Combining Chemical Biology and Forward Genetics to Identify the Target of Anticancer Small Molecules

Oxaboroles exhibit diverse biological activity, including antimalarial and anti-parasitic activity. A benzoxaborole compound was found to exhibit potent anticancer activity, and my work discovered the anticancer mechanism of this compound. To tackle this question, two approaches were employed: chemical biology and forward genetics. The chemical biology approach revealed several binding partners of unknown consequences, but no clear cytotoxic targets. In the forward genetics approach, drug-resistant clones were generated in a mutagenic colorectal cancer cell line. Whole exome sequencing revealed mutations in CPSF3, a pre-mRNA 3'-end-processing endonuclease. Knock-in CRISPR/Cas9 experiments recapitulated resistance to the compound, increasing confidence that CPSF3 is the direct target of the benzoxaboroles. Recombinant CPSF3 initially appeared to cleave pre-mRNA in vitro, but non-specific RNAse activity made it difficult to interpret results. Click chemistry was employed to demonstrate direct binding of benzoxaboroles to CPSF3; competition experiments using this method served as evidence of a structure-activity relationship between the benzoxaboroles' CPSF3 binding and biological activity. Finally, transcription analyses show that CPSF3 inhibition by the benzoxaboroles results in mRNA read-through, and that this inhibition is significantly milder in clones exhibiting CPSF3 mutations. Overall, my work demonstrates that the anticancer benzoxaboroles cause cell death due to mRNA read-through induced by CPSF3 inhibition.