2,3-Dihydroxy-quinoxaline induces ATPase activity of Herpes Simplex Virus thymidine kinase

•2,3-Dihydroxy-quinoxaline inhibits acyclovir phosphorylation by herpesvirus thymidine kinase.•2,3-Dihydroxy-quinoxaline promotes ATPase activity of herpesvirus thymidine kinase.•This promotion is dose-dependent and follows the Michaelis–Menten scheme.•We propose a kinetic scheme for the whole process that is consistent with the experimental data. 2,3-Dihydroxy-quinoxaline, a small molecule that promotes ATPase catalytic activity of Herpes Simplex Virus thymidine kinase (HSV-TK), was identified by virtual screening. This compound competitively inhibited HSV-TK catalyzed phosphorylation of acyclovir with Ki=250μM (95% CI: 106–405μM) and dose-dependently increased the rate of the ATP hydrolysis with KM=112μM (95% CI: 28–195μM). The kinetic scheme consistent with this experimental data is proposed.