Authentication of Hippuristanol-eIF4A1 Target Engagement Facilitates Identification of eIF4A1 Helicase Dependencies within 5' Leader Regions

Hippuristanol (Hipp) is a natural product that selectively inhibits protein synthesis by targeting eukaryotic initiation factor (eIF) 4A, a DEAD-box RNA helicase required for ribosome recruitmentt o mRNA templates. Using a CRISPR/Cas9-based variomics screen, we identify functional eIF4A1 Hipp-resistant alleles, which in turn allow us to link the translation-inhibitory and cytotoxic properties of Hipp to eIF4A1 target-engagement. Genome-wide translational profiling in the absence or presence of Hipp (~EC50) were undertaken and our validation studies provided insight into structure-activity relationships of eIF4A-dependent mRNAs. Overall design: Ribosome Profiling and RNA-Sequencing of DMSO and Hippuristanol treated HAP1 cells.