Table 4.

Tools and challenges in developing small-molecule therapeutics targeting RNA

Development step	Tools	Challenges
Identify druggable RNA targets	Identify regions in an RNA target that are stably folded by using evolution and prediction restrained by structure mapping experiments Hypothesize a MOA for a binding compound Convert a binder into a degrader if binding alone is not functional	Validating a predicted RNA structure in cells and identifying factors to prioritize targets that are more easily drugged
Identify small molecule binders	Complete sequence-based design and identify on- and off-targets and routes for optimization Screen compound collections to find binders, considering screening on- and off-targets	Defining chemotypes in compounds that bind RNA selectively Performing quantitative structure–activity relationships for RNA targets
Evaluate bioactivity of small molecules in cells	Study the effect of RNA target-associated phenotypes and validate by using gain- or loss-of-function Use Chem-CLIP to define cellular on- and off-targets and evaluate the selectivity on the transcriptome and the proteome; correlate Chem-CLIP with -omics studies to define optimization plan	Correlating binding to function is not straightforward as not all binders will be biologically functional
Structure-based lead design and optimization	Solve structure of complex by NMR spectroscopy and X-ray crystallography, etc. Study pharmacophore model to define features in binding compounds and expand chemical space for target by using chemical similarity searching of pharmacophore and structure-based drug design	Structure-based drug design and docking for RNA is different than for protein, i.e., electrostatic interactions are a significant driver Force fields need development, as there are limited examples of driving potency and specificity using these approaches
Evaluate efficacy and PK in animal models	Study disease defect in model organisms, including transgenic models In vitro and in vivo PK assays are well established	Physicochemical properties that potently and specifically affect RNA are unknown; need to be open minded and empirical with compounds, especially those outside rule of five space Studies in model organisms can be challenging especially for noncoding RNAs owing to differences in the human sequence and structure Varied modes of action can complicate PK and pharmacodynamic profiles
Preclinical and clinical studies	Study compound activity in patient tissues, available through many repositories RNA-seq can be incorporated into clinical development pipeline to study efficacy, toxicity, potential off-targets	Should the pipeline of tests used for oligonucleotide-based medicines be altered? Can this process be streamlined by using RNA-seq and other tools? Differences in sequence and structure in human versus mouse or other species should be considered in efficacy and toxicity studies

MOA, mode of action; PK, pharmacokinetic(s); RNA-seq, RNA sequencing.