Specificity, cooperativity, synergy, and mechanisms of splice-modifying drugs.

Abstract

Drugs 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 quantitatively describes the genomic specificity, sequence specificity, 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 drugs developed for treating spinal muscular atrophy and familial dysautonomia. The results quantitatively define the specificities of risdiplam and branaplam for 5’ splice site sequences, strongly suggest that branaplam recognizes 5’ splice sites in two distinct molecular conformations, and disprove the prevailing two-site hypothesis for risdiplam activity at SMN2 exon 7. Remarkably, the results also show that single-drug cooperativity and multi-drug synergy are widespread among splice-modifying drugs more generally. 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.