logic of traditional drug development. Compared with the few hundreds of protein drug targets, RNA can be said to be a blue ocean, and targeting RNA will greatly expand the choice of drug targets and open up a wide world for new drug development.