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内容記述 |
Favipiravir is an anti-influenza prodrug that is metabolized to its phosphoribosylated form, favipiravir-ribofuranosyl-5′-monophosphate (favipiravir-RMP), by human endogenous enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). This enzymatic reaction is the rate-determining step in generating the active form of favipiravir, making it important to understand the molecular mechanisms underlying the HGPRT-catalyzed RMP-modification of favipiravir. However, the pharmacokinetics of this reaction have not been fully elucidated, despite X-ray crystallographic studies on the HGPRT-favipiravir complex. Here, we identified functional hot-spot residues in HGPRT that play important roles in the enzymatic conversion of favipiravir to favipiravir-RMP. Real-time monitoring of the HGPRT reaction via ligand-observed solution NMR experiments, biochemical mutagenesis of HGPRT, and computational calculations and molecular dynamics simulations, allowed us to investigate the free binding energetics and structural properties of the interaction between HGPRT and favipiravir-RMP. This powerful hybrid experimental strategy allows the identification of functional hot-spot residues in the enzyme and provide complementary structural biological information. This approach could be universally applicable to investigating drug-protein interaction modes. |
