Novel Role of 3-Phosphoglycerate Kinase, a Glycolytic Enzyme, in the Activation of L-Nucleoside Analogs, a New Class of Anticancer and Antiviral Agents

Abstract

L-Nucleoside analogs are a new class of clinically active antiviral and anticancer agents. The phosphorylation of these analogs from diphosphate to triphosphate metabolites is crucial for their biological action. We studied the role of 3-phosphoglycerate kinase, a glycolytic enzyme, in the metabolism of L-nucleoside analogs, using small interfering RNAs to down-regulate the amount of this enzyme in HelaS3 and 2.2.15 cells, chosen as models for studying the impact of the enzyme on the anticancer and antihepatitis B virus activities of these analogs. Decrease in the expression of 3-phosphoglycerate kinase led to a corresponding decrease in the formation of the triphosphate metabolites of L-nucleoside analogs (but not D-nucleoside analogs), resulting in detrimental effects on their activity. The enzyme is important for generating as well as maintaining the steady state levels of L-nucleotides in the cells, thereby playing a key role in the activity of L-nucleoside analogs against human immunodeficiency virus, hepatitis B virus, and cancer. This study also indicates a structure-based distinction in the metabolism of L-and D-nucleoside analogs, disputing the classic notion that nucleoside diphosphate kinases are responsible for the phosphorylation of all classes of nucleoside analog diphosphates.L-Nucleoside analogs are an emerging class of anticancer and antiviral agents that are being used clinically or are under advanced clinical trials (1, 2). Structurally, L-nucleoside analogs are mirror images of the natural D-nucleosides. Despite the unnatural L-configuration, these analogs are able to utilize the salvage pathway of deoxynucleoside metabolism to be phosphorylated to the active triphosphate form (3, 4). Similar to the D-nucleoside analog counterparts, L-nucleoside analog triphosphates exert their pharmacological activity through incorporation into cellular or viral DNA, leading to chain termination or inhibition of viral reverse transcriptase or DNA polymerase (5-10). In the past decade, the realization that L-nucleosides are capable of being activated in cells has led to the discovery of several antiviral pyrimidine L-deoxynucleoside analogs. The first two steps in the metabolism of L-nucleoside analogs seem to be similar to those of its D-nucleoside counterparts. The cytidine analogs, 3TC, L-OddC, L-Fd4C, and gemcitabine, are phosphorylated by cytoplasmic deoxycytidine kinase to the monophosphate metabolite, whereas L-FMAU can be phosphorylated by both deoxycytidine kinase and thymidine kinase (4, 10, 23-25). The cytidine analogs are then phosphorylated by cytidine/uridine monophosphate kinase to the respective diphosphates, and thymidine analogs are phosphorylated by thymidylate kinase (4, 26 -28). The last step of pyrimidine nucleoside analog metabolism was assumed to be carried out by nucleoside diphosphate kinase (NDPK); however, its role in the phosphorylation of L-nucleoside analog diphosphates remained doubtful (29 -31). We examined this in further detail by studying the phosphorylation of pyrimidine D...

Nucleoside analogs: molecular mechanisms signaling cell death

“…Although the mono-, di-and triphosphate forms of troxacitabine accumulate in cells, it is interesting that the diphosphate form predominates (Grove et al, 1995). This is likely because of the less efficient phosphorylation of troxacitabine diphosphate to the active metabolite, troxacitabine triphosphate, by 3-phosphoglycerate kinase rather than by nucleoside diphosphate kinase (Krishnan et al, 2002(Krishnan et al, , 2003. Deficiencies in nucleoside transporters do not cause increases in drug resistance, suggesting that cellular uptake of troxacitabine is mainly by passive diffusion (Grove and Cheng, 1996;Gourdeau et al, 2001b).…”

Comparison of the Phosphorylation of 4′-Ethynyl 2′,3′-Dihydro-3′-Deoxythymidine with That of Other Anti-Human Immunodeficiency Virus Thymidine Analogs

“…The metabolism of the radiolabeled compounds was monitored according to previously established procedures (17). Briefly, CEM cells were seeded at 10 6 cells/ml and were incubated with 2 M (75 mCi/mmol) of various radiolabeled compounds.…”

“…Among them, nm23-H1 and nm23-H2, the predominant isoforms residing in the cytoplasm (18), have been characterized in detail with regard to their kinetic properties and catalytic mechanisms (29). Other enzymes that are able to phosphorylate nucleoside diphosphates include creatine kinase (CK), pyruvate kinase (PK), and 3-phosphoglycerate kinase (PGK) (14)(15)(16)(17). Our previous studies demonstrated that TK1 is the enzyme responsible for the phosphorylation of 4Ј-ethynyl D4T to 4Ј-ethynyl D4TMP (6).…”

A Spring-loaded Release Mechanism Regulates Domain Movement and Catalysis in Phosphoglycerate Kinase

“…In addition to its metabolic role, the phosphoryl transfer activity of PGK is important in the processing of antiretroviral prodrugs that take the form of L-nucleoside analogues (2). The rate-limiting step in the in vivo activation of such compounds has been demonstrated to be the addition of a third phosphate by PGK (3). A third activity of PGK is as a signaling molecule in chordates.…”