Discovery of Drug-like Inhibitors of the Human Caf1/CNOT7 poly(A)-Selective Nuclease Using Compound Screening

The human Ccr4-Not complex is a central regulator of post-transcriptional gene regulation, impacting on translation and mRNA degradation. In mRNA degradation, Ccr4-Not participates in the shortening of the mRNA poly(A)-tail via two catalytic subunits. The Caf1 nuclease is encoded by the highly similar paralogues CNOT7 or CNOT8. In addition to its poly(A)-specific ribonuclease activity, this subunit also provides a structural role by binding Ccr4, the second catalytic nuclease subunit encoded by the paralogues CNOT6 or CNOT6L. To facilitate investigations into the roles of the Caf1 subunit, and to complement genetic tools, we set out to identify inhibitors of the enzymatic activity of Caf1/CNOT7. To this end, we screened a library of 10,880 chemically diverse, drug-like compounds using a fluorescence-based biochemical assay. This effort led to the discovery of 15 inhibitors of Caf1/CNOT7 with biochemical IC₅₀ values below 25 μM. Molecular docking was performed to explore potential binding modes of these compounds. The compounds reported here may be useful to differentiate between catalytic and non-catalytic roles of Caf1/CNOT7. In addition, they may be valuable starting points for the development of more potent inhibitors of the Caf1/CNOT7 poly(A)-selective ribonuclease.