ddhCTP produced by the radical‐SAM activity of RSAD2 (viperin) inhibits the NAD+‐dependent activity of enzymes to modulate metabolism

Radical S‐adenosylmethionine (SAM) domain‐containing protein 2 (RSAD2; viperin) is a key enzyme in innate immune responses that is highly expressed in response to viral infection and inflammatory stimuli in many cell types. Recently, it was found that RSAD2 catalyses transformation of cytidine triphosphate (CTP) to its analogue 3′‐deoxy‐3′,4′‐didehydro‐CTP (ddhCTP). The cellular function of this metabolite is unknown. Here, we analysed the extra‐ and intracellular metabolite levels in human induced pluripotent stem cell (hiPSC)‐derived macrophages using high‐resolution LC‐MS/MS. The results together with biochemical assays and molecular docking simulations revealed that ddhCTP inhibits the NAD+‐dependent activity of enzymes including that of the housekeeping enzyme glyceraldehyde 3‐phosphate dehydrogenase (GAPDH). We propose that ddhCTP regulates cellular metabolism in response to inflammatory stimuli such as viral infection, pointing to a broader function of RSAD2 than previously thought.