Specificity, synergy, and mechanisms of splice-modifying drugs

Abstract

AbstractDrugs that target pre-mRNA splicing hold great therapeutic potential, but the quantitative understanding of how these drugs work is limited. Here we introduce a biophysical-modeling framework that can quantitatively describe the sequence-specific and concentration-dependent behavior of splice-modifying drugs. Using massively parallel splicing assays, RNA-seq experiments, and precision dose-response curves, we apply this framework to two small-molecule drugs, risdiplam and branaplam, developed for treating spinal muscular atrophy. The results quantitatively define the specificities of risdiplam and branaplam for 5’ splice site sequences, suggest that branaplam recognizes 5’ splice sites via two distinct interaction modes, and disprove the prevailing two-site hypothesis for risdiplam activity atSMN2exon 7. The results also show, more generally, that single-drug cooperativity and multi-drug synergy are widespread among both small-molecule drugs and antisense-oligonucleotide drugs that promote exon inclusion. Our biophysical-modeling approach thus clarifies the mechanisms of existing splice-modifying treatments and provides a quantitative basis for the rational development of new therapies.