Abstract
Helicases are essential motor enzymes that couple nucleoside-triphosphate hydrolysis with DNA or RNA strand unwinding. Helicases are integral to replication, transcription, splicing, and translation of the genome, play crucial roles in the proliferation of cancer cells and propagation of viral pathogens, and are implicated in neurodegenerative diseases. Despite their therapeutic potential, drug discovery efforts targeting helicases face significant challenges due to their dynamic enzymatic cycles, the transient nature of their conformational states, and the conservation of their active sites. Analysis of cocrystal structures of inhibitor-helicase complexes revealed four distinct mechanisms of inhibition: allosteric, ATP-competitive, RNA-competitive, and interfacial inhibitors. While these static X-ray structures reveal potential binding pockets that may support the development of selective drugs, the application of advanced techniques such as cryo-EM, single-molecule analysis, and computational modeling will be essential for understanding helicase dynamics and designing effective inhibitors.