While proteins have traditionally been the primary target class for drug discovery, the landscape is rapidly evolving. RNA targets have recently garnered significant attention, driven by advances in RNA interference technologies and the development of small molecule drugs like Risdiplam. Despite the progress in RNAi drugs, small molecules remain a crucial component in drug discovery due to their unique advantages such as cell permeability, stability, and the ability to modulate a broad range of biological targets. As a powerful tool for small molecule ligand discovery, DNA-encoded libraries (DELs) for RNA target hit identification presents unique challenges. HitGen's DEL technology is at the forefront of these efforts, uniquely equipped to navigate these challenges and pioneer the discovery of small molecule ligands for RNA targets.
Figure 1. Small molecules targeting RNA (by mechanism of action and by RNA types)
DEL for RNA: Current challenges
• Dynamic nature of RNA structures: RNA molecules are known for their dynamic nature, often forming complex secondary and tertiary structures. These structures can be vulnerable to certain DEL selection buffers, which can stabilize one conformation over others. This can lead to the enrichment of features in the DEL selection based on a single conformation, complicating downstream off-DNA compound confirmation due to the dynamic nature of RNA in physiological conditions.
• Interactions between DNA tags and RNA targets: DELs rely on affinity-based methods for ligand discovery. However, interactions between the DNA tags of the DELs and RNA targets can introduce a significant number of false positive signals. This makes it akin to finding a needle (true positive) in a haystack (false positive signals), adding a layer of complexity to the discovery process.
• Underexplored chemical space for RNA ligands: The knowledge about RNA binders is relatively limited when compared to protein binders. The chemical space for RNA ligands remains largely underexplored, posing a challenge for the identification of effective small molecule ligands for RNA targets.
Pioneering small molecule ligand discovery for RNA targets at HitGen
At HitGen, we endeavor to overcome the above-mentioned challenges and to deliver robust small molecule ligands for RNA targets. Our approach includes:
• Assessment of RNA conformation under various selection conditions beforehand: Prior to screening experiments, we conduct extensive bioinformatic analyses of RNA structures under different selection conditions. This allows us to identify and select the conditions that do not or only minimally impact RNA folding, ensuring the integrity and functionality of the RNA targets during the screening process.
• Innovative screening methods and informatics approaches to control the interference of the DNA:RNA interaction: To minimize false positive signals during DEL screening, we deploy a variety of strategies such as the addition of competing oligonucleotides during both incubation and elution steps. In addition, proprietary algorithms were developed at HitGen to decrease the impact of the DNA:RNA interaction.
• Exploring new frontiers with HitGen's DEL library of one trillion compounds: Our newest DEL library with over one trillion compounds provides an expanded repertoire to identify RNA ligands that may not fit into the traditional chemical space, which significantly increases the likelihood to discover robust small molecule ligands for RNA targets.
• Collaborative and transparent partnership: At HitGen, we believe in the power of collaboration, and we work closely with our partners to ensure the success of their discovery projects. Our commitment to transparency means we share data openly and completely, but exclusively with our collaborators. Therefore, we not only deliver successful scientific findings, but also warrant the safety of their intelligence properties.
Showcasing success in small molecule ligand discovery for RNA targets
Our platform has already shown promising results in small molecule ligand discovery for RNA targets. We also published the detailed methods for applying DEL on RNA targets in Nucleic Acids Research (Nucleic Acids Res. 2022 Jul 8;50(12):e67.) Here are a few examples of our successful projects:
• Novel inhibitors for E. coli FMN riboswitch: The FMN riboswitch is a class of RNA element that plays a crucial role in controlling bacterial growth by regulating gene expression in response to fluctuations in the cellular environment. Our platform has successfully identified novel inhibitors for the E. coli FMN riboswitch that are comparable the reference compound, ribocil C (Figure 2). These findings not only validate our approach but also open up new avenues for the development of effective antibacterial agents.
Figure 2. A-C) Representative features and compounds identified from HitGen’s DEL for RNA selection. D) ITC binding parameters and FMN inhibition potency.
• Allele-specific RNA binders: In a significant breakthrough, our platform has been utilized to identify RNA ligands that demonstrate selective binding to a specific RNA motif, while effectively discriminating against a mutant allele with only a single nucleotide difference. This level of precision is achieved through careful design and incorporation of counter targets, which guide the selection of ligands with the desired specificity. This project is a testament to the power of our platform in addressing complex challenges in RNA-targeted drug discovery. As this project is a collaboration, the detailed data are confidential at this time. However, the success of this endeavor underscores our ability to work in partnership with other entities, respecting confidentiality and data security, while still achieving remarkable results.
With HitGen's DEL technology, the possibilities for small molecule ligand discovery for RNA targets are virtually limitless. Contact us and let our team of experts help you unlock the full potential of RNA targets for your drug discovery programs.
Building C2, NO.8, Huigu 1st East Road
Tianfu International Bio-Town,
Shuangliu District, Chengdu City,
Sichuan Province,P.R. China
Tel : +86-28-8519-7385
HitGen Pharmaceuticals Inc.(US Subsidiary)
Tel : +1-(508)-840-9646
Granta Park,Great Abington
Tel : +44(0)1223-895555