a b s t r a c t 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 K i = 250 lM (95% CI: 106-405 lM) and dose-dependently increased the rate of the ATP hydrolysis with K M = 112 lM (95% CI: 28-195 lM). The kinetic scheme consistent with this experimental data is proposed. Thymidine kinase (TK, EC 2.7.1.21) catalyzes phosphorylation of thymidine to TMP and plays a significant role in the synthesis of DNA and cell division. It operates in most eukaryotic cells as well as in several viruses (HSV, HCMV, EBV) [1]. Viral TKs significantly differ in both specificity and selectivity from the human ones and thus represent an attractive target for antiviral therapy [2].Thymidine kinase of Herpes Simplex Virus type 1 (HSV-TK) was successfully targeted by both selective inhibitors and substrate prodrugs which are enzymatically converted to toxic compounds. One of the most common antiviral drug, Acyclovir (ACV), is a HSV-TK substrate which is converted to acyclo-guanosine monophosphate (Acyclo-GMP). Further phosphorylation converts it to Acyclo-GTP that lacks 3 0 -OH and after incorporation into viral DNA terminates its synthesis [3]. Compounds that possess their antiviral activity through the direct inhibition of HSV-TK gained a lot of attention [4][5][6][7] however none of them is clinically approved.Here we report a novel mode of HSV-TK activity modulation by a small molecule which results in a switch from kinase to ATPase catalytic activity. Initially we aimed at identification of novel fragment HSV-TK inhibitors using virtual fragment screening approach [8][9][10]. Model of the HSV-TK was constructed from PDB ID 1KI3[11] (TK UL23 from type 1 HSV strain 17), while highly homologous recombinant TK UL23 from acyclovir-sensitive strain L2 of type 1 HSV was used in further experiments [12]. The activity of the predicted hits was evaluated in the model reaction of acyclovir phosphorylation by HSV-TK (Supporting information) which closely resembles the properties of the thymidine phosphorylation. Several selected compounds demonstrated IC 50 in submillimolar range which is typical for fragment drug candidates [13]. One of the hits (2,3-dihydroxy-quinoxaline, DHQ, Fig. 1) not only inhibited the phosphorylation of the ACV but also promoted the formation of the additional 32 P-containing spot other than ATP and ACV-P on the chromatogram (Fig. 2). Apparently it was not the phospho-DHQ since non-enzymatic phosphorylation of DHQ with POCl 3 produced a very unstable compound that had different R f . Treatment of the thin layer chromatography (TLC) plate with molybdate solution lead to the specific blue coloring of the unknown spot, clearly indicating that it was the orthophosphate. Incubations of the enzyme-free mixtures containing only DHQ (0-400 lM) and [c-32 P] ATP demonstrated no phosphate formation (Fig. 5) thus indicating...