Specificity, synergy, and mechanisms of splice-modifying drugs

Drugs that target pre-mRNA splicing hold great therapeutic potential, but the mechanistic understanding of how these drugs function 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 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 at SMN2 exon 7. The results also show, more generally, that single-drug cooperativity and multi-drug synergy are widespread among splice-modifying drugs. 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. Overall design: Comparative alternative splicing profiling analysis of RNA-seq data for HeLa cells treated with splice modifying drugs risdiplam and branamplan, and DMSO as control Massively parallel splicing assay of 5'splice site sequences in HeLa cells treated with splice modifying drugs risdiplam and branamplan, and DMSO as control in 3 different genetic contexts (MPSA samples)