Most drugs currently approved or in clinical trials target proteins or DNA. Not many target RNA as there are different types of RNA in the cell and their structure and function are not very clearly linked to disease etiology. RNA has no known endogenous agonists or antagonists and although there are more than 1,500 proteins known to bind RNA, there is little information on what those proteins do and how they do it. Until recently, RNA was regraded merely as a translator of genetic information encoded in the DNA. This notion however is rapidly changing, and there is now evidence that targeting RNA for therapeutic purposes can be very lucrative. The SARS-CoV-2 pandemic and the likelihood of future viral pandemics has further escalated the interest in finding ways to target RNA. “We have 2,000–3,000 proteins that are considered druggable, but by targeting the RNA you can affect many more proteins and diseases,” says Hasane Ratni, Ph.D., Distinguished Scientist and Team Leader in Medicinal Chemistry at F. Hoffmann-La Roche.
Small molecules versus oligos to target RNA
Traditionally, RNA has been modulated using oligonucleotides such as small interfering RNA (siRNA), antisense, aptamers, and other RNA moieties. However, the excitement in the field now is due to the development of small molecule drugs that target RNA. While siRNA and other RNA modalities have been successful in binding and inhibiting RNA, the bioavailability and cellular penetration of these molecules has been quite challenging. Being large negatively charged oligonucleotides, they are highly susceptible to degradation by RNAses and have limited access to intracellular targets. Small molecules that target unique structural sites on the RNA are certainly more attractive from a bioavailability and delivery perspective.
The choice of small molecules versus oligonucleotides to modulate the RNA depends on the disease. “It comes down to the target profile, location, and how quickly you want to get there,” says Natalie Dales, Ph.D., Director, Global Discovery Chemistry, Novartis. “Small molecules are more appropriate for CNS indications where you are crossing the blood-brain barrier, or if you are going after oral or more controlled delivery. The choice also depends on what other drugs the patient is taking and the disease severity.”
Binding is only the first step
Genetic, biochemical, and epidemiology studies are often used to narrow down the RNA targets that may play a role in a certain disease. That role is further validated using loss/gain-of-function studies, phenotypic screening, and by using animal models. Techniques like surface plasmon resonance (SPR), thermal shift assays, nuclear magnetic resonance (NMR), and mass spectrometry (MS) are then used to find small molecules that can bind to that RNA target and inhibit its effects. Or phenotypic screening is performed using an RNA-targeting library to find small molecules that show the desired physiological effect. According to Ratni, many small biotech companies have developed platforms to find RNA binding small molecules but providing a validated RNA target or a clinical candidate is what is most important. “Finding small molecules that can bind to a pocket in the RNA molecule is tricky but demonstrating that the binding translates to changes in protein function is the most challenging part. If there was a methodology to achieve this in a rational way, it will revolutionize drug discovery.”
Jennifer Petter, Ph.D., Founder and CSO of Arrakis Therapeutics, agrees that finding good targets for RNA and finding mechanisms for inhibition is the biggest challenge. “We now need people to tell us what RNA targets to go after and how binding to the target matters.
Arrakis’ approach is to find the gene associated with the disease, then find the sequence on the gene that is functionally relevant, use this to build the RNA with the right structure, perform the screen, and find the molecules that go into the cell and bind the RNA. “We use size exclusion chromatography-mass spectrometry (SEC-MS) to find molecules that bind to RNA,” says Petter. “But the technique doesn’t tell you if it alters the RNA to do what you want it to do. You need secondary assays to tell you that.”
Binding does not imply specificity
Once the RNA target is validated, it’s important to find out whether the target is easily accessible. Is it abundant enough in the disease cells and are there ways (structural motifs) on the RNA for small molecule drugs to bind and cause functional changes? “The very early RNA discovery work involves a lot of biophysical techniques to validate the compound interaction with the RNA,” says Jane Withka, Ph.D., Director and Collaboration Lead in the Discovery Network Group at Pfizer. It is difficult to get an accurate structure of the RNA with tools like NMR that can only be used with small segments of RNA, or with X-ray crystallography, as the RNA molecule is quite floppy. Advanced techniques like cryogenic electron microscopy (CryoEM) are needed to provide high-resolution structural features of RNA to identify 3D pockets for improved small molecule binding. There are also predictive tools like SHAPE (for selective 2′-hydroxyl acylation analyzed by primer extension) that can help identify the 3D pockets and predict the likelihood of the small molecule binding to the RNA, although, some of these predictions yield molecules that are often not suitable for drug discovery.
After binding, the next question is around specificity of that binding. Is it specific enough to avoid other targets in the cell thereby minimizing off-target adverse events? Structurally RNA is very similar to the DNA, in that they are both negatively charged oligonucleotides. Hence, targeting RNA using small molecules can be challenging as these molecules often bind both DNA and RNA. Safety also remains a valid concern when trying to make changes at the DNA or RNA level.
What’s next in RNA targeting
Development of RNA, both as a drug target and as a therapeutic, will depend on advances in the technologies for studying RNA structure and function. Probing the role of non-coding RNA, nonsense-mediated mechanisms, and other aspects of RNA biology that are not directly involved in protein production will also shed more light on the cellular complexities. “We need more insight into the understanding of G-quads and pseudoknots and other structures and the role of those structures in RNA function and stability, which will likely open up other areas for RNA intervention,” says Petter.
Currently the small molecules that bind RNA inhibit its function by blocking the translation of the protein of interest. Soon we could find approaches to target RNA to not only inhibit, but also activate, relevant proteins. There is work underway currently to find ways to degrade RNA, using techniques like ribonuclease targeting chimeras (RIBOTACs), which is similar to proteolysis targeting chimeras (PROTACs) recruiting E3 ligases to degrade proteins. Finding ways to achieve tissue specificity and selectivity will continue to be an area of intense research, whether the target is the RNA or a RNA-binding protein. Targeting RNA is an exciting field to be in, says Withka. “A few years ago, people thought you can’t target a kinase and ultimately we found ways to do it. We are at that stage with RNA today.”