Experimental and structural evidence that herpes 1 kinase and cellular DNA polymerase(s) discriminate on the basis of sugar pucker.
Two isomers of methanocarba (MC) thymidine (T), one an effective antiherpes agent with the pseudosugar moiety locked in the North (N) hemisphere of the pseudorotational cycle (1a, N-MCT) and the other an inactive isomer locked in the antipodean South (S) conformation (1b, S-MCT) were used to determine whether kinases and polymerases discriminate between their substrates on the basis of sugar conformation. A combined solid-state and solution conformational analysis of both compounds, coupled with the direct measurement of mono-, di-, and triphosphate levels in control cells, cells infected with the Herpes simplex virus, or cells transfected with the corresponding viral kinase gene (HSV-tk), suggests that kinases prefer substrates that adopt the S sugar conformation. On the other hand, the cellular DNA polymerase(s) of a murine tumor cell line transfected with HSV-tk incorporated almost exclusively the triphosphate of the locked N conformer (N-MCTTP), notwithstanding the presence of higher triphosphate levels of the S-conformer (S-MCTTP).
Marquez
Victor E
VE
Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer Institute at Frederick, National Institutes of Health, 376 Boyles St., Frederick, MD, 21702, USA. marquezv@dc37a.nci.nih.gov
Ben-Kasus
Tsipi
T
Barchi
Joseph J
JJ
Green
Karen M
KM
Nicklaus
Marc C
MC
Agbaria
Riad
R
D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D000998:Antiviral Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D004247:DNA / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D018259:Herpesvirus 1, Human / Q000201:enzymology* / Q000235:genetics; D051379:Mice; D008958:Models, Molecular; D008968:Molecular Conformation; D019906:Nuclear Magnetic Resonance, Biomolecular; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013936:Thymidine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D013937:Thymidine Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014709:Vero Cells
D000970:Antineoplastic Agents; D000998:Antiviral Agents; D004247:DNA; D013937:Thymidine Kinase; D004259:DNA-Directed DNA Polymerase; C469995:(north)-methanocarbathymidine; D013936:Thymidine